Abstract

摘要

Fibroblast-like synoviocytes (FLSs) and osteoclasts are central cells in the maintenance of joint homeostasis. Rheumatoid arthritis (RA) is a chronic inflammatory disease of joints that induces cytokine-activated FLSs and progressive bone erosion. Interactions between FLSs and other cells, such as T cells and B cells, have been recognized in the development of RA.

成纤维细胞样滑膜细胞（FLS）和破骨细胞是维持关节稳态的中心细胞。类风湿性关节炎（RA）是一种关节慢性炎症性疾病，可诱导细胞因子激活的FLS和进行性骨侵蚀。FLS与其他细胞（例如T细胞和B细胞）之间的相互作用已在RA的发展中得到认可。

Here we hypothesized that calcium released from bone by mature osteoclasts might activate FLSs, which are also affected by inflammatory cytokines in the inflamed synovium. Osteoclastogenesis occurs in the presence of cytokine-stimulated FLS medium, and calcium released from the bone disc activates FLS migration.

在这里，我们假设成熟破骨细胞从骨骼释放的钙可能激活FLS，FLS也受到发炎滑膜中炎性细胞因子的影响。破骨细胞生成发生在细胞因子刺激的FLS培养基的存在下，并且从骨盘释放的钙激活FLS迁移。

We first investigated the calcium and cytokine feedback loop between FLSs and osteoclast maturation. Moreover, by addressing the role of the sodium-bicarbonate cotransporter NBCn1 in osteoclastogenesis, we found that the inhibition of NBCn1 attenuated the infinite calcium and cytokine feedback loop between FLSs and osteoclasts.

我们首先研究了FLS和破骨细胞成熟之间的钙和细胞因子反馈环。此外，通过解决碳酸氢钠共转运蛋白NBCn1在破骨细胞生成中的作用，我们发现NBCn1的抑制减弱了FLS和破骨细胞之间的无限钙和细胞因子反馈环。

In a collagen-induced arthritis mouse model, the inhibition of NBC reduced the RA pathological phenotype and bone resorption area in the femur. These results suggest that modulation of the crosstalk between FLSs and osteoclasts by inhibiting the calcium and cytokine feedback loop could be considered to develop pioneering strategies to combat RA severity and dysregulated bone homeostasis..

在胶原诱导的关节炎小鼠模型中，NBC的抑制降低了股骨中的RA病理表型和骨吸收面积。。。

Introduction

导言

Rheumatoid arthritis (RA) is a systemic inflammatory disease that causes pain, stiffness, inflammation and tissue damage, such as joint swelling and bone destruction. Structural changes in joints have been observed in patients with RA symptoms

类风湿性关节炎（RA）是一种全身性炎症性疾病，可引起疼痛，僵硬，炎症和组织损伤，如关节肿胀和骨质破坏。RA症状患者的关节结构发生了变化

1

1

,

,

2

2

,

,

3

3

. The pathological characteristics of RA include synovial inflammation, joint swelling, fibroblast-like synoviocyte (FLS) hyperplasia, pannus formation and bone destruction

RA的病理特征包括滑膜炎症，关节肿胀，成纤维细胞样滑膜细胞（FLS）增生，血管翳形成和骨质破坏

4

4

. Physiologically, multiple aspects of RA mechanisms have been elucidated, and several clinical advancements have reduced disease symptoms. However, the complexity of RA and the precise mechanism involved have not been fully elucidated.

生理学上，RA机制的多个方面已经阐明，并且一些临床进展已经减少了疾病症状。然而，RA的复杂性和所涉及的确切机制尚未完全阐明。

The inflamed synovium consists of various signaling components, including those involved in the immune response, metabolism, signal transduction, transport and apoptosis

发炎的滑膜由各种信号成分组成，包括参与免疫反应、代谢、信号转导、转运和凋亡的信号成分

5

5

. Bone damage in patients with RA is also related to disease severity. The synovium directly contacts bone, and bone-erosive lesions are observed in patients with RA

RA患者的骨损伤也与疾病的严重程度有关。滑膜直接接触骨骼，RA患者可见骨侵蚀性病变

6

6

,

,

7

7

. Although clinical treatments for RA improve these signaling activities, combination treatments often attenuate disease severity; however, some individuals are nonresponsive to these therapies

尽管RA的临床治疗改善了这些信号传导活性，但联合治疗通常会减轻疾病的严重程度；然而，一些人对这些疗法没有反应

2

2

. Notably, clinical trials have yielded unsatisfactory results, including adverse effects, with combination therapies. Based on the existing knowledge regarding combination treatment and no responsiveness, we determined whether a convergent target exists in the current mechanisms of RA to treat the bone and inflamed synovium..

值得注意的是，联合治疗的临床试验产生了不令人满意的结果，包括不良反应。基于关于联合治疗和无反应性的现有知识，我们确定RA目前治疗骨骼和发炎滑膜的机制中是否存在趋同靶点。。

Ca

加利福尼亚州

2+

2+

is a global signaling messenger. Notably, Ca

是全球信号信使。值得注意的是，Ca

2+

2+

signaling plays a pivotal role in bone remodeling. Bone is a major source of Ca

信号在骨重塑中起着关键作用。骨骼是钙的主要来源

2+

2+

, and cellular Ca

，以及细胞Ca

2+

2+

stores, such as those in the endoplasmic reticulum or nucleus, are sources of signaling. Premature osteoclasts are differentiated upon stimulation with bone remodeling factors, such as macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL), and induce a type of cellular fusion termed osteoclastogenesis.

储存，如内质网或细胞核中的储存，是信号的来源。早产破骨细胞在骨重塑因子（如巨噬细胞集落刺激因子（M-CSF）和核因子-κB受体激活因子（NF-κB）配体（RANKL））刺激下分化，并诱导一种称为破骨细胞生成的细胞融合。

8

8

,

,

9

9

,

,

10

10

,

,

11

11

. Subsequently, multinucleated giant cells of osteoclasts are formed, and the bone is resorbed

随后，破骨细胞的多核巨细胞形成，骨骼被吸收

11

11

. We hypothesized that Ca

我们假设Ca

2+

2+

released from the bone might activate FLSs, which are also affected by various inflammatory cytokines in the inflamed synovium. Activated FLSs cause hyperplasia, cartilage degradation and bone destruction. Thus, in this study, we designed the experimental conditions to demonstrate that free Ca

从骨骼释放可能会激活FLS，FLS也会受到发炎滑膜中各种炎性细胞因子的影响。活化的FLS会导致增生，软骨降解和骨骼破坏。因此，在这项研究中，我们设计了实验条件来证明游离Ca

2+

2+

derived from bone and inflammatory cytokines mediate circulating feedback signaling through the cytokine loops between FLSs and osteoclasts to exacerbate bone damage in individuals with RA.

源自骨和炎性细胞因子的细胞因子通过FLS和破骨细胞之间的细胞因子环介导循环反馈信号传导，从而加剧RA患者的骨损伤。

Previously, we reported that inflammatory cytokine-mediated FLS migration occurs via the involvement of the electroneutral type (1 sodium/1 bicarbonate) of the sodium‒bicarbonate cotransporter (NBCn)1 (ref.

以前，我们报道炎性细胞因子介导的FLS迁移是通过参与碳酸氢钠共转运蛋白（NBCn）1的电中性类型（1钠/1碳酸氢盐）发生的（参考文献）。

12

12

). Treatment with the NBC inhibitor S0859 inhibited cytokine-mediated FLS aggressiveness. In the present study, we investigated a potential strategy against cytokine feedback loops in RA by modulating bicarbonate transporters. We determined the role of NBCn1 in osteoclastogenesis and whether the inhibition of NBC reduces the infinite cytokine feedback loop between FLSs and osteoclasts.

)。用NBC抑制剂S0859治疗可抑制细胞因子介导的FLS侵袭性。在本研究中，我们通过调节碳酸氢盐转运蛋白研究了RA中针对细胞因子反馈环的潜在策略。我们确定了NBCn1在破骨细胞生成中的作用，以及NBC的抑制是否减少了FLS和破骨细胞之间的无限细胞因子反馈环。

Moreover, we verified the inhibitory effect of S0859 on bone damage in a collagen-induced arthritis (CIA) mouse model. Overall, modulation of the crosstalk between FLSs and osteoclasts through NBCn1 could be considered a pioneering strategy to attenuate RA severity and dysregulated bone homeostasis..

此外，我们验证了S0859对胶原诱导的关节炎（CIA）小鼠模型中骨损伤的抑制作用。总体而言，通过NBCn1调节FLS和破骨细胞之间的串扰可以被认为是减轻RA严重程度和骨稳态失调的开创性策略。。

Materials and methods

材料和方法

Patient enrollment and synovial fluid collection

患者登记和滑液收集

A total of 16 patients were enrolled, including 8 patients with RA and 8 patients with knee osteoarthritis (OA), all of whom provided informed consent to participate in the study and had effusion in the knee joints. Patients with RA met the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria.

共有16名患者入选，其中包括8名RA患者和8名膝关节骨关节炎（OA）患者，所有患者均提供知情同意书以参与研究并在膝关节积液。RA患者符合2010年美国风湿病学会/欧洲抗风湿病联盟分类标准。

13

13

; however, those taking biological disease-modifying antirheumatic drugs (DMARDs) were excluded. Synovial fluids were collected from patients with RA or OA, as described in ref.

；然而，那些服用生物疾病缓解抗风湿药物（DMARDs）的人被排除在外。如参考文献所述，从RA或OA患者收集滑液。

12

12

, using procedures approved by the institutional review board of Gil Medical Center (GAIRB-2019-018). The synovial fluid was centrifuged at 1,500 rpm for 15 min and stored at −20 °C until use.

，使用Gil医疗中心机构审查委员会批准的程序（GAIRB-2019-018）。将滑液以1500 rpm离心15分钟，并保存在-20°C直至使用。

Measurement of the Ca

Ca的测量

2+

2+

concentration in human synovial fluids

人体滑液中的浓度

Synovial fluids obtained from patients with OA or RA were used to measure the concentration of Ca

从OA或RA患者获得的滑液用于测量Ca的浓度

2+

2+

with a Ca

使用Ca

2+

2+

colorimetric assay kit (MAK022, Sigma). The Ca

比色测定试剂盒（MAK022，Sigma）。Ca

2+

2+

concentration was measured according to the manufacturer’s instructions. Synovial fluid samples from patients were loaded into a 96-well plate and mixed with a chromogenic reagent. The Ca

根据制造商的说明测量浓度。将来自患者的滑液样品加载到96孔板中并与显色试剂混合。Ca

2+

2+

colorimetric assay buffer was subsequently added to the mixture, and the final mixture was incubated for 10 min at room temperature in the dark. The absorbance of mixture in each sample was measured at 575 nm. The absorbance was calculated using the standard curve shown in Supplementary Fig.

随后将比色测定缓冲液加入混合物中，并将最终混合物在室温下在黑暗中孵育10分钟。在575nm处测量每个样品中混合物的吸光度。使用Supplementary Fig.中所示的标准曲线计算吸光度。

1

1

.

.

Total RNA sequencing, KEGG pathway enrichment analysis and GO enrichment analysis

RNA extracted from RA-FLSs in the presence or absence of tumor necrosis factor-α (TNF-α; MTA00B; R&D Systems, 10 ng/ml) was analyzed via total RNA sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) enrichment analyses were conducted to evaluate the RNA expression patterns.

通过总RNA测序分析在存在或不存在肿瘤坏死因子-α（TNF-α；MTA00B；R＆D Systems，10ng/ml）的情况下从RA FLS中提取的RNA。进行了京都基因和基因组百科全书（KEGG）途径富集和基因本体论（GO）富集分析，以评估RNA表达模式。

The RNA samples were selected for KEGG and GO analyses. The top 12 biological processes in the KEGG analysis are indicated by bars and represent specific genes corresponding to the osteoclast differentiation process. Ion channel- and transporter-related GO terms are indicated by circles, and their statistical significance is indicated by the color of the .

选择RNA样品进行KEGG和GO分析。KEGG分析中的前12个生物学过程由条形图表示，代表与破骨细胞分化过程相对应的特定基因。离子通道和转运蛋白相关的GO术语用圆圈表示，它们的统计显着性用颜色表示。

P

P

value.

价值。

Primary FLS culture

初级FLS培养

Primary human FLSs, which were isolated from the synovial tissues of human patients with RA and OA, were purchased from Cell Applications (human RA-FLSs, 408RA05a; human OA-FLSs, 408OA05a). For the isolation of mouse FLS (Ms-FLS) from C57BL/6N mice, the skin surrounding the knee joints was excised from 43 euthanized C57BL/6N mice.

从患有RA和OA的人类患者的滑膜组织中分离的原代人FLS购自Cell Applications（human RA FLS，408RA05a；human OA FLS，408OA05a）。为了从C57BL/6N小鼠中分离小鼠FLS（Ms FLS），从43只安乐死的C57BL/6N小鼠中切除膝关节周围的皮肤。

Synovial tissue was then collected and rinsed with Dulbecco’s phosphate-buffered saline (DPBS). The tissue was subsequently finely chopped using surgical scissors. The minced tissue fragments were incubated with 1% collagenase type IV at 37 °C and 200 rpm for 1 h. After the incubation, the tissue samples were vortexed and centrifuged at 1,700 rpm for 10 min.

然后收集滑膜组织并用Dulbecco磷酸盐缓冲盐水（DPBS）冲洗。随后使用手术剪刀将组织切碎。将切碎的组织碎片与1%IV型胶原酶在37℃和200 rpm下孵育1小时。孵育后，将组织样品涡旋并以1700 rpm离心10分钟。

The resulting supernatants were removed, and the remaining cell pellet was resuspended in Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% penicillin–streptomycin (p/s; 15140-122, Invitrogen) and 10% fetal bovine serum (FBS; 16000-044, Invitrogen) to culture the cells. The Ms-FLSs were then cultured at 37 °C in a humidified atmosphere of 5% CO.

除去产生的上清液，将剩余的细胞沉淀重悬于补充有1%青霉素-链霉素（p/s；15140-122，Invitrogen）和10%胎牛血清（FBS；16000-044，Invitrogen）的Dulbecco改良Eagle培养基（DMEM）中以培养细胞。然后将Ms FLS在37℃，5%CO的潮湿气氛中培养。

2

2

/95% air. All FLSs were cultured in DMEM (11995-065, Gibco) supplemented with 1% p/s and 10% FBS and maintained at 37 °C in a humidified incubator with 5% CO

/95%空气。所有FLS均在补充有1%p/s和10%FBS的DMEM（11995-065，Gibco）中培养，并在含5%CO的潮湿培养箱中保持在37°C

2

2

and 95% air. Upon reaching approximately 80% confluence, the culture medium was aspirated, and the cells were washed with DPBS (LB001-02, Welgene). The cells were subsequently treated with trypsin–EDTA for 1 min (Ms-FLSs) or 5 min (RA-FLSs and OA-FLSs) to detach the cells. All FLSs were used within five passages for the experiments..

和95%的空气。达到约80%汇合后，吸出培养基，并用DPBS（LB001-02，Welgene）洗涤细胞。随后用胰蛋白酶-EDTA处理细胞1分钟（Ms FLSs）或5分钟（RA FLSs和OA FLSs）以分离细胞。所有FLS均在五代内用于实验。。

Osteoclast differentiation protocols

破骨细胞分化方案

Bone marrow macrophages

骨髓巨噬细胞

Bone marrow was flushed from the femurs and tibias of C57BL/6N mice using alpha-modified Eagle’s medium (α-MEM) supplemented with 1% p/s and 10% FBS. After the removal of erythrocytes using a hypotonic buffer, the cells were cultured in α-MEM supplemented with 1% p/s and 10% FBS for 24 h. Nonadherent cells were then collected and subsequently cultured on Petri dishes in α-MEM supplemented with 1% p/s, 10% FBS and M-CSF for an additional 3 days.

使用添加1%p/s和10%FBS的α改良Eagle培养基（α-MEM）从C57BL/6N小鼠的股骨和胫骨冲洗骨髓。使用低渗缓冲液去除红细胞后，将细胞在添加1%p/s和10%FBS的α-MEM中培养24 h、 然后收集非粘附细胞，随后在补充有1%p/s，10%FBS和M-CSF的α-MEM的培养皿中再培养3天。

Unattached cells were removed by suction, and the remaining adherent cells were collected and used as bone marrow macrophages (BMMs). After this initial period, the cells were further differentiated by the addition of both M-CSF and RANKL for an additional 4 days. Similar to Raw264.7 cells, the culture medium of BMMs was replaced every 2 days..

通过抽吸除去未附着的细胞，收集剩余的贴壁细胞并用作骨髓巨噬细胞（BMM）。在这个初始阶段之后，通过添加M-CSF和RANKL进一步分化细胞4天。与Raw264.7细胞类似，BMM的培养基每2天更换一次。。

Raw264.7 cells

Raw264.7细胞

Raw264.7 cells were differentiated into osteoclasts by culturing them in α-MEM supplemented with 1% p/s, 10% FBS, M-CSF (315-02, PeproTech, 30 ng/ml) and RANKL (390-TN-010/CF, R&D Systems) for 7 days. The culture medium was replaced every 2 days.

通过将Raw264.7细胞在补充有1%p/s，10%FBS，M-CSF（315-02，PeproTech，30ng/ml）和RANKL（390-TN-010/CF，R＆D Systems）的α-MEM中培养7天，将其分化为破骨细胞。培养基每2天更换一次。

Fluorescence-activated cell sorting

荧光激活细胞分选

For the fluorescence-activated cell sorting (FACS) analysis, Ms-FLSs were separated into single cells in DPBS supplemented with 1% FBS. An antibody specific for an FLS marker, mouse anti-CD90.2 (14-0903-82, Invitrogen), was administered to primary cultured FLSs for 30 min at 4 °C to identify FLSs. The cells were washed three times with DPBS, centrifuged at 1,500 rpm for 5 min and incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (IgG) antibody for 30 min at 4 °C.

对于荧光激活细胞分选（FACS）分析，将Ms FLS在补充有1%FBS的DPBS中分离成单细胞。对FLS标记物小鼠抗CD90.2（14-0903-82，Invitrogen）特异的抗体在4℃下给予原代培养的FLS 30分钟以鉴定FLS。将细胞用DPBS洗涤三次，以1500 rpm离心5分钟，并与异硫氰酸荧光素（FITC）缀合的抗兔免疫球蛋白G（IgG）抗体在4℃温育30分钟。

After washes with DPBS, the centrifuged cells were suspended in DPBS supplemented with 1% FBS and passed through a 35-μm filter into a tube. The cell population was analyzed using a flow cytometer (BD LSRII, BD Biosciences), and the data were analyzed using FlowJo software (FlowJo). The population of CD90.2-positive cells was determined by comparing FITC-fluorescent dots with the negative control..

用DPBS洗涤后，将离心的细胞悬浮在补充有1%FBS的DPBS中，并通过35μm过滤器进入管中。使用流式细胞仪（BD LSRII，BD Biosciences）分析细胞群，并使用FlowJo软件（FlowJo）分析数据。通过比较FITC荧光点和阴性对照来确定CD90.2阳性细胞的数量。。

RT‒qPCR

RT‒qPCR

Total RNA was extracted from the cells using the Ribo

使用Ribo从细胞中提取总RNA

Ex

Ex公司

extraction system (GeneAll) according to the manufacturer’s instructions. RNA was quantified using an ND-1000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA was then synthesized using Accupower RocketScript Cycle RT PreMix (Bioneer) according to the manufacturer’s protocol. Reverse-transcription quantitative polymerase chain reaction (RT‒qPCR) was performed using PowerUp SYBR Green Master Mix (#A25741, Applied Biosystems) on a QuantStudio 3 RT‒PCR system (#A28567, Applied Biosystems) with the primers listed in Table .

。使用ND-1000分光光度计（Thermo Fisher Scientific）定量RNA。然后根据制造商的规程，使用Accupower RocketScript Cycle RT PreMix（Bioneer）合成互补DNA。使用Power Up SYBR Green Master Mix（#A25741，Applied Biosystems）在QuantStudio 3 RT-PCR系统（#A28567，Applied Biosystems）上使用表中列出的引物进行逆转录定量聚合酶链反应（RT-qPCR）。

1

1

. The RT‒qPCR cycling conditions were as follows: initial uracil DNA glycosylase activation at 50 °C for 2 min, followed by dual-lock DNA polymerase activation at 95 °C for 2 min. These steps were followed by denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s and elongation at 72 °C for 1 min..

RT-qPCR循环条件如下：在50℃下初始尿嘧啶DNA糖基化酶活化2分钟，然后在95℃下双锁DNA聚合酶活化2分钟。这些步骤之后是在95℃变性15秒，在55℃退火15秒，在72℃延伸1分钟。。

Table 1 Primers.

表1引物。

Full size table

全尺寸表

Transwell membrane migration assay

Transwell膜迁移测定

The migration assay was conducted using a transwell-polycarbonate membrane (6.5-mm insert, 8.0-μm pore size). The inserts were filled with 200 μl of RA-FLSs or Ms-FLSs (5 × 10

使用transwell聚碳酸酯膜（6.5mm插入物，8.0μm孔径）进行迁移测定。插入物中填充200μlRA FLSs或Ms FLSs（5×10

4

4

cells per well, Cell Applications) containing 1% FBS and reagents. TNF and M-CSF + RANKL (M + R) dissolved in 500 μl of DMEM or α-MEM at different pH values were added to the bottom chamber, along with conditioned media from mature osteoclasts cultured on dentin discs, which were stimulated by Raw264.7 cells and BMMs using five distinct FLS-conditioned media.

每孔细胞，细胞应用）含有1%的FBS和试剂。将溶解在不同pH值的500μlDMEM或α-MEM中的TNF和M-CSF+ RANKL（M + R）以及来自在牙本质盘上培养的成熟破骨细胞的条件培养基一起加入底室，其由Raw264.7细胞和BMM使用五种不同的FLS条件培养基刺激。

The cells were incubated for 6 h. Thereafter, the DMEM in the bottom chamber was removed, chilled methanol was added and the inserted membranes were soaked for 1 min at −20 °C. The chilled methanol was removed, and the plate was washed three times with DPBS. A 4′,6-diamidino-2-phenylindole (DAPI) solution was mixed with distilled water and loaded into the bottom chamber.

将细胞孵育6小时。然后，除去底室中的DMEM，加入冷冻的甲醇，并将插入的膜在-20℃下浸泡1分钟。除去冷却的甲醇，用DPBS洗涤板三次。将4'，6-二脒基-2-苯基吲哚（DAPI）溶液与蒸馏水混合并装入底室。

The plates were then incubated for 30 min in the dark. The medium was carefully removed from the insert, and distilled water was added to the bottom chamber at room temperature. DAPI fluorescence was measured at 405 nm using an LSM 700 Zeiss confocal microscope (Fluoview, Carl Zeiss). FLS migration was determined on the basis of the number of nuclei stained with DAPI on the transwell membrane..

然后将板在黑暗中孵育30分钟。小心地从插入物中取出培养基，并在室温下将蒸馏水加入底室。使用LSM 700 Zeiss共聚焦显微镜（Fluoview，Carl Zeiss）在405 nm处测量DAPI荧光。根据在transwell膜上用DAPI染色的细胞核数量确定FLS迁移。。

Immunostaining of the insert membrane in a transwell culture system

transwell培养系统中插入膜的免疫染色

After the migration assay, the inserts were prepared for immunostaining. The membrane of each insert was carefully cut out, isolated and immunostained with an NBCn1 antibody (ab82335, Abcam). The isolated membranes were first placed in a 100 mM glycine solution for 10 min at 4 °C, followed by an incubation with 0.5% bovine serum albumin (BSA) for 1 h at room temperature in the dark.

迁移测定后，准备插入物进行免疫染色。小心地切下每个插入物的膜，分离并用NBCn1抗体（ab82335，Abcam）进行免疫染色。首先将分离的膜在4℃下置于100mM甘氨酸溶液中10分钟，然后在室温下在黑暗中与0.5%牛血清白蛋白（BSA）孵育1小时。

The membranes were then incubated overnight at 4 °C with the primary antibody diluted 1:100 and subsequently incubated with the secondary antibody, rhodamine-tagged goat anti-rabbit IgG, for 1 h at room temperature. Finally, the membranes were mounted on glass slides using Fluoromount-G containing DAPI (Electron Microscopy Sciences).

。最后，使用含有DAPI（电子显微镜科学）的Fluoromount-G将膜安装在载玻片上。

Fluorescence images were acquired using an LSM 700 Zeiss confocal microscope (Fluoview) and analyzed with ZEN (Zeiss analysis software)..

使用LSM 700 Zeiss共聚焦显微镜（Fluoview）获取荧光图像，并使用ZEN（Zeiss分析软件）进行分析。。

MTT assay

MTT测定

Raw264.7 cells, BMMs, Ms-FLSs and RA-FLSs (1 × 10

Raw264.7细胞、BMMs、Ms FLSs和RA FLSs（110

4

4

cells per well) were seeded in 96-well plates and treated with S0859 or CaCl

将细胞接种于96孔板中，用S0859或CaCl处理

2

2

for 24 h. Tetrazolium bromide dye (MTT; 298-93-1, 2 mg/ml; Merck) was mixed with heated DPBS (37 °C). After the addition of the MTT solution, the cells were incubated in the dark at 37 °C in a humidified atmosphere of 5% CO

对于24小时的四唑溴化物染料（MTT；298-93-1,2mg/ml；将默克公司（Merck）与加热的DPBS（37℃）混合。加入MTT溶液后，将细胞在37°C的黑暗中于5%CO的潮湿气氛中孵育

2

2

and 95% air for 2 h. The supernatant was carefully removed from the plates, and dimethyl sulfoxide (100%) was added to the plates. The absorbance was measured at 570 nm using a fluorescence microplate reader (Varioskan LUX, Thermo Fisher Scientific).

和95%空气2小时。小心地从平板上除去上清液，并将二甲基亚砜（100%）加入平板中。使用荧光酶标仪（Varioskan LUX，Thermo Fisher Scientific）在570 nm处测量吸光度。

Measurement of sodium-bicarbonate cotransporter (NBC) activity

碳酸氢钠协同转运蛋白（NBC）活性的测定

2′-7′-Bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM; B1170, Invitrogen) was used to measure intracellular pH changes in RA-FLSs, Raw264.7 cells and BMMs at dual excitation wavelengths (440/495 nm) and a single emission wavelength (530 nm). 5-(

使用2'-7'-双-（羧乙基）-5-（和-6）-羧基荧光素（BCECF-AM；B1170，Invitrogen）测量RA FLSs，Raw264.7细胞和BMM在双激发波长（440/495nm）和单发射波长（530nm）下的细胞内pH变化。五-(

N

N

-ethyl-

-乙基-

N

N

-isopropyl) amiloride (EIPA; A3085, Sigma), an inhibitor of the major pH modulator sodium/hydrogen exchanger, was mixed with the HCO

-将主要pH调节剂钠/氢交换剂的抑制剂异丙基）阿米洛利（EIPA；A3085，Sigma）与HCO混合

3

3

−

−

solution to measure only the NBC activity-mediated pH modulation. RA-FLSs, Raw264.7 cells and BMMs were treated with CaCl

仅测量NBC活性介导的pH调节的解决方案。用CaCl处理RA FLSs，Raw264.7细胞和BMM

2

2

, M-CSF, RANKL and S0859 and loaded onto coverslips with 0.1% pluronic F-127 and 20 μM BCECF-AM perfused with a physiological salt solution (the composition was previously described in ref.

。

14

14

and was called regular solution). NBC activity was measured by incubating the cells with CO

被称为正规溶液）。通过将细胞与CO孵育来测量NBC活性

2

2

-saturated HCO

-饱和HCO

3

3

−

−

-buffered medium with 10 μM EIPA and then acidifying the cells with sodium-free bicarbonate-buffered medium. The emitted fluorescence was monitored using a CCD camera (Photometrics) attached to an inverted microscope (Olympus) and analyzed using a MetaFluor system (Molecular Devices). All BCECF fluorescence images were obtained at 1-s intervals, and the background fluorescence was subtracted from the raw background signals at each wavelength..

-用10μMEIPA缓冲培养基，然后用无钠碳酸氢盐缓冲培养基酸化细胞。使用连接到倒置显微镜（Olympus）的CCD相机（Photometrics）监测发射的荧光，并使用MetaFluor系统（Molecular Devices）进行分析。以1秒的间隔获得所有BCECF荧光图像，并从每个波长的原始背景信号中减去背景荧光。。

Immunofluorescence staining and confocal imaging

免疫荧光染色和共聚焦成像

Raw264.7 cells and BMMs seeded onto cover glasses were treated with M-CSF and RANKL for 24 h. The cells were fixed with chilled methanol (−20 °C) for 10 min, a 100 mM glycine solution was added and incubated for 10 min at 4 °C, and cells were washed three times with cold DPBS. The cells were then blocked with 0.5% BSA in DPBS supplemented with 10% goat serum for 1 h at room temperature in the dark, incubated with NBCn1 and NFATc1 (sc-7294; SANTACRUZ Biotechnology) antibodies at 4 °C overnight, and washed three times with 0.5% BSA in DPBS.

将接种在盖玻片上的Raw264.7细胞和BMM用M-CSF和RANKL处理24小时。将细胞用冷冻甲醇（-20℃）固定10分钟，加入100 mM甘氨酸溶液，并在4℃下孵育10分钟，并用冷DPBS洗涤细胞三次。然后用补充有10%山羊血清的DPBS中的0.5%BSA封闭细胞1 h在室温下在黑暗中，与NBCn1和NFATc1（sc-7294；SANTACRUZ Biotechnology）抗体在4℃孵育过夜，并用0.5%BSA的DPBS溶液洗涤三次。

The cells were treated with a secondary rhodamine-tagged goat anti-rabbit IgG antibody for 1 h at room temperature to detect the primary antibody. After the incubation, the cells were washed three times with DPBS, and the cover glasses were carefully attached and mounted on a glass slide with DAPI-containing Fluoromount-G.

用罗丹明标记的山羊抗兔IgG二抗在室温下处理细胞1小时以检测一抗。温育后，将细胞用DPBS洗涤三次，小心地将盖玻片连接并安装在载玻片上，载玻片上含有DAPI的Fluoromount-G。

Fluorescence images were acquired using a Zeiss LSM700 confocal microscope (Fluoview, Carl Zeiss) and analyzed using ZEN (Carl Zeiss) software..

使用Zeiss LSM700共聚焦显微镜（Fluoview，Carl Zeiss）获取荧光图像，并使用ZEN（Carl Zeiss）软件进行分析。。

CIA mouse model

CIA鼠标模型

All experimental animal procedures were performed in accordance with the Gachon University guidelines and approved by the Animal Care and Use Committee of Gachon University (LCDI-2022-0118, LCDI-2022-0039 and LCDI-2024-0029). DBA/1 (6-week-old male, 22–25 g) mice were purchased from KOATECH and Orient Bio.

所有实验动物程序均按照加川大学指南进行，并经加川大学动物护理和使用委员会（LCDI-2022-0118，LCDI-2022-0039和LCDI-2024-0029）批准。DBA/1（6周龄雄性，22-25g）小鼠购自KOATECH和Orient Bio。

The CIA model was induced by administering 4 μg of bovine type II collagen per gram of body weight (20021, Chondrex) dissolved in complete Freund’s adjuvant (F5881, Sigma) at a 1:1 ratio via intradermal injection for the first immunization. After 2 weeks, 4 μg of bovine type II collagen per gram of body weight was dissolved in incomplete Freund’s adjuvant (F5506, Sigma) at a 1:1 ratio and administered as a booster injection.

通过皮内注射以1:1的比例溶解在完全弗氏佐剂（F5881，Sigma）中的每克体重（20021，Chondrex）给予4μg牛II型胶原诱导CIA模型用于第一次免疫。2周后，将每克体重4μg牛II型胶原蛋白以1:1的比例溶解在不完全弗氏佐剂（F5506，Sigma）中，并作为加强注射给药。

The animals were divided into the CIA, CIA + S0859, CIA + adalimumab, and control (vehicle) groups. At the 4-week mark, adalimumab (HY-P9908A, MCE) was administered via a tail vein injection at a dosage of 0.25 mg/kg (body weight). Concurrently, S0859 was dissolved in dimethyl sulfoxide (19128, Bio Life Solutions), diluted with saline and injected into the tail vein at a concentration of 0.8 mg/kg (body weight).

将动物分为CIA，CIA+S0859，CIA+阿达木单抗和对照（载体）组。。同时，将S0859溶解在二甲基亚砜（19128，Bio-Life Solutions）中，用盐水稀释并以0.8mg/kg（体重）的浓度注入尾静脉。

Two weeks after the initial administration of adalimumab and S0859, a second dose was administered at the same concentration. Sixty mice were divided equally into control and vehicle groups, and 35 mice were used for CIA modeling. The mice in the experimental and control groups were euthanized 6 weeks after the second S0859 injection.

初次服用阿达木单抗和S0859两周后，以相同浓度服用第二剂。将60只小鼠平均分为对照组和载体组，并将35只小鼠用于CIA建模。实验组和对照组的小鼠在第二次S0859注射后6周被安乐死。

The serum, femurs and paws of the animals in all the groups were collected for analysis. Changes in body weight, the paw score and paw thickness were measured every 2 weeks, and paw images were obtained..

收集所有组动物的血清，股骨和爪子进行分析。每2周测量一次体重，爪子评分和爪子厚度的变化，并获得爪子图像。。

Determination of the paw score and thickness

爪痕和厚度的测定

Scoring was performed in a blinded manner to estimate the severity of CIA in mouse paws, which ranged from 0 to 4 for each paw

评分采用盲法，以估计小鼠爪子CIA的严重程度，每个爪子的评分范围为0到4

12

12

: normal paw, 0; one inflamed and swollen toe, 1; more than one inflamed and swollen toe, 2; the entire paw was inflamed and swollen, 3; and severe inflammation and swelling of the ankylosed paw, 4 (relatively extended range, 0–16). The paw score was measured by three independent and blinded observers, and the thickness of the hind paw was measured using calipers..

：正常爪，0；一个发炎和肿胀的脚趾，1；不止一个发炎和肿胀的脚趾，2个；整个爪子发炎肿胀，3；以及强直爪的严重炎症和肿胀，4（相对较大的范围，0-16）。爪子评分由三名独立且不知情的观察者测量，后爪的厚度用卡尺测量。。

Three-dimensional micro-CT

三维显微CT

The dissected femurs from all the groups were fixed with 10% neutral-buffered formalin (FR2013-100-00, Biosesang for 1 h. Micro-computed tomography (CT) was conducted on the joint of the femurs using a micro-CT scanner (SKYSCAN 1276, Bruker) under the following conditions: exposure at 50 kV, 200 μA, 0.25 mm artificial intelligence (AI) filter, 200 μm offset and 1.7 mm analysis height..

所有组的解剖股骨均用10%中性缓冲福尔马林（FR2013-100-00，Biosang 1 h、 使用微型CT扫描仪（SKYSCAN 1276，Bruker）在以下条件下对股骨关节进行微型计算机断层扫描（CT）：暴露于50 kV，200微安，0.25毫米人工智能（AI）过滤器，200μm偏移和1.7 mm分析高度。。

Osteoclast stimulation with FLS medium

用FLS培养基刺激破骨细胞

FLSs isolated from 6-week-old mice were cultured in 24-well plates (5 × 10

将从6周龄小鼠中分离的FLS在24孔板中培养（5×10

4

4

cells per well) and stimulated with TNF for 6 h. Raw264.7 cells and BMMs (1 × 10

细胞/孔），并用TNF刺激6H.Raw264.7细胞和BMM（1×10

4

4

cells per well) were treated with FLS-conditioned medium and then incubated at 37 °C for 4–7 days.

每孔细胞）用FLS条件培养基处理，然后在37°C下孵育4-7天。

TRAP staining

TRAP染色

Raw264.7 cells and BMMs (1 × 10

Raw264.7细胞和BMMs（1×10

4

4

cells per well) were seeded in a 96-well plate and incubated at 37 °C to allow cells to adhere to the bottom of the well. Raw264.7 cells were then cultured in medium containing M-CSF and RANKL for 7 days. BMMs were first cultured in medium containing M-CSF for 3 days, followed by an additional 4 days in medium containing both M-CSF and RANKL.

将每孔细胞接种在96孔板中，并在37℃下孵育以使细胞粘附在孔的底部。然后将Raw264.7细胞在含有M-CSF和RANKL的培养基中培养7天。首先将BMM在含有M-CSF的培养基中培养3天，然后在含有M-CSF和RANKL的培养基中再培养4天。

Thereafter, the cells were cultured in differentiation medium containing RANKL, M-CSF and FLS medium (α-MEM with 1% p/s and 10% FBS). After the incubation, the cells were fixed with a 4% formaldehyde solution (Tech & Innovation) for 10 min. The dissected paws were fixed with 10% neutral-buffered formalin for 1 h, and the skin was removed.

然后，将细胞在含有RANKL，M-CSF和FLS培养基（含有1%p/s和10%FBS的α-MEM）的分化培养基中培养。孵育后，将细胞用4%甲醛溶液（Tech＆Innovation）固定10分钟。将解剖的爪子用10%中性缓冲福尔马林固定1小时，取出皮肤。

The fixed joints were decalcified in Rapid Cal Immuno (6040; BBC Biochemical) for 2 weeks. Tissues were embedded in paraffin and placed on slides. Thereafter, the fixed cells and tissues were stained with a tartrate-resistant acid phosphatase (TRAP)/alkaline phosphatase staining kit (FUJIFILM) according to the manufacturer’s instructions.

固定关节在Rapid Cal Immuno（6040；BBC Biochemical）中脱钙2周。将组织包埋在石蜡中并置于载玻片上。此后，根据制造商的说明，用抗酒石酸酸性磷酸酶（TRAP）/碱性磷酸酶染色试剂盒（FUJIFILM）对固定的细胞和组织进行染色。

Images of the stained cells and paws were obtained using a Nikon DIAPHOT 300 inverted optical microscope (Nikon) equipped with a KOPIC HK6E3 digital camera (SONY). The intensity and number of stained cells were analyzed using MetaMorph software (Molecular Devices). For TRAP staining of bone slices, the numbers of osteoclasts/bone perimeters and osteoclast surface/bone surface were analyzed using Osteo-measure software (Osteometrics)..

使用配备有KOPIC HK6E3数码相机（索尼）的尼康DIAPHOT 300倒置光学显微镜（尼康）获得染色细胞和爪子的图像。使用MetaMorph软件（Molecular Devices）分析染色细胞的强度和数量。对于骨切片的TRAP染色，使用Osteo measure软件（Osteometrics）分析破骨细胞/骨周长和破骨细胞表面/骨表面的数量。。

Soluble TRAP measurement

Raw264.7 cells were cultured for 7 days. Thereafter, the culture medium was collected and stored on ice. An enzymatic analysis was performed in a 96-well microplate. Each sample of medium (30 μl) was added to the plate on ice, followed by the addition of 30 μl of soluble TRAP analysis buffer (95 mM sodium acetate, 47.5 mM sodium tartrate and 5 mg/ml .

将Raw264.7细胞培养7天。此后，收集培养基并储存在冰上。。将每个培养基样品（30μl）加入冰上的平板中，然后加入30μl可溶性TRAP分析缓冲液（95 mM乙酸钠，47.5 mM酒石酸钠和5 mg/ml。

p

p

-nitrophenyl phosphate, pH 4.5). The cells were then incubated at 37 °C for 30 min. The plate was immediately transferred onto ice, and 60 μl of cold 0.5 N NaOH was added to each well to terminate the reaction. The absorbance of the reaction mixture was measured at 405 nm using a VARIOSKAN LUX ELISA reader (Thermo Fisher Scientific)..

-磷酸硝基苯酯，pH 4.5）。然后将细胞在37℃下孵育30分钟。立即将板转移到冰上，并向每个孔中加入60μl冷的0.5N NaOH以终止反应。使用VARIOSKAN LUX ELISA阅读器（Thermo Fisher Scientific）在405 nm处测量反应混合物的吸光度。。

Pit assay

Pit分析

Raw264.7 cells (1 × 10

4

4

cells per well) were seeded into plates with an OsteoSite dentin disc (Immunodiagnosticsystems, United Kingdom) and cultured for 7 days in differentiation medium containing RANKL, M-CSF and FLS medium (α-MEM with 1% p/s and 10% FBS). BMMs (1 × 10

将每孔细胞接种到带有OsteoSite牙本质盘（英国免疫诊断系统）的平板中，培养7 在含有RANKL，M-CSF和FLS培养基（含1%p/s和10%FBS的α-MEM）的分化培养基中培养天。BMMs（110）

4

4

cells per well) were initially treated with M-CSF for 3 days, followed by an additional 4 days of culture in differentiation medium containing RANKL, M-CSF and FLS medium (α-MEM with 1% p/s and 10% FBS). After 7 days of culture, the mature osteoclasts in each well were removed using a 5% sodium hypochlorite solution and washed three times with distilled water.

首先用M-CSF处理每孔细胞3天，然后在含有RANKL，M-CSF和FLS培养基（含有1%p/s和10%FBS的α-MEM）的分化培养基中再培养4天。。

The resorption areas on the dentin surface were captured using a Nikon DIAPHOT 300 inverted optical microscope (Nikon) equipped with a KOPIC HK6E3 digital camera (SONY). The total area and absorption area of each image were calculated using MetaMorph software (Molecular Devices)..

使用配备有KOPIC HK6E3数码相机（索尼）的尼康DIAPHOT 300倒置光学显微镜（尼康）捕获牙本质表面的再吸收区域。使用MetaMorph软件（Molecular Devices）计算每个图像的总面积和吸收面积。。

Western blotting

西方墨点法

Raw264.7 cells and BMMs (2 × 10

Raw264.7细胞和BMMs（210

5

5

cells per well) were seeded into a six-well plate and incubated at 37 °C to allow cells to adhere to the bottom of the wells. Raw264.7 cells were then cultured in medium containing M-CSF and RANKL for either 3 or 7 days. BMMs were initially cultured in medium containing M-CSF for 3 days, followed by an additional 4 days of culture in medium containing both M-CSF and RANKL.

将每孔细胞接种到六孔板中，并在37℃下孵育以使细胞粘附到孔的底部。然后将Raw264.7细胞在含有M-CSF和RANKL的培养基中培养3或7天。BMM最初在含有M-CSF的培养基中培养3天，然后在含有M-CSF和RANKL的培养基中再培养4天。

After these initial treatments, the cells were exposed to differentiation medium containing RANKL, M-CSF and FLS medium (α-MEM with 1% p/s and 10% FBS). Proteins were isolated from the cells using a lysis buffer containing 20 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and a protease inhibitor mixture.

在这些初始处理后，将细胞暴露于含有RANKL，M-CSF和FLS培养基（含有1%p/s和10%FBS的α-MEM）的分化培养基中。使用含有20mM Tris的裂解缓冲液从细胞中分离蛋白质，150 mM NaCl，2 mM EDTA，1%Triton X-100和蛋白酶抑制剂混合物。

The protein concentration was quantified using the Bradford assay (5000207, Quick Start BSA standard, Bio-Rad). Proteins were then denatured in SDS sample buffer at 37 °C for 30 min. Denatured protein samples (30 μg each) were subjected to SDS‒PAGE. Proteins were visualized with NFATc1 (sc-7294, SANTACRUZ Biotechnology), NBCn1 and β-actin (A3854, Sigma) antibodies with an enhanced chemiluminescence solution (Thermo Fisher Scientific).

使用Bradford测定法（5000207，Quick Start BSA standard，Bio-Rad）定量蛋白质浓度。然后将蛋白质在SDS样品缓冲液中于37℃变性30分钟。将变性的蛋白质样品（每个30μg）进行SDS-PAGE。用增强的化学发光溶液（Thermo Fisher Scientific）用NFATc1（sc-7294，SANTACRUZ Biotechnology），NBCn1和β-肌动蛋白（A3854，Sigma）抗体观察蛋白质。

Images were obtained using an Amersham ImageQuant 800 imaging system (29399481, Cytiva)..

使用Amersham ImageQuant 800成像系统（29399481，Cytiva）获得图像。。

ELISA

酶联免疫吸附试验

The levels of M-CSF (MMC00B, R&D Systems) and C-terminal telopeptide of type I collagen (CTX-1; AC-06F1, Immunodiagnosticsystems) in the cell culture supernatants were quantified using their respective enzyme-linked immunosorbent assay (ELISA) kits, according to the protocols provided by the manufacturers..

根据制造商提供的方案，使用各自的酶联免疫吸附测定（ELISA）试剂盒定量细胞培养上清液中M-CSF（MMC00B，R＆D Systems）和I型胶原C末端端肽（CTX-1；AC-06F1，Immunodiagnosticsystems）的水平。。

Statistical analysis

统计分析

The experimental results are presented as the mean ± standard error of the mean (s.e.m.). Differences between the mean values of two samples were analyzed using analysis of variance (ANOVA). The significance of differences in each experiment was determined using ANOVA. Significance is indicated by an asterisk for comparison with the control group and a number sign for comparison with the CIA group..

实验结果表示为平均值的平均值±标准误差（s.e.m.）。使用方差分析（ANOVA）分析两个样本的平均值之间的差异。使用ANOVA确定每个实验中差异的显着性。用星号表示与对照组的比较，用数字符号表示与CIA组的比较。。

Results

结果

Cytokine- and high-Ca

细胞因子和高钙

2+

2+

-mediated FLSs activation induces the expression of bone destruction factors and NBC activation

-介导的FLSs激活诱导骨破坏因子的表达和NBC激活

Patients with RA exhibit features of bone and cartilage erosion. Thus, we hypothesized that Ca

RA患者表现出骨和软骨侵蚀的特征。因此，我们假设Ca

2+

2+

released from destroyed bones increases synovial Ca

从破坏的骨骼中释放出来会增加滑膜Ca

2+

2+

levels. Compared with those of patients with OA, the synovial fluids of patients with RA presented mildly increased Ca

级别。与OA患者相比，RA患者的滑液中Ca轻度升高

2+

2+

levels; however, no statistically significant difference was found (Fig.

水平；然而，没有发现统计学上的显着差异（图）。

1a

1a级

; the standard curve for the Ca

；Ca的标准曲线

2+

2+

concentration is shown in Supplementary Fig.

浓度如补充图所示。

1

1

). The synovial Ca

)。滑膜Ca

2+

2+

concentration was approximately 60 ppm, which equates to 1.5 mM. Several studies have addressed the robust expression of cytokines and chemokines in inflamed tissues, including RA joints

浓度约为60ppm，相当于1.5mM。一些研究已经解决了炎症组织（包括RA关节）中细胞因子和趋化因子的强烈表达

15

15

. Thus, we verified the differential gene expression levels in FLSs between the control group and group treated with RA-associated inflammatory cytokine TNF. The expression of genes related to cytokine signaling, chemokine signaling, adhesion, T cell differentiation and osteoclastogenesis was increased in TNF-treated human RA-FLSs (Fig.

因此，我们验证了对照组和RA相关炎性细胞因子TNF治疗组之间FLS中的差异基因表达水平。在TNF处理的人RA FLS中，与细胞因子信号传导，趋化因子信号传导，粘附，T细胞分化和破骨细胞生成相关的基因的表达增加（图）。

.

.

1b

1b级

). We focused on the upregulated osteoclastogenesis-related gene M-CSF (Supplementary Fig.

)。我们专注于上调破骨细胞生成相关基因M-CSF（Supplementary Fig.）。

2

2

). The GO enrichment analysis revealed that treatment with TNF induced significant changes in the expression of ion channel- and transporter-related genes (Fig.

)。GO富集分析显示，用TNF处理诱导离子通道和转运蛋白相关基因表达的显着变化（图）。

1c

1c级

). The Ca

)。Ca

2+

2+

concentration in the bone marrow markedly varies from 0.1 to 4.5 mM (ref.

骨髓中的浓度显着从0.1到4.5mM不等（参考文献）。

16

16

). In this study, cells were stimulated with 3 mM extracellular Ca

)。在这项研究中，用3毫米细胞外钙刺激细胞

2+

2+

for high Ca

对于高Ca

2+

2+

stimulation, and the levels of factors involved in osteoclastogenesis were examined in the presence of inflammatory cytokines and increased extracellular Ca

在存在炎性细胞因子和细胞外钙增加的情况下，检测破骨细胞生成相关因子的水平

2+

2+

levels. Stimulation with TNF and 3 mM Ca

级别。用TNF和3mM Ca刺激

2+

2+

increased the mRNA expression of

增加了mRNA的表达

RANKL

RANKL

and

和

M-CSF

M-CSF

, but not that of osteoprotegerin (

，但不是骨保护素(

OPG

OPG公司

), in human RA-FLSs (Fig.

)，在人类RA FLS中（图）。

1d

1维

). In human OA-FLSs, stimulation with TNF and 3 mM Ca

)。在人类OA FLS中，用TNF和3mM Ca刺激

2+

2+

did not increase the expression of these genes (Fig.

没有增加这些基因的表达（图）。

1e

1e级

). Increased Ca

)。增加Ca

2+

2+

levels did not attenuate cell viability or mildly induce cellular proliferation (Fig.

水平不减弱细胞活力或轻度诱导细胞增殖（图）。

1f

1层

). Extracellular Ca

)。细胞外钙

2+

2+

is sensed by the Ca

由Ca感知

2+

2+

-sensing receptor (CaSR), which is expressed in epithelial cells and is involved in various cellular functions, such as differentiation, proliferation and bone metastatic homeostasis

-传感受体（CaSR），在上皮细胞中表达，参与多种细胞功能，如分化、增殖和骨转移稳态

17

17

. The activation of CaSR induces an increase in the intracellular Ca

CaSR的激活诱导细胞内Ca的增加

2+

2+

concentration, ERK1/2 activation and cellular proliferation

浓度，ERK1/2激活和细胞增殖

8

8

. Notably, stimulation with TNF and 3 mM Ca

值得注意的是，用TNF和3mM Ca刺激

2+

2+

increased the expression of the

增加了

CaSR

CaSR

mRNA in human RA-FLSs (Fig.

人RA FLS中的mRNA（图）。

1g

1克

). According to our previous study, inflammatory cytokines enhance FLS migration

)。根据我们之前的研究，炎性细胞因子增强FLS迁移

12

12

. Stimulation with TNF and 3 mM Ca

.用TNF和3mM Ca刺激

2+

2+

in the extracellular medium increased human FLS migration (Fig.

在细胞外培养基中增加了人FLS的迁移（图）。

1h, i

1小时，我

). Ca

)。加利福尼亚州

2+

2+

-mediated ERK1/2 activation is assumed to promote NBC activity

-假定介导的ERK1/2激活可促进NBC活性

18

18

; therefore, we verified the expression and activity of a migration-related module, the NBC encoded by

因此，我们验证了与迁移相关的模块NBC的表达和活性

SLC4A7

SLC4A7

, in the presence of 3 mM Ca

，在3毫米Ca的存在下

2+

2+

. An increase in

.增加

SLC4A7

SLC4A7

mRNA expression was induced by 3 mM Ca

3 mM Ca诱导mRNA表达

2+

2+

in human RA-FLSs (Fig.

在人类RA FLS中（图）。

1j

1j

). In addition, NBC activity was increased by treatment with 3 mM Ca

)。此外，用3mM Ca处理可提高NBC活性

2+

2+

in human RA-FLSs (Fig.

在人类RA FLS中（图）。

1k, l

1k，l

). Costimulation with TNF and 3 mM Ca

)。与TNF和3mM Ca共刺激

2+

2+

had additive effects on the expression and activity of NBC in human RA-FLSs (Fig.

对人RA FLS中NBC的表达和活性具有累加效应（图）。

1j–

1j–

l

l

). These results indicate that cytokine- and extracellular high-Ca

)。这些结果表明，细胞因子和细胞外高钙

2+

2+

-mediated RA-FLS activation induces the expression of bone destruction factors and the migration-related module, NBCn1.

-介导的RA-FLS激活诱导骨破坏因子和迁移相关模块NBCn1的表达。

Fig. 1: Cytokine- and high-Ca

图1：细胞因子和高Ca

2+

2+

-mediated FLSs activation induces the expression of bone destruction factors and NBC activation.

-介导的FLSs激活诱导骨破坏因子的表达和NBC激活。

a

一

Measurement of the Ca

Ca的测量

2+

2+

concentration in the synovial fluid of patients with OA or RA.

。

b

b类

KEGG pathway analysis of genes with upregulated expression in TNF-treated human RA-FLSs.

在TNF处理的人RA FLS中表达上调的基因的KEGG途径分析。

c

c级

Significant changes in the expression of ion channel and transporter genes in TNF-treated human RA-FLSs were observed on the basis of the enriched GO terms for molecular function.

基于分子功能的富集GO术语，观察到TNF处理的人RA FLS中离子通道和转运蛋白基因表达的显着变化。

d

d

Relative mRNA expression levels (

相对mRNA表达水平(

OPG

OPG公司

,

,

RANKL

RANKL

and

和

M-CSF

M-CSF

) in human RA-FLSs treated with TNF or 3 mM CaCl

)

2

2

for 24 h. The bars represent the mean ± s.e.m. (

24小时。条形代表平均值±s.e.m(

n

n

= 3–6).

（3-6）。

e

e

Relative mRNA expression levels (

相对mRNA表达水平(

OPG

OPG公司

,

,

RANKL

RANKL

and

和

M-CSF

M-CSF

) in human OA-FLSs treated with TNF or 3 mM CaCl

)在用TNF或3mM CaCl处理的人OA FLS中

2

2

for 24 h. The bars represent the mean ± s.e.m. (

24小时。条形代表平均值±s.e.m(

n

n

= 2–3).

（2-3）。

f

f级

The viability of human RA-FLSs treated with different doses of CaCl

不同剂量CaCl处理人RA FLS的活力

2

2

(0, 1, 1.5, 3 or 4.5 mM) for 24 h (

(0, 1, 1.5, 3 or 4.5 mM) for 24 h (

n

n

= 3).

（3）。

g

克

The mRNA expression levels of the

mRNA表达水平

CaSR

CaSR

in human RA-FLSs cultured under the indicated conditions for 24 h. The bars represent the mean ± s.e.m. (

在指定条件下培养24小时的人RA FLS中。条形代表平均值±s.e.m(

n

n

= 4).

4.

h

小时

A migration assay of human RA-FLSs cultured under the indicated conditions for 6 h. Fluorescence staining with DAPI (blue). Scale bar, 50 μm.

在指定条件下培养6小时的人RA FLS的迁移测定。用DAPI（蓝色）进行荧光染色。比例尺，50微米。

i

我

An analysis of the total DAPI intensity to examine human RA-FLS migration. The bars represent the mean ± s.e.m. (

分析总DAPI强度以检查人类RA-FLS迁移。条形代表平均值±s.e.m(

n

n

= 4).

（4）。

j

j

The mRNA expression level of

mRNA表达水平

SLC4A7

SLC4A7

in human RA-FLSs cultured under the indicated conditions for 24 h. The bars represent the mean ± s.e.m. (

在指定条件下培养24小时的人RA FLS中。条形代表平均值±s.e.m(

n

n

= 4).

（4）。

k

k

NBC activity was assessed by measuring changes in the pH

通过测量pH值的变化来评估NBC活性

i

我

values of human RA-FLSs cultured under the indicated conditions for 24 h.

在指定条件下培养24小时的人RA FLS的值。

l

l

An analysis of the NBC activity of human RA-FLSs cultured under the indicated conditions for 24 h. The bars represent the mean ± s.e.m. (

在指定条件下培养24小时的人RA FLS的NBC活性分析。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

Full size image

全尺寸图像

Media from FLSs exposed to cytokines and high Ca

来自FLS的培养基暴露于细胞因子和高钙

2+

2+

levels induce NFATc1 activation in osteoclasts

水平诱导破骨细胞中的NFATc1活化

We determined the effects of high Ca

我们确定了高钙的影响

2+

2+

levels in FLSs on the expression of inflammatory cytokines, which were selected according to the major cytokines involved in RA. The experimental system was generated in mice to maintain the strain in subsequent experiments with isolated osteoclasts; thus, we isolated FLSs from the joints of the mice.

FLS中的水平对炎性细胞因子的表达有影响，这些细胞因子是根据RA中涉及的主要细胞因子选择的。在小鼠中产生实验系统，以在随后用分离的破骨细胞进行的实验中维持菌株；因此，我们从小鼠的关节中分离出FLS。

We confirmed that treatment with 3 mM extracellular Ca.

我们证实用3mM细胞外Ca治疗。

2+

2+

increased the mRNA expression of the inflammatory cytokines

增加炎性细胞因子的mRNA表达

Tnf, Il-1β

Tnf，1β

,

,

Il-6

Il-6

and

和

Il-8

Il-8

in isolated primary Ms-FLSs (Fig.

在孤立的初级Ms FLS中（图）。

2a

2a级

). Cytokine-exposed FLSs, but not IL-6-exposed FLSs, increased the expression of the M-CSF mRNA in Ms-FLSs (Fig.

)。细胞因子暴露的FLS，而不是IL-6暴露的FLS，增加了Ms FLS中M-CSF mRNA的表达（图）。

2b

2b级

). As a method to elucidate the cytokine feedback loops involved in RA, we designed and illustrated an experimental scheme to support the understanding of the experimental approach shown in Fig.

)。作为阐明RA中涉及的细胞因子反馈环的方法，我们设计并说明了一种实验方案，以支持对Fig.所示实验方法的理解。

2c

2摄氏度

. Isolated mouse primary FLSs were stimulated with or without TNF. Isolated Ms-FLSs were verified by staining with an antibody against the FLS marker CD90.2 (ref.

.用或不用TNF刺激分离的小鼠原代FLS。通过用针对FLS标记CD90.2的抗体染色来验证分离的Ms FLS（参考文献）。

19

19

), and CD90.2(+) FLSs were used in subsequent experiments (Fig.

)，CD90.2（+）FLSs用于随后的实验（图）。

2d

二维

). Conditioned or stimulated FLS media were administered to Raw264.7 cells and primary BMMs. Notably, osteoclastogenesis requires factors involved in differentiation and proliferation, such as M-CSF and RANKL

)。将条件化或刺激的FLS培养基施用于Raw264.7细胞和原代BMM。值得注意的是，破骨细胞生成需要参与分化和增殖的因素，例如M-CSF和RANKL

11

11

. The expression of the master transcription factor nuclear factor of activated T cells (NFATc1)

.活化T细胞的主要转录因子核因子（NFATc1）的表达

11

11

was determined to assess osteoclast differentiation. Raw264.7 cells treated with M-CSF and RANKL exhibited increased expression of the NFATc1 protein (Fig.

确定评估破骨细胞分化。。

2e, f

2e，f

). Similarly, Raw264.7 cells treated with FLS medium containing TNF (Fm-T, termed activated FLS medium) exhibited increased expression of NFATc1 (Fig.

)。类似地，用含有TNF的FLS培养基（Fm-T，称为活化的FLS培养基）处理的Raw264.7细胞表现出增加的NFATc1表达（图）。

2e, f

2e，f

). Treatment with FLS control medium (Fm-C, termed no stimulation FLS medium) or TNF alone (T) slightly increased NFATc1 expression in Raw264.7 cells, but the difference was not statistically significant. The nuclear localization of NFATc1 was also increased in M-CSF- and RANKL-treated (+ M + R) and activated FLS medium-treated (Fm-T) BMMs (Fig.

)。用FLS对照培养基（Fm-C，称为无刺激FLS培养基）或单独的TNF（T）处理略微增加Raw264.7细胞中NFATc1的表达，但差异无统计学意义。在M-CSF和RANKL处理的（+M+R）和活化的FLS培养基处理的（Fm-T）BMM中，NFATc1的核定位也增加（图）。

.

.

2g, h

2克，小时

). These results indicate that increased cytokines and Ca

)。这些结果表明，细胞因子和钙的增加

2+

2+

levels induce increased M-CSF expression in FLSs and that TNF-exposed FLS media potentially contain cytokines and osteoclastogenic factors. Thus, osteoclasts exhibit increased NFATc1 activation in the presence of TNF-exposed FLS media, similar to the activation observed upon M-CSF + RANKL stimulation..

水平诱导FLS中M-CSF表达增加，并且TNF暴露的FLS培养基可能含有细胞因子和破骨细胞生成因子。因此，破骨细胞在TNF暴露的FLS培养基存在下表现出增加的NFATc1活化，类似于在M-CSF+ RANKL刺激下观察到的活化。。

Fig. 2: Medium from FLSs exposed to cytokines and high Ca

图2:FLS暴露于细胞因子和高Ca的培养基

2+

2+

levels induces NFATc1 activation in osteoclasts.

水平诱导破骨细胞中的NFATc1活化。

a

一

Relative mRNA levels of inflammatory cytokines (

炎性细胞因子的相对mRNA水平(

Tnf

肿瘤坏死因子

,

,

Il-1β

1β

,

,

Il-6

Il-6

and

和

Il-8

Il-8

) in isolated primary Ms-FLSs treated with 3 mM CaCl

)

2

2

for 3 h. The bars represent the mean ± s.e.m. (

对于3小时。条形代表平均值±s.e.m(

n

n

= 4).

4.

b

b类

Relative mRNA levels of M-CSF in Ms-FLSs subjected to the indicated conditions (10 ng/ml TNF, IL-1β, IL-6, IL-8 and EGF) for 6 h. The bars represent the mean ± s.e.m. (

在指定条件（10ng/ml TNF，IL-1β，IL-6，IL-8和EGF）下6小时，Ms FLS中M-CSF的相对mRNA水平。条形代表平均值±s.e.M(

n

n

= 8).

（8）。

c

c级

A schematic of osteoclast maturation and subsequent assays conducted in the presence of the indicated stimuli. FLS medium without or with TNF is indicated as Fm-C or Fm-T, respectively. These media were administered to Raw264.7 cells and BMMs.

破骨细胞成熟的示意图以及在指定刺激物存在下进行的后续测定。不含或含有TNF的FLS培养基分别表示为Fm-C或Fm-T。将这些培养基施用于Raw264.7细胞和BMM。

d

d

FACS scatter plots showing the CD90.2-positive cell population, marked as FITC-positive, among Ms-FLSs. N.C., negative control.

FACS散点图显示Ms FLS中标记为FITC阳性的CD90.2阳性细胞群。N、 C.，阴性对照。

e

e

NFATc1 protein levels in Raw264.7 cells stimulated with the indicated cytokines for 3 days. β-Actin was used as a loading control.

用指定的细胞因子刺激3天的Raw264.7细胞中的NFATc1蛋白水平。β-肌动蛋白用作上样对照。

f

f级

An analysis of the band intensity of the NFATc1 protein in Raw264.7 cells. The bars represent the mean ± s.e.m. (

Raw264.7细胞中NFATc1蛋白条带强度的分析。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

g

克

Immunofluorescence staining for NFATc1 (red) and DAPI (blue) in BMMs subjected to the indicated conditions. Scale bar, 20 μm.

在指定条件下对BMM中的NFATc1（红色）和DAPI（蓝色）进行免疫荧光染色。比例尺，20微米。

h

小时

An analysis of the normalized intensity (total intensity/measuring area) of NFATc1 in cells cultured under the indicated conditions. The bars represent the mean ± s.e.m. (

在指定条件下培养的细胞中NFATc1的归一化强度（总强度/测量面积）的分析。条形代表平均值±s.e.m(

n

n

= 4). F, FLSs; T, TNF; M, M-CSF; R, RANKL; C (R), Raw264.7; C (B), BMMs; C, control.

=4）.F，FLSs；T、 TNF；M、 M-CSF；R、 兰克尔；C（R），Raw264.7；C（B），BMMs；C、 控制。

Full size image

全尺寸图像

Cytokine-exposed FLS medium stimulates osteoclasts maturation, which exhibits positive TRAP staining and increased resorption of the bone matrix

细胞因子暴露的FLS培养基刺激破骨细胞成熟，表现出TRAP阳性染色和骨基质吸收增加

Osteoclasts were incubated with M-CSF, RANKL and FLS medium to determine their osteoclastogenic activity. Osteoclastogenic activity, which represents multinucleated cells formation of osteoclasts, was determined via TRAP (osteoclast marker) staining and a pit assay, which verified the activity of bone resorption.

将破骨细胞与M-CSF，RANKL和FLS培养基一起温育以确定它们的破骨细胞生成活性。破骨细胞生成活性代表破骨细胞的多核细胞形成，通过TRAP（破骨细胞标记物）染色和pit测定来确定，其验证了骨吸收的活性。

20

20

. M-CSF + RANKL-treated (+ M + R) and activated FLS medium-treated (Fm-T) Raw264.7 cells were markedly positive for TRAP and presented an increased number of osteoclasts (Fig.

M-CSF+RANKL处理的（+M+R）和活化的FLS培养基处理的（Fm-T）Raw264.7细胞对TRAP呈明显阳性，破骨细胞数量增加（图）。

3a–d

). Although the TNF-treated (+T) and control medium-treated (Fm-C) groups were also TRAP positive, the differences were not significant compared with those observed in the M + R- and Fm-T-treated groups. The M + R-treated group was used as a positive control for osteoclast activation. The level of the soluble form of TRAP was increased in the M + R-treated and Fm-T-treated groups (Fig.

)。尽管TNF处理的（+T）和对照培养基处理的（Fm-C）组也呈TRAP阳性，但与M++R和Fm-T处理组相比，差异不显着。M+R治疗组用作破骨细胞活化的阳性对照。在M+R处理组和Fm-T处理组中，TRAP的可溶性形式水平增加（图）。

.

.

3e

3e公司

). Notably, activated osteoclasts possess bone-absorbing functions. The pit area (bone-absorbing area) was increased in the M + R-treated and Fm-T-treated Raw264.7 cells (Fig.

)。。M+ R处理和Fm-T处理的Raw264.7细胞的凹坑面积（骨吸收面积）增加（图）。

3f

3f级

). We confirmed TRAP staining and pit formation under the same conditions in BMMs. M + R- and Fm-T-treated BMMs were markedly TRAP positive, with increased numbers of osteoclasts (Fig.

)。我们在BMM中在相同条件下证实了TRAP染色和凹坑形成。M + R和Fm-T处理的BMM显着呈TRAP阳性，破骨细胞数量增加（图）。

3g–j

3g–j

) and pit areas (Fig.

)和凹坑区域（图）。

3k

3公里

). We assumed that the Fm-T group contained osteoclastogenic factors, such as M-CSF. Thus, the Fm-T group was incubated with an M-CSF antibody to remove the osteoclastogenic signals. The intensity and number of formed multinucleated cells of osteoclast were markedly reduced by treatment with the M-CSF antibody (Fig.

)。我们假设Fm-T组含有破骨细胞生成因子，如M-CSF。因此，将Fm-T组与M-CSF抗体孵育以去除破骨细胞生成信号。通过用M-CSF抗体处理，破骨细胞形成的多核细胞的强度和数量显着降低（图）。

.

.

3l–n

3l–n

). These results indicate that cytokine-exposed FLS medium contains osteoclastogenic stimuli, which promote osteoclast activity and induce the formation of resorption pits.

)。这些结果表明，暴露于细胞因子的FLS培养基含有破骨细胞生成刺激物，其促进破骨细胞活性并诱导再吸收坑的形成。

Fig. 3: Cytokine-exposed FLS media stimulates osteoclasts maturation, which exhibits positive TRAP staining and increased resorption of the bone matrix.

图3：暴露于细胞因子的FLS培养基刺激破骨细胞成熟，其表现出阳性TRAP染色和骨基质的再吸收增加。

a

一

Representative images of TRAP staining of osteoclasts derived from Raw264.7 cells subjected to the indicated conditions for 7 days. Scale bar, 200 μm.

来自Raw264.7细胞的破骨细胞在指定条件下7天的TRAP染色的代表性图像。比例尺，200微米。

b

b类

Representative images of the bone resorption pit assay of Raw264.7 cells cultured with dentin discs and subjected to the indicated conditions for 7 days. Scale bar, 200 μm.

用牙本质盘培养并在指定条件下放置7天的Raw264.7细胞的骨吸收坑测定的代表性图像。比例尺，200微米。

c

c级

An analysis of the relative TRAP intensity in osteoclasts derived from Raw264.7 cells. The bars represent the mean ± s.e.m. (

来自Raw264.7细胞的破骨细胞中相对陷阱强度的分析。条形代表平均值±s.e.m(

n

n

= 6).

（6）。

d

d

An analysis of the number of osteoclasts per well derived from Raw264.7 cells. The bars represent the mean ± s.e.m. (

来自Raw264.7细胞的每孔破骨细胞数量的分析。条形代表平均值±s.e.m(

n

n

= 6).

（6）。

e

e

An analysis of the relative soluble TRAP (sTRAP) intensity in Raw264.7 cells. The bars represent the mean ± s.e.m. (

。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

f

f级

An analysis of lacunae (dark area) formed by Raw264.7 cells. The bars represent the mean ± s.e.m. (

Raw264.7细胞形成的腔隙（暗区）分析。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

g

克

Representative images of TRAP staining of osteoclasts derived from BMMs subjected to the indicated conditions. Scale bar, 100 μm.

来自BMM的破骨细胞在指定条件下的TRAP染色的代表性图像。比例尺，100微米。

h

小时

Representative images of the bone resorption pit assay of BMMs cultured with dentin discs and subjected to the indicated conditions. Scale bar, 200 μm.

用牙本质盘培养并在指定条件下进行的BMM骨吸收坑测定的代表性图像。比例尺，200微米。

i

我

An analysis of the relative TRAP intensity in osteoclasts derived from BMMs. The bars represent the mean ± s.e.m. (

来自BMM的破骨细胞中相对陷阱强度的分析。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

j

j

An analysis of the number of osteoclasts per well derived from BMMs. The bars represent the mean ± s.e.m. (

分析来自BMM的每孔破骨细胞的数量。条形代表平均值±s.e.m(

n

n

= 3).

3.

k

k

Analysis of lacunae (dark area) formed by BMMs. The bars represent the mean ± s.e.m. (

BMMs形成的腔隙（暗区）分析。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

l

l

Representative images of TRAP staining in osteoclasts derived from Raw264.7 cells treated with or without the M-CSF antibody and subjected to the indicated conditions for 7 days. Scale bar, 200 μm.

在用或不用M-CSF抗体处理并在指定条件下处理7天的Raw264.7细胞衍生的破骨细胞中TRAP染色的代表性图像。比例尺，200微米。

m

m

An analysis of the relative TRAP intensity in osteoclasts derived from Raw264.7 cells. The bars represent the mean ± s.e.m. (

来自Raw264.7细胞的破骨细胞中相对陷阱强度的分析。条形代表平均值±s.e.m(

n

n

= 4).

4.

n

n

An analysis of the relative number of TRAP-positive osteoclasts derived from Raw264.7 cells. The bars represent the mean ± s.e.m. (

分析来自Raw264.7细胞的TRAP阳性破骨细胞的相对数量。条形代表平均值±s.e.m(

n

n

= 4).

（4）。

Full size image

全尺寸图像

Components released from mature osteoclasts enhance FLS migration

成熟破骨细胞释放的成分增强FLS迁移

Mature osteoclasts possess bone resorption abilities. The cells were cultured in dentin disc-containing medium to verify bone resorption by osteoclasts. The dentin disc contains various components, including Ca

成熟的破骨细胞具有骨吸收能力。将细胞在含有牙本质盘的培养基中培养以验证破骨细胞的骨吸收。牙本质盘包含各种成分，包括Ca

2+

2+

, phosphorus and ions, such as Na

，磷和离子，例如Na

+

+

, K

，K

+

+

and Mg

和Mg

2+

2+

(ref.

（参考。

21

21

). Thus, we determined the bone resorption activity of osteoclasts in dentin disc-containing medium with or without FLS-associated osteoclastogenic stimuli. We subsequently investigated whether the mobility of FLSs was affected by factors released by activated osteoclasts. We illustrated the experimental approach in Fig.

)。因此，我们确定了含或不含FLS相关破骨细胞生成刺激的含牙本质盘培养基中破骨细胞的骨吸收活性。随后，我们研究了FLS的迁移率是否受到活化破骨细胞释放的因素的影响。我们在Fig.中说明了实验方法。

.

.

4a

4a级

to provide a better understanding. FLSs were treated with M-CSF and RANKL to verify the effect of factors released by activated osteoclasts on FLS migration. FLSs exhibited increased migration in the presence of TNF, but their migratory ability was not affected by M-CSF + RANKL, suggesting that FLS migration was not affected by M-CSF + RANKL stimulation (Fig.

提供更好的理解。用M-CSF和RANKL处理FLS，以验证活化的破骨细胞释放的因子对FLS迁移的影响。FLS在TNF存在下表现出增加的迁移，但它们的迁移能力不受M-CSF+RANKL的影响，表明FLS迁移不受M-CSF+RANKL刺激的影响（图）。

.

.

4b, c

4b，c

). Raw264.7 cells were exposed to five different FLS media and cultured for 8–9 days until they reached the mature state. New FLSs were exposed to dentin disc-containing Raw264.7 cells medium in the lower chamber of the transwell system to determine the effects of the factors released by mature osteoclasts on FLS migration.

)。将Raw264.7细胞暴露于五种不同的FLS培养基中，培养8-9天，直到达到成熟状态。将新的FLS暴露于transwell系统下室中含有Raw264.7细胞培养基的牙本质盘，以确定成熟破骨细胞释放的因子对FLS迁移的影响。

FLS migration was increased in the mature Raw264.7-conditioned medium after exposure to M-CSF + RANKL (termed Rm-M + R) and TNF (termed Rm-Fm-T) (Fig. .

暴露于M-CSF+ RANKL（称为Rm-M+ R）和TNF（称为Rm-Fm-T）后，成熟Raw264.7条件培养基中FLS迁移增加（图）。

4d, e

4d，e

). Thus, we hypothesized that the biological factors released from Raw264.7 cells into the medium influence FLS migration. Briefly, the cells were treated with deactivated medium (termed deactivated Rm-Fm-T), which was boiled and chilled. Compared with Rm-Fm-T medium, deactivated Rm-Fm-T medium did not increase FLS migration (Fig.

)。因此，我们假设从Raw264.7细胞释放到培养基中的生物因子会影响FLS迁移。简而言之，用失活的培养基（称为失活的Rm-Fm-T）处理细胞，将其煮沸并冷却。与Rm-Fm-T培养基相比，失活的Rm-Fm-T培养基不增加FLS迁移（图）。

.

.

4f

4f级

and Supplementary Fig.

和补充图。

3

3

). The dentin disc contains a Ca

)。牙本质盘包含一个Ca

2+

2+

component

成分

22

22

. Therefore, to determine the effect of Ca

因此，为了确定Ca的影响

2+

2+

released from the dentin disc on FLS migration, the Ca

在FLS迁移时从牙本质盘中释放出来，Ca

2+

2+

-chelating drug BAPTA was added to remove Ca

-加入螯合药物BAPTA以去除Ca

2+

2+

from the dentin-extracted medium. The increase in FLS migration caused by the released Ca

从牙本质提取的培养基中提取。释放的Ca导致FLS迁移的增加

2+

2+

in the Rm-Fm-T medium was inhibited by BAPTA treatment through the Ca

在Rm-Fm-T培养基中，通过Ca进行BAPTA处理可抑制

2+

2+

chelation (Fig.

螯合（图）。

4g, h

4g，小时

). These results indicate that the components, including Ca

)。这些结果表明，包括Ca在内的成分

2+

2+

, release from dentin-containing mature osteoclast medium increase FLS migration.

，从含牙本质的成熟破骨细胞培养基中释放会增加FLS的迁移。

Fig. 4: Components released from mature osteoclasts enhance FLS migration.

图4：成熟破骨细胞释放的成分增强FLS迁移。

a

一

The schematic procedure of Ms-FLS migration cultured in the presence of the Raw264.7 cell medium-mediated stimuli.

在Raw264.7细胞培养基介导的刺激存在下培养的Ms FLS迁移的示意图。

b

b类

Migration assays of isolated Ms-FLSs treated with TNF or M-CSF + RANKL for 6 h. Fluorescence staining with DAPI (blue). Scale bar, 200 μm.

用TNF或M-CSF+RANKL处理6小时的分离的Ms FLS的迁移测定。用DAPI（蓝色）进行荧光染色。比例尺，200微米。

c

c级

An analysis of the total DAPI intensity was performed to assess Ms-FLS migration under the indicated conditions. The bars represent the mean ± s.e.m. (

进行了总DAPI强度的分析，以评估在指定条件下Ms FLS的迁移。条形代表平均值±s.e.m(

n

n

= 13).

（13）。

d

d

Migration assays of isolated Ms-FLSs subjected to the indicated conditions for 6 h. Fluorescence staining with DAPI (blue). Scale bar, 200 μm.

分离的Ms FLS的迁移测定在指定条件下进行6小时。用DAPI（蓝色）进行荧光染色。比例尺，200微米。

e

e

An analysis of the total DAPI intensity was performed to assess Ms-FLS migration under the indicated conditions. The bars represent the mean ± s.e.m. (

进行了总DAPI强度的分析，以评估在指定条件下Ms FLS的迁移。条形代表平均值±s.e.m(

n

n

= 13).

（13）。

f

f级

Deactivated media were prepared from Rm-Fm-T (conditioned medium from Raw264.7 cells treated with FLS medium containing TNF). An analysis of the total DAPI intensity was performed to assess Ms-FLS migration in the presence or absence of deactivated media. The bars represent the mean ± s.e.m. (

由Rm-Fm-T（来自用含有TNF的FLS培养基处理的Raw264.7细胞的条件培养基）制备失活的培养基。进行了总DAPI强度的分析，以评估在存在或不存在失活培养基的情况下Ms FLS的迁移。条形代表平均值±s.e.m(

n

n

= 10).

（10）。

g

克

Migration assays of isolated Ms-FLSs subjected to the indicated conditions in the presence or absence of TNF and BAPTA for 6 h. Fluorescence staining with DAPI (blue). Scale bar, 200 μm.

在存在或不存在TNF和BAPTA的情况下，在指定条件下对分离的Ms FLS进行迁移测定6小时。用DAPI（蓝色）进行荧光染色。比例尺，200微米。

h

小时

An analysis of the total DAPI intensity was performed to assess Ms-FLS migration under the indicated conditions. The bars represent the mean ± s.e.m. (

进行了总DAPI强度的分析，以评估在指定条件下Ms FLS的迁移。条形代表平均值±s.e.m(

n

n

= 6).

6.

Full size image

全尺寸图像

Osteoclast activation reveals the enhanced expression of migratory module, NBC

破骨细胞活化揭示了迁移模块NBC的表达增强

Previously, we reported that the migratory function of FLSs and the severity of RA in CIA-induced mice were attenuated by NBC inhibition

此前，我们报道了NBC抑制减弱了CIA诱导的小鼠FLSs的迁移功能和RA的严重程度

12

12

. We hypothesized that osteoclast fusion and maturation are modulated by the migratory module to facilitate fusion and migration. We verified the role of the migration-related protein in osteoclasts by measuring the expression of the

我们假设破骨细胞的融合和成熟受迁移模块的调节，以促进融合和迁移。我们通过测量破骨细胞中迁移相关蛋白的表达来验证其在破骨细胞中的作用

Slc4a

Slc4a

family gene set encoding NBC,

编码NBC的家族基因集，

Slc4a4

Slc4a4

,

,

Slc4a7

Slc4a7

and

和

Slc4a10

Slc4a10

, in Raw264.7 cells. Treatment with M-CSF and RANKL increased the expression of the

，在Raw264.7细胞中。用M-CSF和RANKL治疗可增加

Slc4a7

Slc4a7

mRNA (Fig.

mRNA（图）。

5a

5a级

). The protein expression of NBCn1, which is encoded by

)。NBCn1的蛋白质表达，由

Slc4a7

Slc4a7

, was also increased in the presence of RANKL (Fig.

，在RANKL存在下也增加了（图）。

5b, c

5b，c

). NBCn1 is a plasma-membrane-associated protein

)。NBCn1是一种质膜相关蛋白

23

23

. Increased membrane expression of NBCn1 was observed in Raw264.7 cells and BMMs in the presence of M-CSF and RANKL (Fig.

在M-CSF和RANKL存在下，在Raw264.7细胞和BMM中观察到NBCn1的膜表达增加（图）。

5d–g

5d–g

). The increased expression of the NBCn1 protein reflects increased NBC activity. NBC activity was also increased in Raw264.7 cells and BMMs after treatment with M-CSF + RANKL (Fig.

)。NBCn1蛋白表达的增加反映了NBC活性的增加。在用M-CSF+ RANKL处理后，Raw264.7细胞和BMM中的NBC活性也增加（图）。

5h–k

5h–k

). These results indicate that osteoclast activation by M-CSF and RANKL increases NBC expression and activity.

)。这些结果表明，M-CSF和RANKL激活破骨细胞可增加NBC的表达和活性。

Fig. 5: Osteoclast activation reveals the enhanced expression of migratory module, NBC.

图5：破骨细胞活化揭示了迁移模块NBC的增强表达。

a

一

Relative mRNA levels of

相对mRNA水平

Slc4a4

Slc4a4

,

,

Slc4a7

Slc4a7

and

和

Slc4a10

Slc4a10

in Raw264.7 cells treated with or without M-CSF and RANKL for 24 h. The bars represent the mean ± s.e.m. (

在用或不用M-CSF和RANKL处理24小时的Raw264.7细胞中。条形代表平均值±s.e.M(

n

n

= 4).

4.

b

b类

NBCn1 protein levels in Raw264.7 cells treated with or without M-CSF and RANKL for 24 h.

用或不用M-CSF和RANKL处理24小时的Raw264.7细胞中的NBCn1蛋白水平。

c

c级

An analysis of the NBCn1 intensity. The bars represent the mean ± s.e.m. (

NBCn1强度的分析。条形代表平均值±s.e.m(

n

n

= 3).

（3）。

d

d

,

,

f

f级

Immunofluorescence staining for NBCn1 (red) and DAPI (blue) in Raw264.7 cells (

Raw264.7细胞中NBCn1（红色）和DAPI（蓝色）的免疫荧光染色(

d

d

) and BMMs (

)和BMMs(

f

f级

) treated with or without M-CSF and RANKL for 24 h. Scale bar, 10 μm.

)用或不用M-CSF和RANKL治疗24小时。比例尺，10μm。

e

e

,

,

g

克

An analysis of the normalized intensity (total intensity/measuring area) of NBCn1 in Raw264.7 cells (

Raw264.7细胞中NBCn1归一化强度（总强度/测量面积）的分析(

e

e

) and BMM (

)和BMM(

g

克

) cells. The bars represent the mean ± s.e.m. (

)细胞。条形代表平均值±s.e.m(

n

n

= 5–9).

（5-9）。

h

小时

NBC activity was assessed by measuring the changes in the pH

通过测量pH值的变化来评估NBC活性

i

我

values of Raw264.7 cells stimulated with or without M-CSF and RANKL for 24 h.

用或不用M-CSF和RANKL刺激24小时的Raw264.7细胞的值。

i

我

An analysis of NBC activity in Raw264.7 cells (R). The bars represent the mean ± s.e.m. (

Raw264.7细胞中NBC活性的分析（R）。条形代表平均值±s.e.m(

n

n

= 17).

（17）。

j

j

NBC activity was assessed by measuring changes in the pH

通过测量pH值的变化来评估NBC活性

i

我

values of BMMs stimulated with or without M-CSF and RANKL for 24 h.

用或不用M-CSF和RANKL刺激24小时的BMM值。

k

k

An analysis of NBC activity in BMMs under the indicated conditions. The bars represent the mean ± s.e.m. (

。条形代表平均值±s.e.m(

n

n

= 3).

3.

Full size image

全尺寸图像

NBC inhibition attenuates osteoclastogenesis and bone resorption in Raw264.7 cells

NBC抑制作用减弱Raw264.7细胞的破骨细胞生成和骨吸收

NBC activity was measured in Raw264.7 cells treated with or without the NBC inhibitor S0859 to determine the role of NBC in osteoclast activation

在用或不用NBC抑制剂S0859处理的Raw264.7细胞中测量NBC活性，以确定NBC在破骨细胞活化中的作用

12

12

. Treatment with S0859 attenuated M-CSF + RANKL-mediated NBC activity in Raw264.7 cells (Fig.

用S0859处理减弱了Raw264.7细胞中M-CSF+ RANKL介导的NBC活性（图）。

6a, b

6a，b

). Treatment with 40 μM S0859 did not affect the viability of Raw264.7 cells (Supplementary Fig.

)。用40μMS0859处理不影响Raw264.7细胞的活力（Supplementary Fig.）。

4a

4a级

). We determined whether the attenuated NBC activity of Raw264.7 cells induces cellular fusion and bone resorption. Treatment with S0859 markedly attenuated M + R-stimulated multinucleated cell fusion and decreased the number of mature osteoclasts (Fig.

)。我们确定了Raw264.7细胞的NBC活性减弱是否诱导细胞融合和骨吸收。用S0859处理显着减弱M+R刺激的多核细胞融合并减少成熟破骨细胞的数量（图）。

6c–e

6c–e

) and bone resorption in Raw264.7 cells (Fig.

)和Raw264.7细胞中的骨吸收（图）。

6f, g

6f，g

). NFATc1 protein expression in Raw264.7 cells treated with or without S0859 was evaluated to determine the master transcriptional regulator of osteoclastogenesis. M-CSF + RANKL-treated Raw264.7 cells expressed NFATc1; however, treatment with S0859 reduced NFATc1 protein expression (Fig.

)。评估用或不用S0859处理的Raw264.7细胞中NFATc1蛋白的表达，以确定破骨细胞生成的主要转录调节因子。M-CSF+ RANKL处理的Raw264.7细胞表达NFATc1；然而，用S0859处理降低了NFATc1蛋白的表达（图）。

6h, i

6小时，我

).

).

Fig. 6: NBC inhibition attenuates osteoclastogenesis and bone resorption in Raw264.7 cells.

图6:NBC抑制减弱Raw264.7细胞中的破骨细胞生成和骨吸收。

a

一

NBC activity was assessed by measuring the changes in the pH

通过测量pH值的变化来评估NBC活性

i

我

values of M-CSF- and RANKL-stimulated Raw264.7 cells treated with or without 20 μΜ S0859 for 24 h.

。

b

b类

An analysis of NBC activity in Raw264.7 cells (R). The bars represent the mean ± s.e.m. (

Raw264.7细胞中NBC活性的分析（R）。条形代表平均值±s.e.m(

n

n

= 13–30).

== 13–30).

c

c级

Representative images of TRAP staining in Raw264.7 cells subjected to the indicated conditions in the presence or absence of 20 μΜ S0859 for 7 days. Scale bar, 200 μm.

在存在或不存在20μlS0859的情况下，在指定条件下处理7天的Raw264.7细胞中TRAP染色的代表性图像。比例尺，200微米。

d

d

An analysis of the relative TRAP intensity in osteoclasts derived from Raw264.7 cells subjected to the indicated conditions for 7 days. The bars represent the mean ± s.e.m. (

分析来自Raw264.7细胞的破骨细胞在指定条件下7天的相对陷阱强度。条形代表平均值±s.e.m(

n

n

= 16).

（16）。

e

e

An analysis of the number of osteoclasts derived from Raw264.7 cells subjected to the indicated conditions for 7 days. The bars represent the mean ± s.e.m. (

分析来自Raw264.7细胞的破骨细胞在指定条件下7天的数量。条形代表平均值±s.e.m(

n

n

= 16).

== 16).

f

f级

Bone resorption pit assay of Raw264.7 cells cultured with dentin discs and subjected to the indicated conditions in the presence or absence of 20 μΜ S0859 for 7 days. Scale bar, 200 μm.

用牙本质盘培养的Raw264.7细胞的骨吸收坑测定，并在存在或不存在20μlS0859的情况下在指定条件下进行7天。比例尺，200微米。

g

克

An analysis of lacunae (dark areas) formed by Raw264.7 cells. The bars represent the mean ± s.e.m. (

Raw264.7细胞形成的腔隙（暗区）分析。条形代表平均值±s.e.m(

n

n

= 16).

（16）。

h

小时

NFATc1 protein levels in Raw264.7 cells exposed to the indicated stimuli for 7 days. β-Actin was used as a loading control.

暴露于指定刺激7天的Raw264.7细胞中的NFATc1蛋白水平。β-肌动蛋白用作上样对照。

i

我

An analysis of the band intensity of the NFATc1 protein in Raw264.7 cells. The bars represent the mean ± s.e.m. (

Raw264.7细胞中NFATc1蛋白条带强度的分析。条形代表平均值±s.e.m(

n

n

= 6).

（6）。

Full size image

全尺寸图像

NBC inhibition attenuates osteoclastogenesis and bone resorption in BMMs

NBC抑制作用减弱BMM中的破骨细胞生成和骨吸收

We determined the role of NBC in primary cultured BMMs treated with or without the NBC inhibitor S0859. Treatment with M-CSF and RANKL increased NBC activity; however, in the presence of S0859, M-CSF + RANKL-mediated NBC activity was reduced in BMMs (Fig.

我们确定了NBC在用或不用NBC抑制剂S0859处理的原代培养BMM中的作用。用M-CSF和RANKL治疗增加了NBC活性；然而，在S0859存在下，BMM中M-CSF+ RANKL介导的NBC活性降低（图）。

7a, b

7a，b

). Treatment with 40 μM S0859 did not affect the viability of BMMs (Supplementary Fig.

)。用40μMS0859处理不影响BMMs的活力（Supplementary Fig.）。

4b

4b级

). We determined whether the attenuated NBC activity in BMMs induces cellular fusion and bone resorption. Treatment with S0859 markedly attenuated M-CSF + RANKL-stimulated multinucleated cell fusion and decreased the number of mature osteoclasts (Fig.

)。我们确定BMM中减弱的NBC活性是否诱导细胞融合和骨吸收。用S0859处理显着减弱M-CSF+ RANKL刺激的多核细胞融合并减少成熟破骨细胞的数量（图）。

7c–e

7c–e

) and bone resorption (Fig.

)和骨吸收（图）。

7f, g

7f，g

). However, treatment with S0859 did not affect the viability of FLSs (Supplementary Fig.

)。然而，用S0859处理不影响FLSs的活力（Supplementary Fig.）。

5

5

). BMMs treated with or without S0859 were immunostained for NFATc1 to assess NFATc1 expression. M-CSF + RANKL-treated BMMs presented increased nuclear localization and increased protein expression of NFATc1; however, treatment with S0859 reduced NFATc1 expression (Fig.

)。用或不用S0859处理的BMM对NFATc1进行免疫染色以评估NFATc1的表达。M-CSF+ RANKL处理的BMM表现出增加的核定位和增加的NFATc1蛋白表达；然而，用S0859处理降低了NFATc1的表达（图）。

7h–k

7h–k

). These results indicate that NBC inhibition attenuates osteoclast differentiation and bone resorption activity.

)。这些结果表明NBC抑制减弱破骨细胞分化和骨吸收活性。

Fig. 7: NBC inhibition attenuates osteoclastogenesis and bone resorption in BMMs.

图7:NBC抑制减弱BMM中的破骨细胞生成和骨吸收。

a

一

NBC activity was assessed by measuring the changes in the pH

通过测量pH值的变化来评估NBC活性

i

我

values of M-CSF- and RANKL-stimulated BMMs treated with or without 20 μΜ S0859 for 24 h.

用或不用20μlS0859处理24小时的M-CSF和RANKL刺激的BMM的值。

b

b类

An analysis of NBC activity in BMMs. The bars represent the mean ± s.e.m. (

BMM中NBC活性的分析。条形代表平均值±s.e.m(

n

n

= 5).

（5）。

c

c级

Representative images of TRAP staining in osteoclasts derived from BMMs under the indicated conditions; M-CSF was administered for 3 days, followed by an additional 4 days of M-CSF treatment. The experiments were conducted in the presence or absence of 20 μΜ S0859 for 3 days. Scale bar, 100 μm.

在指定条件下，来自BMM的破骨细胞中TRAP染色的代表性图像；M-CSF给药3天，然后再进行4天的M-CSF治疗。实验在存在或不存在20μlS0859的情况下进行3天。比例尺，100微米。

d

d

An analysis of the relative TRAP intensity in osteoclasts derived from BMMs. The bars represent the mean ± s.e.m. (

来自BMM的破骨细胞中相对陷阱强度的分析。条形代表平均值±s.e.m(

n

n

= 16).

（16）。

e

e

An analysis of the relative number of TRAP-positive osteoclasts derived from BMMs. The bars represent the mean ± s.e.m. (

分析来自BMM的TRAP阳性破骨细胞的相对数量。条形代表平均值±s.e.m(

n

n

= 16).

（16）。

f

f级

Bone resorption pit assay of BMMs cultured with dentin discs and subjected to the indicated conditions. BMMs were cultured with dentin discs and treated with M-CSF for 4 days, followed by exposure to 20 μM S0859 for 3 days. Scale bar, 100 μm.

用牙本质盘培养并在指定条件下进行BMM的骨吸收坑测定。将BMM与牙本质盘一起培养并用M-CSF处理4天，然后暴露于20μMS0859 3天。比例尺，100微米。

g

克

An analysis of lacunae (dark areas) formed by BMMs. The bars represent the mean ± s.e.m. (

BMM形成的腔隙（暗区）分析。条形代表平均值±s.e.m(

n

n

= 16).

== 16).

h

小时

Immunofluorescence staining for NFATc1 (red) and DAPI (blue) in BMMs subjected to the indicated stimuli. BMMs were first treated with M-CSF for 3 days, followed by an additional 4 days of treatment with M-CSF in the presence or absence of 20 μM S0859. Scale bar, 10 μm.

受指定刺激的BMM中NFATc1（红色）和DAPI（蓝色）的免疫荧光染色。BMM首先用M-CSF治疗3天，然后在存在或不存在20μMS0859的情况下用M-CSF再治疗4天。比例尺，10μm。

i

我

, An analysis of the normalized intensity (total intensity/measuring area) of NFATc1 in BMMs. The bars represent the mean ± s.e.m. (

，分析BMM中NFATc1的归一化强度（总强度/测量面积）。条形代表平均值±s.e.m(

n

n

= 4).

（4）。

j

j

NFATc1 protein levels in BMMs subjected to the indicated conditions. The cells were first treated with M-CSF for 3 days, followed by an additional 4 days of treatment with M-CSF in the presence or absence of 20 μM S0859. β-Actin was used as a loading control.

在指定条件下，BMM中的NFATc1蛋白水平。首先用M-CSF处理细胞3天，然后在存在或不存在20μMS0859的情况下用M-CSF处理另外4天。β-肌动蛋白用作上样对照。

k

k

An analysis of the band intensity of the NFATc1 protein in BMMs. The bars represent the mean ± s.e.m. (

BMM中NFATc1蛋白条带强度的分析。条形代表平均值±s.e.m(

n

n

= 4).

4.

Full size image

全尺寸图像

NBC inhibitor S0859-exposed osteoclast medium does not induce FLS migration

NBC抑制剂S0859暴露的破骨细胞培养基不诱导FLS迁移

BMMs were exposed to FLS medium obtained under five different conditions and cultured for 8–9 days until they reached the mature state to verify the role of NBC in BMM fusion and bone resorption after treatment with or without S0859. New FLSs were exposed to dentin disc-containing BMM medium in the lower chamber of the transwell system to determine the effects of the factors released by mature osteoclasts on FLS migration.

。将新的FLS暴露于transwell系统下室中含有BMM培养基的牙本质盘中，以确定成熟破骨细胞释放的因子对FLS迁移的影响。

We illustrated the experimental approach in Fig. .

我们在图中说明了实验方法。

8a

8a个

. FLS migration was increased in response to mature BMM-conditioned medium, Bm-M + R and Bm-Fm-T; however, FLS migration, mediated by the dentin components released from mature BMMs into the medium, was inhibited in the presence of S0859 (Fig.

响应于成熟的BMM条件培养基，Bm-M + R和Bm-Fm-T，FLS迁移增加；然而，在S0859存在下，由成熟BMM释放到培养基中的牙本质成分介导的FLS迁移受到抑制（图）。

8b, c

8b，c

). As shown in Fig.

)。如图所示。

4f, h

4f，h

, Ca

，加利福尼亚州

2+

2+

chelation or deactivated medium Bm-Fm-T reduced FLS migration (Fig.

螯合或失活培养基Bm-Fm-T减少FLS迁移（图）。

8b, c

8b，c

). Furthermore, we determined whether NBC inhibition affects the expression of the osteoclast differentiation factor M-CSF by stimulating primary cultures of Ms-FLSs with various cytokines. Treatment with S0859 attenuated the cytokine-mediated M-CSF increases (Fig.

)。此外，我们通过用各种细胞因子刺激Ms FLS的原代培养物来确定NBC抑制是否影响破骨细胞分化因子M-CSF的表达。用S0859治疗减弱了细胞因子介导的M-CSF增加（图）。

8d

8天

). These results indicate that NBC inhibition has inhibitory effects on cytokine-mediated FLS migration and osteoclast maturation.

)。这些结果表明NBC抑制对细胞因子介导的FLS迁移和破骨细胞成熟具有抑制作用。

Fig. 8: NBC inhibitor S0859-exposed osteoclast medium does not induce FLS migration.

图8:NBC抑制剂S0859暴露的破骨细胞培养基不诱导FLS迁移。

a

一

The schematic procedure of primary Ms-FLS migration cultured in the presence of the BMM medium-mediated stimuli.

在BMM培养基介导的刺激存在下培养的原代Ms FLS迁移的示意图。

b

b类

Migration assays of isolated Ms-FLSs subjected to the indicated conditions for 6 h. Fluorescence staining with DAPI (blue). Scale bar, 200 μm.

分离的Ms FLS的迁移测定在指定条件下进行6小时。用DAPI（蓝色）进行荧光染色。比例尺，200微米。

c

c级

An analysis of the total DAPI intensity was performed to assess Ms-FLS migration under the indicated conditions. The bars represent the mean ± s.e.m. (

进行了总DAPI强度的分析，以评估在指定条件下Ms FLS的迁移。条形代表平均值±s.e.m(

n

n

= 12).

（12）。

d

d

The concentration of M-CSF secreted from Ms-FLSs subjected to the indicated conditions. The bars represent the mean ± s.e.m. (

在指定的条件下，从Ms FLS分泌的M-CSF浓度。条形代表平均值±s.e.m(

n

n

= 3, *

= 3， *

P

P

< 0.05, **

< 0.05， **

P

P

< 0.01,

< 0.01，

#

#

P

P

< 0.05,

< 0.05，

##

##

P

P

< 0.01).

（0.01）

Full size image

全尺寸图像

Inhibitory effect of S0859 on bone resorption in the CIA mouse model

S0859对CIA小鼠模型骨吸收的抑制作用

We determined whether the inhibition of NBC disrupted the infinite cytokine feedback loop and bone damage in a mouse model of CIA. The process used to establish the CIA mouse model is shown in Supplementary Fig.

我们确定了NBC的抑制是否破坏了CIA小鼠模型中无限的细胞因子反馈环和骨损伤。用于建立CIA小鼠模型的过程如Supplementary Fig.所示。

6

6

. The CIA model mice presented increased paw thickness and paw scores, with gradually increasing body weights observed 4 weeks after arthritis development (Fig.

CIA模型小鼠的爪子厚度和爪子评分增加，关节炎发展后4周观察到体重逐渐增加（图）。

9a–d

9a–d

and Supplementary Fig.

和补充图。

7a–d

7a–d

). Paw swelling was observed in the CIA group; however, this swelling was reduced at 14 weeks in the S0859-injected or adalimumab-injected CIA groups (Fig.

)。在CIA组中观察到爪肿胀；然而，在注射S0859或注射阿达木单抗的CIA组中，这种肿胀在14周时减少（图）。

9a–c

9a–c

). Adalimumab, which is a monoclonal TNF antibody widely used to treat RA

)。阿达木单抗是一种广泛用于治疗RA的单克隆TNF抗体

24

24

,

,

25

25

, was used as a positive control. The serum levels of C-terminal telopeptide of type I collagen (CTX-1), a biomarker of bone resorption

，用作阳性对照。骨吸收的生物标志物I型胶原C末端端肽（CTX-1）的血清水平

26

26

, were measured to verify bone resorption in the mouse model. Elevated CTX-1 levels were detected in the CIA group, indicating increased bone resorption, whereas CTX-1 levels were significantly reduced in the S0859-injected or adalimumab-injected CIA groups (Fig.

，以验证小鼠模型中的骨吸收。在CIA组中检测到CTX-1水平升高，表明骨吸收增加，而注射S0859或注射阿达木单抗的CIA组中CTX-1水平显着降低（图）。

9e

9e级

). In addition, bone slice samples were stained with TRAP to verify whether NBC inhibition modulates osteoclastogenesis in mouse bones. The CIA group presented increased numbers of osteoclast nuclei and osteoclastogenic areas, whereas the S0859-injected or adalimumab-injected CIA groups presented reduced osteoclastogenic areas (Fig.

)。。CIA组破骨细胞核和破骨细胞生成区数量增加，而注射S0859或注射阿达木单抗的CIA组破骨细胞生成区减少（图）。

.

.

9f–h

9f–h

and Supplementary Fig.

和补充图。

7e–g

7e–g

). Moreover, bone porosity was measured using micro-CT to verify the inhibitory effect of S0859 on bone remodeling in the CIA mouse model. The bone density of the CIA group was reduced, whereas that of the S0859-injected or adalimumab-injected CIA group recovered to control levels (Fig.

)。此外，使用micro-CT测量骨孔隙率，以验证S0859对CIA小鼠模型中骨重塑的抑制作用。CIA组的骨密度降低，而注射S0859或注射阿达木单抗的CIA组的骨密度恢复到对照水平（图）。

9i, j

and Supplementary Fig.

和补充图。

7h, i

7小时，我

). The effects of S0859 alone on the paw thickness, paw score, body weight, TRAP staining of bone slices, and micro-CT images in control mice are shown in Supplementary Fig.

)。补充图显示了单独使用S0859对对照小鼠爪子厚度，爪子评分，体重，骨切片TRAP染色和显微CT图像的影响。

7

7

. Treatment of control mice with S0859 resulted in outcomes similar to untreated control mice (Supplementary Fig.

用S0859处理对照小鼠的结果与未处理的对照小鼠相似（Supplementary Fig.）。

7

7

). These results indicate that NBC inhibition reduces swelling, pathological changes associated with RA, and bone-absorbing osteoclastogenic function.

)。这些结果表明，NBC抑制可减轻肿胀，与RA相关的病理变化以及骨吸收破骨细胞生成功能。

Fig. 9: Inhibitory effect of S0859 on bone resorption in the CIA mouse model.

图9:S0859对CIA小鼠模型中骨吸收的抑制作用。

a

一

Representative images of the paws of mice treated with the indicated stimuli captured at 0, 8 and 14 weeks. Scale bar, 5 mm.

在0,8和14周时捕获的用指定刺激处理的小鼠爪子的代表性图像。比例尺，5毫米。

b

b类

The paw thickness (mm) of mice in all groups was analyzed every 2 weeks. The bars represent the mean ± s.e.m. (

每2周分析所有组小鼠的爪子厚度（mm）。条形代表平均值±s.e.m(

##

##

P

P

< 0.01,

< 0.01，

###

###

P

P

< 0.001).

（0.001）

c

c级

Paw scores of all the groups were analyzed every 2 weeks. The bars represent the mean ± s.e.m. (

每2周分析所有组的Paw评分。条形代表平均值±s.e.m(

##

##

P

P

< 0.01,

< 0.01，

###

###

P

P

< 0.001).

（0.001）

d

d

Body weight was analyzed every 2 weeks in all groups. The bars represent the mean ± s.e.m. In

所有组每2周分析一次体重。条形代表中的平均值±s.e.m

b

b类

–

–

d

d

the arrowheads represent the drug administration points.

箭头代表药物管理点。

e

e

The serum concentration of secreted CTX-1 in mice subjected to the indicated treatments. The bars represent the mean ± s.e.m.

接受指定治疗的小鼠中分泌的CTX-1的血清浓度。条形代表平均值±s.e.m。

f

f级

Representative images of TRAP-stained plantar bones obtained from mice subjected to the indicated treatments after 14 weeks. Scale bar, 500 μm.

14周后，从接受指定治疗的小鼠获得的TRAP染色的足底骨的代表性图像。比例尺，500微米。

g

克

,

,

h

小时

An analysis of the number of osteoclasts (N.Oc)/bone perimeter (B.Pm) (

破骨细胞数量（N.Oc）/骨周长（B.Pm）的分析(

g

克

) and the osteoclast surface (Oc.S)/bone surface (BS) (

)(

h

小时

) in the indicated groups. The bars represent the mean ± s.e.m. compared with the control group.

)在指定的组中。条形图表示与对照组相比的平均值±s.e.m。

i

我

Representative micro-CT images of trabecular bone in the femur of mice from the indicated groups. The top images show vertical sections, whereas the bottom images show horizontal sections. Scale bars, 500 μm.

来自所示组的小鼠股骨中小梁骨的代表性显微CT图像。顶部图像显示垂直部分，而底部图像显示水平部分。比例尺，500微米。

j

j

An analysis of the bone density of the trabecular region of the femur from mice in the indicated groups. The bars represent the mean ± s.e.m. compared with the control group. C, control; S, S0859; A, adalimumab.

分析所示组小鼠股骨小梁区域的骨密度。条形图表示与对照组相比的平均值±s.e.m。C、 控制；S、 ；A、 阿达木单抗。

Full size image

全尺寸图像

Discussion

讨论

In this study, we investigated the Ca

在这项研究中，我们调查了Ca

2+

2+

and cytokine feedback loop between inflammation and bone resorption in RA-associated cellular and mouse models. Inflammatory cytokines and Ca

RA相关细胞和小鼠模型中炎症和骨吸收之间的细胞因子反馈回路。炎性细胞因子和Ca

2+

2+

released from the bone increased the level of the bone resorption factor M-CSF in RA-FLSs. Factors released from RA-FLSs mediate the increased bone resorption activity of osteoclasts. The outputs of bone resorption subsequently induce FLS migration through the modulation of NBCn1. Secreted factors related to RA, such as inflammatory cytokines and Ca.

从骨骼释放增加了RA FLS中骨吸收因子M-CSF的水平。RA FLS释放的因子介导破骨细胞骨吸收活性的增加。骨吸收的输出随后通过调节NBCn1诱导FLS迁移。与RA相关的分泌因子，如炎性细胞因子和Ca。

2+

2+

, mediated osteoclastogenesis, which was attenuated by inhibition of NBC activity. Furthermore, we verified the inhibitory effect of S0859 on bone damage and osteoclastogenic activity in the bones of a CIA mouse model. Previously, we reported that the aggressiveness of RA-FLSs is mediated by the recruitment of NBCn1 to the plasma membrane.

介导的破骨细胞生成，其通过抑制NBC活性而减弱。此外，我们验证了S0859对CIA小鼠模型骨骼中骨损伤和破骨细胞生成活性的抑制作用。以前，我们报道了RA FLS的侵袭性是通过将NBCn1募集到质膜来介导的。

12

12

. NBCn1, a bicarbonate transporter, provides a mechanical driving module for migration toward inflammatory signals

.NBCn1是一种碳酸氢盐转运蛋白，为向炎症信号的迁移提供了机械驱动模块

12

12

and metastatic modulation in cancers

和癌症的转移调节

27

27

. Within the scope of inflammation, the inhibition of NBCn1 via treatment with S0859 improves RA severity in a CIA mouse model without changes in blood count, hepatic and renal toxicities

在炎症范围内，通过S0859治疗抑制NBCn1可改善CIA小鼠模型中RA的严重程度，而不会改变血细胞计数，肝脏和肾脏毒性

12

12

. In the present study, NBC inhibition by S0859 revealed dual inhibitory effects on the cytokine feedback loop and osteoclast differentiation. Our results also provide experimental insights into the direct interaction between FLSs and osteoclasts through a Ca

在本研究中，S0859对NBC的抑制作用揭示了对细胞因子反馈环和破骨细胞分化的双重抑制作用。我们的结果还提供了通过Ca研究FLS与破骨细胞之间直接相互作用的实验见解

2+

2+

and cytokine feedback loop. A schematic illustration of the Ca

和细胞因子反馈回路。Ca的示意图

2+

2+

and cytokine feedback loop in both FLSs and osteoclasts is shown in Fig.

FLS和破骨细胞中的细胞因子反馈环如图所示。

10

10

.

.

Fig. 10: Schematic illustration of the cytokine feedback loop-mediated crosstalk between FLSs and osteoclasts.

图10:FLS和破骨细胞之间细胞因子反馈环介导的串扰的示意图。

The released inflammatory cytokines and Ca

释放的炎性细胞因子和Ca

2+

2+

, which are main components in inflammatory synovium enhanced the bone resorption factor M-CSF from RA-FLSs. M-CSF derived from RA-FLSs promoted osteoclast-induced bone resorption, which increased the release of Ca

，它们是炎性滑膜中的主要成分，可增强RA FLSs的骨吸收因子M-CSF。源自RA FLSs的M-CSF促进破骨细胞诱导的骨吸收，从而增加Ca的释放

2+

2+

from the bone. Increased Ca

从骨头上。增加Ca

2+

2+

and cytokine levels simultaneously increased FLS activation, suggesting that a cytokine feedback loop is continuously generated, which subsequently exacerbates RA. The open-dotted curved arrows represent the cytokine feedback loop between FLSs and osteoclasts through the involvement of releasing components such as cytokines and Ca.

。开放的虚线弯曲箭头表示FLS和破骨细胞之间的细胞因子反馈环，通过释放成分如细胞因子和Ca的参与。

2+

2+

. OC, osteoclast.

OC，破骨细胞。

Full size image

全尺寸图像

Inflammatory cytokines and acidic stimuli increase ion transporter activity. Previously, we reported that NBCn1 is involved in the aggressiveness of FLSs

炎性细胞因子和酸性刺激增加离子转运蛋白活性。此前，我们报道了NBCn1参与了FLS的攻击性

12

12

. Interestingly, inflammatory cytokine production and FLS migration are stimulated by increased Ca

有趣的是，Ca的增加刺激了炎性细胞因子的产生和FLS的迁移

2+

2+

levels, suggesting that the crosstalk between FLSs and osteoclasts is mediated by these factors. The factors released from the dentin disc, which mainly include Ca

水平，表明FLS和破骨细胞之间的串扰是由这些因素介导的。牙本质盘释放的因素主要包括Ca

2+

2+

, are involved in FLS aggressiveness through the increased activity of the migratory module NBC. These FLSs and osteoclasts possess the same migratory apparatus, NBCn1. Inhibition of NBCn1 attenuates osteoclastogenesis, decreases the bone resorption area and inhibits the invasive phenotype of FLSs. Therefore, the modulation of NBCn1 in osteoclasts might be a new strategy to regulate bone homeostasis..

，通过迁移模块NBC的活动增加而参与FLS的攻击性。这些FLS和破骨细胞具有相同的迁移装置NBCn1。NBCn1的抑制减弱破骨细胞生成，减少骨吸收面积并抑制FLS的侵袭性表型。因此，破骨细胞中NBCn1的调节可能是调节骨稳态的新策略。。

RA is a complex chronic inflammatory disease. Studies are ongoing to develop an optimized therapeutic target, recover individual variability and systemic comorbidities, reduce incomplete responses to medication and pathological complications, minimize side effects and reduce treatment costs related to RA.

RA是一种复杂的慢性炎症性疾病。正在进行研究以开发优化的治疗靶点，恢复个体变异性和全身合并症，减少对药物和病理并发症的不完全反应，最大程度地减少副作用并降低与RA相关的治疗成本。

28

28

,

,

29

29

. The treatment strategies for RA involve various targets, including the inhibition of inflammatory cytokines by agents such as biological DMARDs

类风湿关节炎的治疗策略涉及多种靶点，包括生物DMARDs等药物对炎性细胞因子的抑制

28

28

. In addition, synthetic DMARDs have been proposed to treat RA. For instance, Janus kinase inhibitors, such as upadacitinib, tofacitinib and baricitinib, offer advantages for patients who are nonresponsive to biologic DMARDs in RA treatment; however, some drugs have increased long-term safety concerns, such as cancer and cardiovascular risks, and comparisons of these drugs are still ongoing.

此外，已经提出合成DMARD来治疗RA。例如，Janus激酶抑制剂，如upadacitinib，tofacitinib和baricitinib，为RA治疗中对生物DMARDs无反应的患者提供了优势；然而，一些药物增加了长期安全性问题，如癌症和心血管风险，这些药物的比较仍在进行中。

30

30

,

,

31

31

,

,

32

32

. Nonsteroidal anti-inflammatory drugs or corticosteroids are also commonly used to treat pain and inflammation; however, their long-term application should be carefully considered due to cardiovascular issues

非甾体类抗炎药或皮质类固醇也常用于治疗疼痛和炎症；但是，由于心血管问题，应仔细考虑其长期应用

33

33

,

,

34

34

. In addition, therapies targeting TNF, a predominant cytokine, have been successfully developed over the past two decades

此外，在过去的二十年中，已经成功开发了针对TNF（一种主要的细胞因子）的疗法

25

25

. However, patients who have contraindications or fail to respond to TNF therapy are recommended to receive other types of DMARD. Combination therapies using biologic or synthetic DMARDs such as methotrexate are also recommended

但是，建议有禁忌症或对TNF治疗无效的患者接受其他类型的DMARD。还建议使用生物或合成DMARD（例如甲氨蝶呤）进行联合治疗

35

35

,

,

36

36

,

,

37

37

. Recently, combination approaches involving both the proteasome inhibitor delanzomib and the TNF inhibitor adalimumab have been proposed to treat RA

最近，已经提出了涉及蛋白酶体抑制剂德兰佐米和TNF抑制剂阿达木单抗的联合方法来治疗RA

25

25

. Although DMARDs and their combination have been developed, the number of patients whose RA is difficult to treat is still as high as 20% (ref.

虽然DMARDs及其组合已经开发出来，但RA难以治疗的患者数量仍高达20%（参考文献）。

38

38

). Thus, research on appropriate RA therapies and new targets are still ongoing, as identifying a complete cure remains a challenge. From this perspective, our study highlights the rationale for the need for an appropriate treatment for RA as an alternative to TNF therapy, such as Adalimumab therapy..

)。因此，关于适当的RA疗法和新靶点的研究仍在进行中，因为确定完全治愈仍然是一个挑战。从这个角度来看，我们的研究强调了需要适当治疗RA作为TNF治疗替代方案的基本原理，如阿达木单抗治疗。。

In this study, we determined that both inflammatory cytokines and Ca

在这项研究中，我们确定炎性细胞因子和Ca

2+

2+

release must be controlled to prevent the activation of FLSs, which are involved in RA pathology. In addition to Ca

必须控制释放以防止参与RA病理的FLS的激活。除了Ca

2+

2+

, FLS migration-associated components of the synovial fluid, such as dickkopf-1 (ref.

，FLS迁移相关的滑液成分，如dickkopf-1（参考文献）。

39

39

), gastrin-releasing peptide

)，胃泌素释放肽

11

11

, class 3 semaphorins

，第3类Semaphorins

40

40

and anti-cyclic citrullinated peptide antibody

和抗环瓜氨酸肽抗体

41

41

, have been detected in the RA synovium. Accordingly, the roles of these RA-related components in the Ca

，已在RA滑膜中检测到。

2+

2+

and cytokine feedback loop should be characterized to develop new therapies for RA in the near future. In addition to the suppression strategy for each inflammatory factor in patients with RA, modulation of the crosstalk between FLSs and osteoclasts should be considered a pioneering strategy to attenuate RA severity and bone erosion and develop an appropriate drug treatment strategy..

并且应该表征细胞因子反馈回路，以在不久的将来开发RA的新疗法。除了RA患者中每种炎症因子的抑制策略外，调节FLS和破骨细胞之间的串扰应被视为减轻RA严重程度和骨侵蚀并制定适当药物治疗策略的开创性策略。。

Animal studies

动物研究

All experimental animal procedures were performed in accordance with the Gachon University guidelines and were approved by the Animal Care and Use Committee of Gachon University (LCDI-2022-0118).

所有实验动物程序均按照加川大学指南进行，并得到加川大学动物护理和使用委员会（LCDI-2022-0118）的批准。

References

参考文献

McGonagle, D. et al. The relationship between synovitis and bone changes in early untreated rheumatoid arthritis—a controlled magnetic resonance imaging study.

。

Arthritis Rheum.

。

42

42

, 1706–1711 (1999).

, 1706–1711 (1999).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Lee, D. M. & Weinblatt, M. E. Rheumatoid arthritis.

Lee，D.M。和Weinblatt，M.E。类风湿性关节炎。

Lancet

柳叶刀

358

358

, 903–911 (2001).

, 903–911 (2001).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Aletaha, D. & Smolen, J. S. Diagnosis and management of rheumatoid arthritis: a review.

Aletaha，D。＆Smolen，J.S。类风湿性关节炎的诊断和治疗：综述。

JAMA

JAMA

320

320

, 1360–1372 (2018).

, 1360–1372 (2018).

Article

文章

PubMed

PubMed

Google Scholar

谷歌学者

Nanki, T. et al. Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

趋化因子调节类风湿性关节炎患者成纤维细胞样滑膜细胞产生的IL-6和IL-8。

J. Immunol.

J、 免疫。

167

167

, 5381–5385 (2001).

, 5381–5385 (2001).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Bhattacharjee, M. et al. Synovial fluid proteome in rheumatoid arthritis.

Bhattacharjee，M。等人。类风湿性关节炎中的滑液蛋白质组。

Clin. Proteomics

临床。蛋白质组学

13

13

, 12 (2016).

, 12 (2016).

Article

文章

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

McInnes, I. B. & Schett, G. Cytokines in the pathogenesis of rheumatoid arthritis.

McInnes，I.B。＆Schett，G。细胞因子在类风湿性关节炎的发病机制中。

Nat. Rev. Immunol.

国家免疫修订版。

7

7

, 429–442 (2007).

, 429–442 (2007).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Schett, G. & Gravallese, E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment.

Schett，G。＆Gravallese，E。类风湿性关节炎的骨侵蚀：机制，诊断和治疗。

Nat. Rev. Rheumatol.

风湿病杂志。

8

8

, 656–664 (2012).

, 656–664 (2012).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Fuller, K. et al. Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.

巨噬细胞集落刺激因子刺激分离的破骨细胞的存活和趋化行为。

J. Exp. Med.

实验医学学报

178

178

, 1733–1744 (1993).

, 1733–1744 (1993).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Yasuda, H. et al. Osteoclast differentiation factor is a ligand for osteoprotegerin osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.

Yasuda，H。等人。破骨细胞分化因子是骨保护素破骨细胞生成抑制因子的配体，与TRANCE/RANKL相同。

Proc. Natl Acad. Sci. USA

普罗克。阿卡德之夜滑雪。美国

95

95

, 3597–3602 (1998).

, 3597–3602 (1998).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Maruotti, N. et al. Osteoclastogenesis and arthritis.

Maruotti， N. et al. 骨髓形成与关节炎。

Clin. Exp. Med.

克林Exp. Med。

11

11

, 137–145 (2011).

, 137–145 (2011).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Kang, J. Y. et al. The role of Ca

Kang，J.Y.等人，《CA的作用》

2+

2+

-NFATc1 signaling and its modulation on osteoclastogenesis.

-NFATc1信号传导及其对破骨细胞生成的调节。

Int. J. Mol. Sci.

Int.J.Mol.Sci。

21

21

, 3646 (2020).

, 3646 (2020).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Ji, M. et al. Dynamic synovial fibroblasts are modulated by NBCn1 as a potential target in rheumatoid arthritis.

Ji，M。等人。动态滑膜成纤维细胞被NBCn1调节为类风湿性关节炎的潜在靶标。

Exp. Mol. Med.

实验分子医学。

54

54

, 503–517 (2022).

, 503–517 (2022).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Aletaha, D. et al. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative.

Aletaha，D。等人，2010类风湿性关节炎分类标准：美国风湿病学会/欧洲抗风湿联盟合作倡议。

Arthritis Rheum.

。

62

62

, 2569–2581 (2010).

, 2569–2581 (2010).

Article

文章

PubMed

PubMed

Google Scholar

谷歌学者

Ji, M. J. et al. Cellular channelopathy mediated by hypergravity: IL-6-mediated Nkcc1 activation and enhanced Trpm2 expression in rat atrium.

Ji，M.J.等人。超重力介导的细胞通道病：IL-6介导的Nkcc1激活和大鼠心房Trpm2表达增强。

Cell Tissue Res.

细胞组织研究。

383

383

, 1017–1024 (2021).

, 1017–1024 (2021).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Moschovakis, G. L. et al. The chemokine receptor CCR7 is a promising target for rheumatoid arthritis therapy.

Moschovakis，G.L。等人。趋化因子受体CCR7是类风湿性关节炎治疗的有希望的靶标。

Cell. Mol. Immunol.

细胞。分子免疫。

16

16

, 791–799 (2019).

, 791–799 (2019).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Yeh, S. A. et al. Quantification of bone marrow interstitial pH and calcium concentration by intravital ratiometric imaging.

Yeh，S.A.等人。通过活体比率成像定量骨髓间质pH和钙浓度。

Nat. Commun.

Nat.普通。

13

13

, 393 (2022).

, 393 (2022).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Joeckel, E. et al. High calcium concentration in bones promotes bone metastasis in renal cell carcinomas expressing calcium-sensing receptor.

Joeckel，E。等人。骨骼中高钙浓度促进表达钙敏感受体的肾细胞癌的骨转移。

Mol. Cancer

分子癌症

13

13

, 42 (2014).

, 42 (2014).

Article

文章

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

De Giusti, V. C., Garciarena, C. D. & Aiello, E. A. Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na

De Giusti，V.C.，Garciarena，C.D。和Aiello，E.A。活性氧（ROS）在血管紧张素II诱导的心脏Na刺激中的作用

+

+

/HCO

HCO

3

3

−

−

cotransport.

共同运输。

J. Mol. Cell Cardiol.

J、 分子细胞心脏病学。

47

47

, 716–722 (2009).

, 716–722 (2009).

Article

文章

PubMed

PubMed

Google Scholar

谷歌学者

Hardy, R. S. et al. Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation.

Hardy，R.S.等人。关节炎症小鼠模型中成纤维细胞样滑膜细胞的表征。

Arthritis Res. Ther.

关节炎研究。

15

15

, R24 (2013).

，R24（2013）。

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Susa, M. et al. Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay.

Susa，M。等人。人原代破骨细胞：体外产生和作为药理学和临床测定的应用。

J. Transl. Med.

J. 特兰斯尔麦德

2

2

, 6 (2004).

, 6 (2004).

Article

文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Kono, T. et al. Confusion between carbonate apatite and biological apatite (carbonated hydroxyapatite) in bone and teeth.

Kono，T.等人。骨骼和牙齿中碳酸盐磷灰石和生物磷灰石（碳酸羟基磷灰石）之间的混淆。

Minerals

矿物

12

12

, 170 (2022).

, 170 (2022).

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Magnus, L. et al. Biochemical composition of carious dentin and different layers of sound dentin.

Magnus，L.等人。龋齿牙本质和不同健康牙本质层的生化组成。

J. Oral Sci.

J、 口腔科学。

55

55

, 133–137 (2013).

, 133–137 (2013).

Article

文章

PubMed

PubMed

Google Scholar

谷歌学者

Hong, J. H. et al. Convergence of IRBIT, phosphatidylinositol (4,5) bisphosphate, and WNK/SPAK kinases in regulation of the Na

Hong，J.H.等人。IRBIT，磷脂酰肌醇（4,5）二磷酸和WNK/SPAK激酶在调节Na中的融合

+

+

–HCO

HCO

3

3

−

−

cotransporters family.

。

Proc. Natl Acad. Sci. USA

Proc。国家科学院。滑雪。美国

110

110

, 4105–4110 (2013).

, 4105–4110 (2013).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Rau, R. Adalimumab (a fully human anti-tumour necrosis factor α monoclonal antibody) in the treatment of active rheumatoid arthritis:: the initial results of five trials.

Rau，R。阿达木单抗（一种全人抗肿瘤坏死因子α单克隆抗体）治疗活动性类风湿性关节炎：五项试验的初步结果。

Ann. Rheum. Dis.

安。瑞姆。Dis。

61

61

, 70–73 (2002).

, 70–73 (2002).

Article

文章

Google Scholar

谷歌学者

Wang, L. et al. Delanzomib, a novel proteasome inhibitor, combined with adalimumab drastically ameliorates collagen-induced arthritis in rats by improving and prolonging the anti-TNF-α effect of adalimumab.

。

Front. Pharmacol.

正面。药理学。

12

12

, 782385 (2021).

, 782385 (2021).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Chubb, S. A. Measurement of C-terminal telopeptide of type I collagen (CTX) in serum.

Chubb，S.A.测量血清中I型胶原（CTX）的C末端端肽。

Clin. Biochem.

克林我去过

45

45

, 928–935 (2012).

, 928–935 (2012).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Hwang, S., Shin, D. M. & Hong, J. H. Protective role of IRBIT on sodium bicarbonate cotransporter-n1 for migratory cancer cells.

Hwang，S.，Shin，D.M。和Hong，J.H。IRBIT对迁移癌细胞的碳酸氢钠共转运蛋白-n1的保护作用。

Pharmaceutics

药剂学

12

12

, 816 (2020).

, 816 (2020).

Article

文章

CAS

中科院

PubMed

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Prasad, P. et al. Rheumatoid arthritis: advances in treatment strategies.

Prasad，P。等。类风湿性关节炎：治疗策略的进展。

Mol. Cell Biochem.

分子细胞生物化学。

478

478

, 69–88 (2023).

, 69–88 (2023).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Tan, Y. & Buch, M. H. ‘Difficult to treat’ rheumatoid arthritis: current position and considerations for next steps.

Tan，Y.＆Buch，M.H.“难以治疗”类风湿性关节炎：目前的立场和下一步的考虑因素。

RMD Open

RMD打开

8

8

, e002387 (2022).

，e002387（2022年）。

Article

文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Ozdede, A. & Yazici, H. Cardiovascular and cancer risk with tofacitinib in rheumatoid arthritis.

Ozdede，A。＆Yazici，H。tofacitinib在类风湿性关节炎中的心血管和癌症风险。

N. Engl. J. Med.

恩格尔。J·梅德

386

386

, 1766 (2022).

, 1766 (2022).

Article

文章

PubMed

PubMed

Google Scholar

谷歌学者

Roskoski, R. Jr. Properties of FDA-approved small molecule protein kinase inhibitors: a 2024 update.

Roskoski，R.Jr。FDA批准的小分子蛋白激酶抑制剂的性质：2024年更新。

Pharmacol. Res.

药理学。研究。

200

200

, 107059 (2024).

, 107059 (2024).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Fleischmann, R. et al. Long-term safety and efficacy of upadacitinib versus adalimumab in patients with rheumatoid arthritis: 5-year data from the phase 3, randomised SELECT-COMPARE study.

Fleischmann，R。等人。upadacitinib与阿达木单抗治疗类风湿性关节炎的长期安全性和有效性：来自3期随机选择比较研究的5年数据。

RMD Open

RMD打开

10

10

, e004007 (2024).

，e004007（2024）。

Article

文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Roubille, C. et al. The effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis.

肿瘤坏死因子抑制剂、甲氨蝶呤、非甾体抗炎药和皮质类固醇对类风湿性关节炎、银屑病和银屑病关节炎心血管事件的影响：系统综述和荟萃分析。

Ann. Rheum. Dis.

安。瑞姆。Dis。

74

74

, 480–489 (2015).

, 480–489 (2015).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Agca, R. et al. EULAR recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update.

Agca，R。等人。EULAR对类风湿性关节炎和其他形式的炎性关节疾病患者心血管疾病风险管理的建议：2015/2016更新。

Ann. Rheum. Dis.

安。瑞姆。Dis。

76

76

, 17–28 (2017).

, 17–28 (2017).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Garcia-Gonzalez, C. M. & Baker, J. Treatment of early rheumatoid arthritis: methotrexate and beyond.

Garcia-Gonzalez，C.M。和Baker，J。早期类风湿性关节炎的治疗：甲氨蝶呤及其后。

Curr. Opin. Pharmacol.

货币。奥平。药理学。

64

64

, 102227 (2022).

, 102227 (2022).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Xiong, H. H. et al. Coassembly nanomedicine mediated by intermolecular interactions between methotrexate and baricitinib for improved rheumatoid arthritis treatment.

Xiong，H.H.等人。通过甲氨蝶呤和巴利替尼之间的分子间相互作用介导的共组装纳米医学，用于改善类风湿性关节炎的治疗。

ACS Nano

美国化学会纳米

18

18

, 8337–8349 (2024).

, 8337–8349 (2024).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Bhavsar, S. V. et al. Retention of triple therapy with methotrexate, sulfasalazine, and hydroxychloroquine compared to combination methotrexate and leflunomide in rheumatoid arthritis.

Bhavsar，S.V.等人。与甲氨蝶呤和来氟米特联合治疗类风湿性关节炎相比，保留甲氨蝶呤，柳氮磺吡啶和羟氯喹的三联疗法。

Joint Bone Spine

关节骨脊柱

91

91

, 105732 (2024).

, 105732 (2024).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Zhang, N. et al. Efficacy and safety of the modified Zhiwang decoction combined with methotrexate in early rheumatoid arthritis: study protocol for a randomised controlled trial.

Zhang，N.等。加味知旺汤联合甲氨蝶呤治疗早期类风湿性关节炎的疗效和安全性：一项随机对照试验的研究方案。

BMJ Open

英国医学杂志

14

14

, e076571 (2024).

，e076571（2024年）。

Article

文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Smolen, J. S., Aletaha, D. & McInnes, I. B. Rheumatoid arthritis.

Smolen，J.S.，Aletaha，D。和McInnes，I.B。类风湿性关节炎。

Lancet

柳叶刀

388

388

, 2023–2038 (2016).

, 2023–2038 (2016).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Tang, M. W. et al. Class 3 semaphorins modulate the invasive capacity of rheumatoid arthritis fibroblast-like synoviocytes.

Tang，M.W。等人。3类信号素调节类风湿性关节炎成纤维细胞样滑膜细胞的侵袭能力。

Rheumatology

风湿病学

57

57

, 909–920 (2018).

, 909–920 (2018).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Sun, M. et al. Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts.

Sun，M。等人。抗瓜氨酸蛋白抗体促进滑膜组织来源的成纤维细胞的迁移。

Ann. Rheum. Dis.

安。瑞姆。Dis。

78

78

, 1621–1631 (2019).

, 1621–1631 (2019).

Article

文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Download references

下载参考资料

Acknowledgements

致谢

We developed all the figures. We did not use generative artificial intelligence (AI) or AI-assisted technology. All confocal images were acquired at the Cell to In Vivo Imaging Core Facility Research Center (CII, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, South Korea). This research was funded by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2022R1A2C1003890 to J.H.H.

我们得出了所有的数字。我们没有使用生成人工智能（AI）或AI辅助技术。所有共聚焦图像均在Cell-to-Vivo Imaging Core Facility Research Center（CII，Lee Gil Ya Cancer and Diabetes Institute，Gachon University，仁川，韩国）获得。这项研究由韩国政府（MSIT）资助的韩国国家研究基金会（NRF）资助（2022R1A2C1003890给J.H.H。

and RS-2023-00217709 to D.M.S.)..

和RS-2023-00217709至D.M.S.）。。

Author information

作者信息

Authors and Affiliations

作者和隶属关系

Department of Physiology, College of Medicine, Gachon University, Lee Gil Ya Cancer and Diabetes Institute, Incheon, South Korea

韩国仁川李吉亚癌症与糖尿病研究所，嘉川大学医学院生理学系

Eun Sun Lee, Hyeong Jae Kim & Jeong Hee Hong

Eun Sun Lee、Hyeong Jae Kim 和 Jeong Hee Hong

Department of Health Sciences and Technology, GAIHST, Lee Gil Ya Cancer and Diabetes Institute, Incheon, South Korea

韩国仁川Lee Gil Ya癌症与糖尿病研究所GAIHST健康科学与技术系

Dongun Lee & Jeong Hee Hong

李东云

Department of Dental Hygiene, College of Software Digital Healthcare Convergence, Yonsei University, Wonju, South Korea

韩国元州延世大学软件数字医疗融合学院牙科卫生系

Jung Yun Kang

康正云

Department of Oral Biology, Yonsei University College of Dentistry, Seoul, South Korea

Dong Min Shin

董敏信

Authors

作者

Eun Sun Lee

孙 李

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

PubMed

Google Scholar

谷歌学者

Hyeong Jae Kim

Hyeong Jae Kim

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

PubMed

Google Scholar

谷歌学者

Dongun Lee

李东云

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

PubMed

Google Scholar

谷歌学者

Jung Yun Kang

康正云

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

PubMed

Google Scholar

谷歌学者

Dong Min Shin

董敏信

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

PubMed

Google Scholar

谷歌学者

Jeong Hee Hong

郑熙红

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

PubMed

Google Scholar

谷歌学者

Contributions

捐款

E.S.L., J.Y.K., D.M.S. and J.H.H. conceived and designed the study and acquired, analyzed and interpreted the data. E.S.L., H.J.K. and D.L. drafted the manuscript, performed the animal experiments and acquired the data. D.L., J.H.H., D.M.S. and J.Y.K. critically revised the manuscript. E.S.L. and D.L.

E、 S.L.，J.Y.K.，D.M.S.和J.H.H.构思并设计了这项研究，并获得，分析和解释了数据。E、 S.L.，H.J.K.和D.L.起草了手稿，进行了动物实验并获得了数据。D、 L.，J.H.H.，D.M.S.和J.Y.K.批判性地修改了手稿。E、 S.L.和D.L。

generated the schematic illustrations. J.H.H. and D.M.S. provided final approval of the version to be published, agreed to be accountable for all aspects of the work and ensured that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved..

生成了示意图。J、 H.H.和D.M.S.最终批准了要发布的版本，同意对工作的各个方面负责，并确保与工作任何部分的准确性或完整性有关的问题得到适当调查和解决。。

Corresponding authors

通讯作者

Correspondence to

通信对象

Dong Min Shin

董敏信

or

或

Jeong Hee Hong

琼 赫 洪

.

.

Ethics declarations

道德宣言

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Ethics

伦理学

All procedures related to synovial fluid collection were performed in accordance with the Declaration of Helsinki. The study was approved by the institutional review board of Gil Medical Center (GAIRB-2019-018).

。该研究得到Gil医学中心机构审查委员会（GAIRB-2019-018）的批准。

Additional information

其他信息

Publisher’s note

出版商注释

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

Supplementary information

补充信息

Supplementary Information

补充信息

Rights and permissions

权限和权限

Open Access

开放存取

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

本文根据知识共享署名4.0国际许可证进行许可，该许可证允许以任何媒体或格式使用，共享，改编，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出是否进行了更改。

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

本文中的图像或其他第三方材料包含在文章的知识共享许可中，除非在材料的信用额度中另有说明。如果材料未包含在文章的知识共享许可中，并且您的预期用途不受法律法规的许可或超出许可用途，则您需要直接获得版权所有者的许可。

To view a copy of this licence, visit .

要查看此许可证的副本，请访问。

http://creativecommons.org/licenses/by/4.0/

http://creativecommons.org/licenses/by/4.0/

.

.

Reprints and permissions

重印和许可

About this article

关于本文

Cite this article

引用本文

Lee, E.S., Kim, H.J., Lee, D.

李，E.S.，金，H.J.，李，D。

et al.

等人。

Rheumatoid arthritis severity is mediated by crosstalk between synoviocytes and mature osteoclasts through a calcium and cytokine feedback loop.

类风湿性关节炎的严重程度是由滑膜细胞和成熟破骨细胞之间通过钙和细胞因子反馈环的串扰介导的。

Exp Mol Med

Exp Mol Med公司

(2025). https://doi.org/10.1038/s12276-025-01401-8

(2025).https://doi.org/10.1038/s12276-025-01401-8

Download citation

下载引文

Received

已接收

:

:

04 March 2024

2024年3月4日

Revised

修订

:

:

22 October 2024

2024年10月22日

Accepted

已接受

:

:

05 November 2024

2024年11月5日

Published

已发布

:

:

03 February 2025

2025年2月3日

DOI

DOI

:

:

https://doi.org/10.1038/s12276-025-01401-8

https://doi.org/10.1038/s12276-025-01401-8

Share this article

分享这篇文章

Anyone you share the following link with will be able to read this content:

与您共享以下链接的任何人都可以阅读此内容：

Get shareable link

获取可共享链接

Sorry, a shareable link is not currently available for this article.

很抱歉，本文目前没有可共享的链接。

Copy to clipboard

复制到剪贴板

Provided by the Springer Nature SharedIt content-sharing initiative

由Springer Nature SharedIt内容共享计划提供