丙酮酸脱氢酶激酶2缺乏导致的氧化磷酸化增加改善了手术诱导的骨关节炎小鼠的软骨退化-动脉网

Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis

Nature 等信源发布 2025-02-03 12:31

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Abstract

摘要

Chondrocytes can shift their metabolism to oxidative phosphorylation (OxPhos) in the early stages of osteoarthritis (OA), but as the disease progresses, this metabolic adaptation becomes limited and eventually fails, leading to mitochondrial dysfunction and oxidative stress. Here we investigated whether enhancing OxPhos through the inhibition of pyruvate dehydrogenase kinase (PDK) 2 affects the metabolic flexibility of chondrocytes and cartilage degeneration in a surgical model of OA.

软骨细胞可以在骨关节炎（OA）的早期阶段将其代谢转变为氧化磷酸化（OxPhos），但随着疾病的进展，这种代谢适应变得有限并最终失败，导致线粒体功能障碍和氧化应激。在这里，我们研究了通过抑制丙酮酸脱氢酶激酶（PDK）2来增强OxPhos是否会影响OA手术模型中软骨细胞的代谢灵活性和软骨变性。

Among the PDK isoforms, PDK2 expression was increased by IL-1β in vitro and in the articular cartilage of the DMM model in vivo, accompanied by an increase in phosphorylated PDH. Mice lacking PDK2 showed significant resistance to cartilage damage and reduced pain behaviors in the DMM model. PDK2 deficiency partially restored OxPhos in IL-1β-treated chondrocytes, leading to increases in APT and the NAD.

在PDK同种型中，IL-1β在体外和体内DMM模型的关节软骨中增加了PDK2的表达，并伴随着磷酸化PDH的增加。缺乏PDK2的小鼠在DMM模型中显示出对软骨损伤的显着抵抗力和减轻的疼痛行为。PDK2缺陷部分恢复了IL-1β处理的软骨细胞中的OxPhos，导致APT和NAD增加。

/NADH ratio. These metabolic changes were accompanied by a decrease in reactive oxygen species and senescence in chondrocytes, as well as an increase in the expression of antioxidant proteins such as NRF2 and HO-1 after IL-1β treatment. At the signaling level, PDK2 deficiency reduced p38 signaling and maintained AMPK activation without affecting the JNK, mTOR, AKT and NF-κB pathways.

/NADH比率。。在信号水平上，PDK2缺乏减少了p38信号传导并维持了AMPK活化，而不影响JNK，mTOR，AKT和NF-κB途径。

p38 MAPK signaling was critically involved in reactive oxygen species production under glycolysis-dominant conditions in chondrocytes. Our study provides a proof of concept for PDK2-mediated metabolic reprogramming toward OxPhos as a new therapeutic strategy for OA..

在软骨细胞糖酵解显性条件下，p38 MAPK信号传导与活性氧的产生密切相关。我们的研究为PDK2介导的OxPhos代谢重编程作为OA的新治疗策略提供了概念证明。。

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Kindlin-2保持关节软骨的完整性，以防止骨关节炎

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Introduction

导言

Osteoarthritis (OA) is the most common form of degenerative joint disease and is characterized by chondrocyte apoptosis and degradation of the cartilage extracellular matrix (ECM), which ultimately leads to joint failure

骨关节炎（OA）是最常见的退行性关节疾病，其特征是软骨细胞凋亡和软骨细胞外基质（ECM）降解，最终导致关节衰竭

. Chondrocytes, the only cell type found in cartilage tissues, produce cartilage-specific ECM proteins such as type 2 collagen (Col2) and aggrecan, and they become trapped within the ECM proteins they produce, resembling a state of hibernation

软骨细胞是软骨组织中唯一发现的细胞类型，它产生软骨特异性ECM蛋白，如2型胶原蛋白（Col2）和聚集蛋白聚糖，它们被困在它们产生的ECM蛋白中，类似于冬眠状态

. As cartilage is an avascular tissue, chondrocytes exist in hypoxic conditions despite the diffusion of oxygen from synovial fluid or subchondral bone

由于软骨是一种无血管组织，尽管滑液或软骨下骨中的氧气扩散，软骨细胞仍在低氧条件下存在

. In such a hypoxic milieu, chondrocytes rely predominantly on glycolysis rather than oxidative phosphorylation (OxPhos), which accounts for less than 10% of total cellular ATP production

在这种低氧环境中，软骨细胞主要依赖糖酵解而不是氧化磷酸化（OxPhos），氧化磷酸化占细胞ATP总产量的不到10%

. However, chondrocytes also exhibit a notable level of metabolic flexibility in the catabolic state of early OA, where they can alter their metabolic machinery toward OxPhos

然而，软骨细胞在早期OA的分解代谢状态下也表现出显着的代谢灵活性，可以改变其向OxPhos的代谢机制

. This allows them to adapt to catabolic conditions, thereby enhancing their survival and function. As OA progresses and mitochondrial dysfunction occurs

这使它们能够适应分解代谢条件，从而增强其生存和功能。随着OA的进展和线粒体功能障碍的发生

, however, the metabolic adaptation of OA chondrocytes reaches its limit, leading to a metabolic shift toward the glycolytic pathway

然而，OA软骨细胞的代谢适应达到了极限，导致代谢向糖酵解途径转变

. Chondrocytes in early-stage OA of Kellgren and Lawrence grade 1, compared with those in advanced-stage OA of Kellgren and Lawrence grade 4, exhibit a metabolic phenotype characterized by increased OxPhos and decreased glycolysis

与Kellgren和Lawrence 4级晚期OA相比，Kellgren和Lawrence 1级早期OA的软骨细胞表现出以OxPhos增加和糖酵解减少为特征的代谢表型

. This metabolic shift not only makes it increasingly difficult for chondrocytes to meet their energy demands but also amplifies oxidative stress in a lactate dehydrogenase-mediated manner

这种代谢转变不仅使软骨细胞越来越难以满足其能量需求，而且还以乳酸脱氢酶介导的方式放大了氧化应激

. The increase in oxidative stress, in turn, worsens mitochondrial dysfunction, including increased mitochondrial DNA damage and membrane permeability, eventually contributing to chondrocyte apoptosis and senescence

氧化应激的增加反过来会加重线粒体功能障碍，包括线粒体DNA损伤和膜通透性增加，最终导致软骨细胞凋亡和衰老

. In this way, the glycolysis-prone metabolism of chondrocytes interacts with and amplifies both mitochondrial dysfunction and oxidative stress during OA progression

通过这种方式，软骨细胞易于糖酵解的代谢在OA进展过程中与线粒体功能障碍和氧化应激相互作用并放大

. Despite the understanding that metabolic imbalances in chondrocytes play a crucial role in the pathogenesis of OA, we still lack conclusive evidence on whether redirecting chondrocyte metabolism toward OxPhos could indeed impact its progression.

尽管人们认识到软骨细胞的代谢失衡在OA的发病机制中起着至关重要的作用，但我们仍然缺乏确凿的证据来证明将软骨细胞代谢重定向到OxPhos是否确实会影响其进展。

Glucose that enters chondrocytes mainly through glucose transporter (GLUT) 1 and GLUT3 is metabolized into pyruvate, the end product of glucose catabolism

主要通过葡萄糖转运蛋白（GLUT）1和GLUT3进入软骨细胞的葡萄糖被代谢成丙酮酸，丙酮酸是葡萄糖分解代谢的最终产物

. This pyruvate moves to the mitochondria, where it is converted to acetyl-coenzyme A (acetyl-CoA) by pyruvate dehydrogenase (PDH), the enzyme that links the cytoplasmic glycolysis pathway to the mitochondrial tricarboxylic acid (TCA) cycle. As a canonical input to the TCA cycle, an increase in acetyl-CoA levels leads to an increase in the rate of the TCA cycle and, consequently, OxPhos.

丙酮酸转移到线粒体，在那里它被丙酮酸脱氢酶（PDH）转化为乙酰辅酶A（乙酰辅酶A），PDH是一种将细胞质糖酵解途径与线粒体三羧酸（TCA）循环联系起来的酶。作为TCA循环的典型输入，乙酰辅酶a水平的增加导致TCA循环速率的增加，从而导致OxPhos的增加。

. Therefore, the activity of PDH, which converts pyruvate into acetyl-CoA, is the overall rate-limiting and gatekeeping factor in pyruvate-driven OxPhos

因此，将丙酮酸转化为乙酰辅酶A的PDH活性是丙酮酸驱动的OxPhos的总体限速和守门因子

. The activity of PDH is regulated by PDH kinase (PDK), a serine/threonine kinase that phosphorylates the α subunit of PDH, thereby inactivating it

PDH的活性受PDH激酶（PDK）调节，PDH激酶是一种丝氨酸/苏氨酸激酶，可磷酸化PDH的α亚基，从而使其失活

. The activity of PDK, in turn, is controlled by pyruvate, NAD

PDK的活性又受丙酮酸NAD的控制

, ATP, acetyl-CoA and nuclear transcription factors such as FoxO, PPAR and PGC1α

，ATP，乙酰辅酶A和核转录因子，例如FoxO，PPAR和PGC1α

. From the perspective of the metabolic shift toward glycolysis during OA progression, a potential therapeutic strategy might be derived from metabolic reprogramming to enhance OxPhos through the inhibition of PDK

从OA进展过程中代谢向糖酵解转变的角度来看，一种潜在的治疗策略可能来自代谢重编程，通过抑制PDK来增强OxPhos

. PDK has four isoforms, PDK1 through PDK4. Among them, PDK2 is expressed ubiquitously, while the remaining isoforms have a tissue-specific distribution: PDK1 is found primarily in heart tissue, pancreatic islets and skeletal muscle; PDK3 has a relatively limited tissue distribution in the testis, kidney and brain; and PDK4 has a relatively limited distribution in the heart, skeletal muscle, liver, kidney and pancreatic islets.

PDK有四种同工型，PDK1到PDK4。其中，PDK2无处不在，而其余同工型具有组织特异性分布：PDK1主要存在于心脏组织，胰岛和骨骼肌中；PDK3在睾丸，肾脏和大脑中的组织分布相对有限；。

. However, the expression of PDKs in chondrocytes and their functional role in OA have yet to be investigated.

然而，PDK在软骨细胞中的表达及其在OA中的功能作用尚待研究。

The objective of this study was to determine whether PDK inhibition affects the metabolic flexibility of chondrocytes and contributes to cartilage degeneration in a surgical model of OA. First, we verified the expression of PDK isoforms in both in vitro catabolic conditions and in vivo OA cartilage, which revealed an increase in PDK2 under catabolic conditions.

这项研究的目的是确定PDK抑制是否影响软骨细胞的代谢灵活性，并导致OA手术模型中的软骨变性。首先，我们验证了PDK同种型在体外分解代谢条件和体内OA软骨中的表达，这表明在分解代谢条件下PDK2增加。

The role of PDK2 in OA progression was investigated via a surgical OA model in .

通过手术OA模型研究了PDK2在OA进展中的作用。

Pdk2

-deficient mice, and we subsequently focused on its metabolic phenotype, expression of anabolic and catabolic factors, reactive oxygen species (ROS) production and cellular senescence. From a mechanistic perspective, we identified the signaling pathways affected by

-缺陷小鼠，我们随后关注其代谢表型，合成代谢和分解代谢因子的表达，活性氧（ROS）的产生和细胞衰老。从机理的角度来看，我们确定了受

Pdk2

deficiency and elucidated their role in the production of ROS and cellular senescence.

缺乏并阐明了它们在ROS产生和细胞衰老中的作用。

Materials and methods

材料和方法

Ethics and isolation of primary chondrocytes

伦理与原代软骨细胞的分离

This study was conducted in accordance with the guidelines of the National Research Council (US) Committee for the Care and Use of Laboratory Animals

这项研究是根据美国国家研究委员会实验动物护理和使用委员会的指导方针进行的

and was approved by the institutional review board of the Kyungpook National University School of Medicine (Daegu, Korea) under approval number KNU 2022-0203. Mice that were genetically deficient in the PDK2 gene (PDK2 KO) in the C57BL/6J background were kindly provided by Dr. In-Kyu Lee (Kyungpook National University, Daegu, Korea).

并得到了庆浦国立大学医学院（韩国大邱）机构审查委员会的批准，批准号为KNU 2022-0203。C57BL/6J背景下PDK2基因（PDK2 KO）遗传缺陷的小鼠由in Kyu Lee博士（韩国大邱庆北国立大学）提供。

Primary chondrocytes were isolated from the articular cartilage of the femurs and tibias of 5-day-old C57BL/6J mice. The isolated cartilage pieces were minced into small fragments, incubated for 15 min in 0.25% trypsin–EDTA at 37 °C with gentle shaking and digested in a solution containing 0.2% collagenase type II, which was prepared in Dulbecco’s modified Eagle medium (DMEM), at 37 °C with gentle agitation for 4 h.

从5日龄C57BL/6J小鼠的股骨和胫骨的关节软骨中分离出原代软骨细胞。将分离的软骨碎片切成小块，在0.25%胰蛋白酶-EDTA中于37℃孵育15分钟 °C轻轻摇动，并在含有0.2%II型胶原酶的溶液中消化，该溶液在Dulbecco改良的Eagle培养基（DMEM）中于37°C温和搅拌4小时制备。

After digestion, the cell suspension was filtered through a 40-µm cell strainer and washed twice with phosphate-buffered saline (PBS). The isolated chondrocytes were resuspended in complete 3:2 F12:DMEM-based culture medium supplemented with 0.25% .

消化后，将细胞悬液通过40µm细胞过滤器过滤，并用磷酸盐缓冲盐水（PBS）洗涤两次。将分离的软骨细胞重悬于补充有0.25%的完全3:2 F12:DMEM基培养基中。

-glutamine and 0.25% penicillin–streptomycin, seeded in six-well plates at a concentration of 1 × 10

-谷氨酰胺和0.25%青霉素-链霉素，以1x的浓度接种在六孔板中 10

cells per well and incubated at 37 °C in a humidified atmosphere containing 5% CO

每孔细胞，并在含有5%CO的潮湿气氛中于37°C孵育

. To reduce dedifferentiation risk, only chondrocytes at passage 0 were used in the experiments.

为了降低去分化风险，实验中仅使用第0代的软骨细胞。

RNA and protein expression analysis

RNA和蛋白质表达分析

To induce catabolic conditions in primary chondrocytes, the cells were treated with 10 ng/ml IL-1β and incubated for 6 h for RNA isolation and 24 h for protein isolation. For signaling analysis, the cells were starved overnight and then stimulated with 20 ng/ml IL-1β for the indicated times. Total RNA was extracted using TRIzol (Invitrogen), after which first-strand cDNA was generated with Superscript III reverse transcriptase (Invitrogen).

为了诱导原代软骨细胞的分解代谢条件，将细胞用10ng/ml IL-1β处理，并孵育6小时用于RNA分离，孵育24小时用于蛋白质分离。为了进行信号分析，将细胞饥饿过夜，然后用20ng/ml IL-1β刺激指定的时间。使用TRIzol（Invitrogen）提取总RNA，然后用Superscript III逆转录酶（Invitrogen）产生第一链cDNA。

A ViiA 7 Real-Time PCR System (Applied Biosystems) and SYBR Green Master Mix (Applied Biosystems) were used for qRT‒PCR. The primers used are listed in Supplementary Table .

使用ViiA 7实时PCR系统（Applied Biosystems）和SYBR Green Master Mix（Applied Biosystems）进行qRT-PCR。使用的引物列于补充表中。

. Target gene expression levels were calculated via the 2 − ΔΔCT method and normalized to the geometric mean of GAPDH. All qRT‒PCR analyses were carried out in triplicate and repeated three to five times, and the average results for each sample are presented.

通过2 - ΔΔCT方法计算靶基因表达水平，并归一化为GAPDH的几何平均值。所有qRT-PCR分析一式三份进行，重复三至五次，并给出每个样品的平均结果。

Total proteins were extracted via 300 μl of RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics). Total cell lysates containing 10–20 μg protein were subjected to 10% SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore).

通过补充有蛋白酶和磷酸酶抑制剂（Roche Diagnostics）的300μlRIPA缓冲液提取总蛋白。将含有10–20μg蛋白质的总细胞裂解液进行10%SDS-聚丙烯酰胺凝胶电泳，并转移到聚偏二氟乙烯膜（Immobilon-P；Millipore）上。

The membranes were blocked with 5% skim milk in PBS with 0.25% Tween-20 (PBST) and incubated with primary antibodies against PDK1 (Abcam, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDH-E1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, #sc-377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), p-AMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272) and β-actin (Sigma-Aldrich, #A1978) overnight at 4 °C.

用含0.25%吐温-20（PBST）的PBS中的5%脱脂奶封闭膜，并与抗PDK1（Abcam，#ab202468）、PDK2（#ab68164）、PDK3（#ab154549）、PDK4（#ab214938）、磷酸PDH-E1α（Ser232，Millipore，#AP1063）、PDH-E1（Santa Cruz Biotechnology，#sc-377092）、Col2（#ab34712）的一抗孵育），Sirt1（Cell Signaling Technology，#9475），MMP13（#ab51072），p-p38（#4631），p38（#9212），p-JNK（#9251），JNK（#9252），p-AMPKα（#2531），AMPKα（#2532），p-FoxO3（#9466），FoxO3a（#2497），p-mTOR（#2971），mTOR（#2972），p-p65（#3033），p65（#8242），p-Akt（#9271），Akt（#9272）和β-肌动蛋白（Sigma-Aldrich，#A1978）在4℃过夜。

After being washed with PBST, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies at room temperature for 2 h, and then the blots were developed using enhanced chemiluminescence western blotting detection reagent (Thermo Fisher Scientific) and examined via the MicroChemi system (DNR Bio-Imaging Systems).

用PBST洗涤后，将膜与辣根过氧化物酶偶联的二抗在室温下孵育2 h、然后使用增强的化学发光蛋白质印迹检测试剂（Thermo Fisher Scientific）开发印迹，并通过MicroChemi系统（DNR Bio Imaging Systems）进行检查。

All analyses were performed in biological triplicate, and the western blot band intensities were quantified via ImageJ software (version 1.8.0, National Institutes of Health). The band intensities of the target proteins were normalized to the β-actin band intensity of the respective lanes. The full western blot data are included in Supplementary Fig.

所有分析均一式三份进行，并通过ImageJ软件（版本1.8.0，National Institutes of Health）定量蛋白质印迹条带强度。将靶蛋白的条带强度标准化为各个泳道的β-肌动蛋白条带强度。完整的蛋白质印迹数据包含在Supplementary Fig.中。

Surgical OA induction

手术OA诱导

OA was surgically induced in 12-week-old male C57BL/6J mice, including 7 PDK2 KO mice and 7 of their littermates, through surgical destabilization of the medial meniscus (DMM) under general anesthesia. The sample size of each group was calculated on the basis of the assumptions of a type I error (

在全身麻醉下，通过手术破坏内侧半月板（DMM），在12周龄雄性C57BL/6J小鼠（包括7只PDK2 KO小鼠及其7只同窝小鼠）中手术诱导OA。每组的样本量是根据I型错误的假设计算出来的(

啊

) of 0.1, a standard deviation (

)0.1，标准差(

) of 10, an effect size (

)10，效果大小(

) of 15 and a power of 0.8. The ligaments of the medial meniscus were dissected from the right knee to induce OA, and a sham operation was conducted on the left knee as a control. After surgery, the mice were housed under controlled conditions at 23 ± 1 °C, 50% humidity and a 12-h light/dark cycle. They were kept under specific pathogen-free conditions in the animal facilities of Kyungpook National University Chilgok Hospital..

)为15，幂为0.8。从右膝解剖内侧半月板的韧带以诱导OA，并在左膝上进行假手术作为对照。手术后，将小鼠在23±1℃，50%湿度和12小时光照/黑暗循环的受控条件下饲养。它们被保存在庆北国立大学奇尔库克医院的动物设施中，处于无特定病原体的条件下。。

Eight weeks after surgery, the mice were euthanized via cervical dislocation, the knee joints were collected for histological examination, and all specimens were used for analysis. This study was conducted in accordance with ARRIVE guidelines 2.0, and the checklist is provided in the Supplementary Materials..

手术后8周，通过颈椎脱位对小鼠实施安乐死，收集膝关节进行组织学检查，并将所有标本用于分析。这项研究是根据ARRIVE指南2.0进行的，补充材料中提供了清单。。

Pain behavior analysis

疼痛行为分析

Beginning 2 weeks after surgery, pain behavior was assessed weekly via the spontaneous weight-bearing asymmetry test (incapacitance test), the hot plate test and threshold punctate mechanical stimulation (the von Frey test). To assess spontaneous weight bearing on the hind limbs, an incapacitance meter (Sang Chung Commercial) was used to measure the downward force applied by each hind limb.

从手术后2周开始，通过自发负重不对称测试（失能测试），热板测试和阈值点状机械刺激（von Frey测试）每周评估疼痛行为。为了评估后肢的自发负重，使用失能仪（Sang Chung Commercial）测量每个后肢施加的向下力。

The mice were briefly placed in a restraint, with their hind limbs resting on two weight-averaging platform pads. Measurements of the paw pressure of each hind limb were taken for 10 s approximately ten times, and the results were averaged. The data are expressed as the percentage of weight distributed on the ipsilateral hind limb.

将小鼠短暂放置在约束装置中，后肢休息在两个平均重量的平台垫上。测量每个后肢的爪压力约10次，每次10秒，并将结果取平均值。数据表示为分布在同侧后肢上的体重百分比。

Next, thermal pain sensitivity was assessed with the hot plate test.

接下来，用热板测试评估热痛敏感性。

. The mice were placed on a heated plate (plantar test infrared emitter, Ugo Basile), and the time until they displayed pain behaviors such as licking or shaking their paws was recorded. Finally, tactile allodynia was assessed via the von Frey test. Calibrated monofilaments (von Frey hairs; Stoelting) were applied to the plantar surface of both the ipsilateral and contralateral hind paws, and the mice were placed in an elevated maze in an acrylic cage.

将小鼠置于加热板（足底测试红外发射器，Ugo Basile）上，记录直到它们表现出舔或抖动爪子等疼痛行为的时间。最后，通过von Frey测试评估触觉异常性疼痛。将校准的单丝（von Frey毛发；Stoelting）应用于同侧和对侧后爪的足底表面，并将小鼠置于丙烯酸笼中的高架迷宫中。

Paw withdrawal was considered a positive response. The 50% withdrawal threshold was determined upon six repeated applications of varying force with a von Frey filament via the up–down method.

缩爪被认为是积极的反应。50%的撤离阈值是通过上下方法用冯·弗雷细丝重复施加六次不同的力来确定的。

Safranin-O and immunofluorescence staining

番红O和免疫荧光染色

Mouse knee joints were fixed in 4% paraformaldehyde for 24 h, decalcified in 10% EDTA for 3 weeks and then embedded in paraffin. The embedded blocks were sectioned at a thickness of 6 μm. After deparaffinization and rehydration, the sections were stained with 0.1% Safranin-O solution for 5 min and counterstained with Fast Green solution for 1 min.

将小鼠膝关节在4%多聚甲醛中固定24小时，在10%EDTA中脱钙3周，然后包埋在石蜡中。将嵌入的块切成6μm的厚度。脱蜡和再水化后，将切片用0.1%番红-O溶液染色5分钟，并用Fast Green溶液复染1分钟。

Cartilage destruction was scored in all four quadrants of the joint (grades 0–24) and on the medial tibial plateau (grades 0–6) by two observers under blinded conditions via the Osteoarthritis Research Society International (OARSI) scoring system.

两名观察者在盲法条件下通过国际骨关节炎研究学会（OARSI）评分系统对关节的所有四个象限（0-24级）和胫骨内侧平台（0-6级）的软骨破坏进行评分。

. For immunofluorescence staining, rehydrated sections were subjected to antigen retrieval in sodium citrate buffer (10 mM sodium citrate and 0.05% Tween-20, pH 6.0). The sections were then blocked with 2% bovine serum albumin in PBS for 1 h, followed by overnight incubation at 4 °C with primary antibodies against PDK1 (#ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDH-E1α (Ser232, #AP1063), PDH-E1 (#sc-377092), MMP13 (#ab51072), 8-oxo-dG (#sc-66036) or normal rabbit IgG in 1% bovine serum albumin.

为了进行免疫荧光染色，将再水化的切片在柠檬酸钠缓冲液（10mM柠檬酸钠和0.05%吐温-20，pH 6.0）中进行抗原修复。然后将切片用PBS中的2%牛血清白蛋白封闭1小时，然后在4 用1%牛血清白蛋白中的PDK1（#ab202468），PDK2（#ab68164），PDK3（#ab154549），PDK4（#ab214938），磷酸PDH-E1α（Ser232，#AP1063），PDH-E1（#sc-377092），MMP13（#ab51072），8-oxo-dG（#sc-66036）或正常兔IgG的一抗。

For immunofluorescence, the sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 2 h and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the sections were mounted with anti-fade mounting solution (Vector Labs) and imaged and quantified via a KI-3000F fluorescence microscope..

对于免疫荧光，将切片与Alexa Fluor 488或594偶联的二抗（Jackson ImmunoResearch Laboratories）孵育2小时，然后用4'，6-二脒基-2-苯基吲哚（DAPI）复染。最后，将切片用抗褪色固定溶液（Vector Labs）固定，并通过KI-3000F荧光显微镜成像和定量。。

Seahorse real-time cell metabolic analysis

海马实时细胞代谢分析

Chondrocyte metabolism was analyzed using an XF96 Extracellular Flux Analyzer (Agilent Technologies). Primary chondrocytes from PDK2 KO mice and their littermates were plated in Seahorse XF96 plates at a density of 50,000 cells per well. Confluent chondrocytes were incubated with or without IL-1β (10 ng/ml) for 24 h.

使用XF96细胞外通量分析仪（Agilent Technologies）分析软骨细胞代谢。将来自PDK2 KO小鼠及其同窝仔的原代软骨细胞以每孔50000个细胞的密度接种在Seahorse XF96平板中。将汇合的软骨细胞与或不与IL-1β（10ng/ml）一起温育24小时。

For the mitochondrial stress test, the cells were equilibrated for 1 h in serum-free Seahorse XF Base Medium. Basal cellular respiration was initially measured, and mitochondrial respiration inhibitors, including 1 μM oligomycin, 1 μM FCCP and a 1:1 mixture of 2 μM antimycin A with 1 μM rotenone, were sequentially injected into the assay wells.

对于线粒体应激测试，将细胞在无血清海马XF基础培养基中平衡1小时。最初测量了基础细胞呼吸，线粒体呼吸抑制剂，包括1m寡霉素，将1μM FCCP和2μM抗霉素a与1μM鱼藤酮的1:1混合物依次注入测定孔中。

Mitochondrial ATP production was expressed as the oxygen consumption rate (OCR; pmol of O.

线粒体ATP产生表示为耗氧率（OCR；O的pmol）。

/min). For the glycolysis stress test, the cells were starved for 1 h in glucose-free Seahorse XF DMEM, followed by sequential treatment with 20 mM glucose, 1 μM oligomycin and 100 mM 2-deoxy-D-glucose. Real-time measurements of proton accumulation in the media were taken and quantified as the extracellular acidification rate (ECAR; mpH/min).

/分钟）。对于糖酵解应激测试，将细胞在无葡萄糖的海马XF DMEM中饥饿1小时，然后依次用20 mM葡萄糖处理，1μM寡霉素和100 mM 2-脱氧-D-葡萄糖。实时测量培养基中质子的积累，并将其定量为细胞外酸化率（ECAR；mpH/min）。

. After completion of either the mitochondrial or glycolysis stress test, the cell nuclei were counted after in situ DAPI staining. Analysis data were then normalized on the basis of the cell count per well, using a normalization unit of 5,000 cells.

完成线粒体或糖酵解应激测试后，在原位DAPI染色后计数细胞核。。

ATP and NAD

ATP和NAD

/NADH measurements

/NADH测量

Chondrocytes from PDK2 KO mice and their littermates were seeded at 5,000 cells per well in 96-well plates for ATP measurement and at 20,000 cells per well in six-well plates for NAD

将PDK2 KO小鼠及其同窝小鼠的软骨细胞以每孔5000个细胞的速度接种在96孔板中进行ATP测量，并以每孔20000个细胞的速度接种在六孔板中进行NAD测量

/NADH measurement and treated with or without 10 ng/ml IL-1β for 24 h. The ATP concentration was measured via a commercial ATP detection kit (Abcam, #ab83355) following the manufacturer’s protocol. In brief, chondrocytes were homogenized in 15 μl of ATP assay lysis buffer, and aliquots of the cell lysates were incubated with ATP Reaction Mix for 30 min in the dark.

/NADH测量并用或不用10ng/ml IL-1β处理24小时。按照制造商的方案，通过商业ATP检测试剂盒（Abcam，#ab83355）测量ATP浓度。简而言之，将软骨细胞在15μlATP测定裂解缓冲液中匀浆，并将等分试样的细胞裂解物与ATP反应混合物在黑暗中孵育30分钟。

The absorbance of each mixture was quantified via a spectrophotometer at a wavelength of 570 nm. ATP levels were calculated from a standard curve, which was plotted via the serial dilution of authentic ATP..

通过分光光度计在570nm波长下定量每种混合物的吸光度。ATP水平是从标准曲线计算出来的，该曲线是通过真实ATP的连续稀释绘制的。。

The NAD

全国广告部

/NADH ratio was determined using a commercial NAD

/NADH比率是使用商业NAD确定的

/NADH assay kit (Abcam, ab65348) following the manufacturer’s instructions. In brief, chondrocytes were collected by scraping, washed three times with precooled PBS and lysed with extraction buffer solution. After centrifugation at 12,000 rpm for 5 min at 4 °C, the supernatant was collected. The protein concentration was determined via a bicinchoninic acid assay kit (Beyotime Biotechnology).

/NADH分析试剂盒（Abcam，ab65348）遵循制造商的说明。简而言之，通过刮擦收集软骨细胞，用预冷的PBS洗涤三次，并用提取缓冲液裂解。在4°C下以12000 rpm离心5分钟后，收集上清液。通过二辛可宁酸测定试剂盒（Beyotime Biotechnology）测定蛋白质浓度。

The samples were then heated at 60 °C for 30 min to completely decompose the NAD.

然后将样品在60℃加热30分钟以完全分解NAD。

in the sample. Then, 50 μl of each sample was mixed with 100 μl of Reaction Mix and incubated at room temperature for 5 min. Subsequently, 10 μl of NADH Developer was added to each well and incubated at room temperature for 2 h. The absorbance of each mixture was quantified via a spectrophotometer at a wavelength of 450 nm..

在样本中。然后，将50μl每个样品与100μl反应混合物混合，并在室温下孵育5分钟。随后，向每个孔中加入10μlNADH显影剂，并在室温下孵育2小时。通过分光光度计在450 nm波长下定量每种混合物的吸光度。。

Detection of intracellular ROS and oxidative DNA damage in the assessment of oxidative stress

氧化应激评估中细胞内活性氧和氧化性DNA损伤的检测

Intracellular ROS and mitochondrial ROS were assessed with dihydroethidium (DHE) and MitoSOX Red (Thermo Fisher Scientific, #M36008) fluorescent dyes, respectively. Primary chondrocytes were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100 and then rinsed three times with PBS.

分别用二氢乙锭（DHE）和MitoSOX Red（Thermo Fisher Scientific，#M36008）荧光染料评估细胞内ROS和线粒体ROS。将原代软骨细胞用4%多聚甲醛固定10分钟，用0.25%Triton X-100透化，然后用PBS冲洗三次。

To assess the level of intracellular ROS, the chondrocytes were incubated with 5 μM DHE for 30 min at 37 °C, washed with PBS and then counterstained with DAPI. Images were captured using a KI-3000F fluorescence microscope (Korea Lab Tech)..

为了评估细胞内ROS的水平，将软骨细胞与5μMDHE在37℃温育30分钟，用PBS洗涤，然后用DAPI复染。。。

DNA damage caused by oxidative stress was evaluated by immunofluorescence staining with an 8-oxo-dG antibody. Fixed and permeabilized cells were incubated with the primary 8-oxo-dG antibody overnight at 4 °C. The cells were subsequently incubated with a secondary antibody for 2 h, counterstained with DAPI and imaged using a fluorescence microscope.

通过用8-氧代-dG抗体进行免疫荧光染色来评估由氧化应激引起的DNA损伤。。随后将细胞与二抗孵育2小时，用DAPI复染并使用荧光显微镜成像。

Fluorescence-positive cells were quantified via ImageJ software..

通过ImageJ软件对荧光阳性细胞进行定量。。

Senescence β-galactosidase staining

衰老β-半乳糖苷酶染色

β-Galactosidase activity was detected using a β-galactosidase staining kit (Cell Signaling Technology, #9860). The cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then incubated for 2 h in staining solution containing 1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide and 2 mM MgCl.

使用β-半乳糖苷酶染色试剂盒（Cell Signaling Technology，#9860）检测β-半乳糖苷酶活性。将细胞在室温下用4%多聚甲醛固定10分钟，然后在含有1mg/ml X-gal的染色溶液中孵育2小时，5mM亚铁氰化钾，5 mM铁氰化钾和2 mM MgCl。

in pH 7.4 PBS. Then, the cells were washed with PBS and visualized under a light microscope, and the percentage of blue-colored cells was calculated by counting at least five random fields per sample.

在pH 7.4 PBS中。然后，用PBS洗涤细胞并在光学显微镜下观察，并通过计数每个样品至少五个随机场来计算蓝色细胞的百分比。

Statistical analysis

统计分析

All data are presented herein as the mean ± standard error of the mean (s.e.m.). Statistical analysis to compare the mean values of two groups was performed via the Mann–Whitney

所有数据在此表示为平均值的平均值±标准误差（s.e.m.）。通过Mann-Whitney进行统计分析以比较两组的平均值

test, which is a nonparametric test, because the sample size is small and cannot be assumed to have a normal distribution.

检验，这是一种非参数检验，因为样本量很小，不能假设具有正态分布。

values ≤0.05 were considered statistically significant. Statistical analyses were performed with Prism software version 8.0 (GraphPad Software).

值≤0.05被认为具有统计学意义。。

Results

结果

Among PDK isoforms, PDK2 increases under IL-1β-induced catabolic conditions and in the cartilage of mice with surgically induced OA

在PDK亚型中，PDK2在IL-1β诱导的分解代谢条件下以及在手术诱导的OA小鼠的软骨中增加

To understand PDK-mediated metabolic modulation in OA chondrocytes, we first investigated the expression of PDK isoforms under IL-1β-mediated catabolic conditions. Treatment with IL-1β increased the mRNA levels of

为了了解PDK介导的OA软骨细胞代谢调节，我们首先研究了IL-1β介导的分解代谢条件下PDK亚型的表达。用IL-1β处理增加了

PDK2

and

和

PDK4

, but not

PDK1

或

PDK3

, in primary chondrocytes (Fig.

，在原代软骨细胞中（图）。

1a级

). At the protein level, however, only PDK2 increased after IL-1β treatment. Moreover, the level of the phosphorylated, inactive form of PDH (p-S

)。然而，在蛋白质水平上，IL-1β处理后只有PDK2增加。此外，PDH（p-S）的磷酸化，无活性形式的水平

293

-PDH) increased, suggesting that PDK2 may be involved in the phosphorylation of PDH under IL-1β-mediated catabolic conditions (Fig.

-PDH）增加，表明PDK2可能参与IL-1β介导的分解代谢条件下PDH的磷酸化（图）。

1b级

). To confirm this increase in PDK2, we examined the expression of PDK isoforms in the articular cartilage over time after DMM surgery in mice. Phosphorylated PDH, which indicates the inactivation of PDH, increased at 2 weeks post-DMM surgery. This increase was accompanied by an increase in PDK2, which continued to increase over time.

)。为了证实PDK2的这种增加，我们检测了小鼠DMM手术后关节软骨中PDK亚型的表达。表明PDH失活的磷酸化PDH在DMM手术后2周增加。这种增加伴随着PDK2的增加，PDK2随着时间的推移继续增加。

PDK4 tended to increase at approximately 8 weeks, but this increase was not statistically significant, whereas PDK1 and PDK3 did not increase in the DMM-induced model of OA (Fig. .

PDK4在大约8周时趋于增加，但这种增加没有统计学意义，而PDK1和PDK3在DMM诱导的OA模型中没有增加（图）。

1c级

Fig. 1: Expression of PDK under IL-1β-induced catabolic conditions and in murine cartilage from surgically induced OA.

。

一

The expression of

表达

Pdk

isoform (

同工型(

Pdk1–4

Pdk1-4

) and

)和

Mmp13

mRNAs in primary chondrocytes at 6, 12 and 24 h after 10 ng/ml IL-1β treatment was assessed via qRT‒PCR. mRNA expression is presented as the fold increase in gene expression normalized to

通过qRT-PCR评估10ng/ml IL-1β处理后6,12和24小时原代软骨细胞中的mRNA。mRNA表达表示为基因表达的倍数增加，归一化为

Gapdh

. **

< 0.01 compared with the PBS control group. Mean ± s.e.m.; Mann–Whitney

与PBS对照组相比<0.01。平均±s.e.m。；曼-惠特尼

test;

试验；

= 4 from biological replicates.

来自生物学重复的4。

b类

After 24 h of treatment with 10 ng/ml IL-1β, the protein levels of the PDK isoforms, phosphorylated PDH (p-S293-PDH) and PDH-E1 in primary chondrocytes were assessed by western blot analysis. Western blot band quantification for PDK1-4 was normalized to β-actin, while that for p-S293-PDH was normalized to PDH-E1.

用10ng/ml IL-1β处理24小时后，通过蛋白质印迹分析评估原代软骨细胞中PDK同种型，磷酸化PDH（p-S293-PDH）和PDH-E1的蛋白质水平。将PDK1-4的蛋白质印迹条带定量标准化为β-肌动蛋白，而将p-S293-PDH的蛋白质印迹条带定量标准化为PDH-E1。

The results are displayed in the form of bar graphs. *.

结果以条形图的形式显示。*。

< 0.05 compared with the PBS control group. Mean ± s.e.m.; Mann–Whitney

。平均±s.e.m。；曼-惠特尼

test;

试验；

= 3 from technical replicates among 2 biological replicates.

来自2个生物学重复中的技术重复。

c级

Immunofluorescence analyses revealed increases in MMP13 and PDK2 in articular chondrocytes at 2, 4 and 8 weeks after DMM surgery. The numbers of MMP13- and PDK isoform-positive chondrocytes above the tidemark were quantified and are expressed as ratios to the number of DAPI-positive cells. Scale bar, 100 μm.

免疫荧光分析显示DMM手术后2,4和8周关节软骨细胞中MMP13和PDK2的增加。定量潮标上方MMP13和PDK同种型阳性软骨细胞的数量，并表示为与DAPI阳性细胞数量的比率。比例尺，100微米。

***.

< 0.001, compared with control mice. Mean ± s.e.m.; Mann–Whitney

<0.001，与对照小鼠相比。平均±s.e.m。；曼-惠特尼

test;

试验；

= 6; ns, not significant.

=6；。

Full size image

全尺寸图像

PDK2 deficiency attenuates the severity of OA, oxidative stress and pain-related behaviors in a DMM-induced murine OA model

PDK2缺乏可减轻DMM诱导的小鼠OA模型中OA的严重程度，氧化应激和疼痛相关行为

To assess the impact of chondrocyte metabolic reprogramming on OA progression, we compared the phenotypes of DMM-induced OA in wild-type (WT) and

为了评估软骨细胞代谢重编程对OA进展的影响，我们比较了DMM诱导的OA在野生型（WT）和

Pdk2

KO mice. Compared with WT mice, genetic deletion of PDK2 significantly decreased the progression of DMM-induced OA. This was indicated by the decrease in cartilage degradation, as quantified by the OARSI score, and reduced osteophyte maturation and subchondral bone thickness (Fig.

KO小鼠。与WT小鼠相比，PDK2的基因缺失显着降低了DMM诱导的OA的进展。。

2a, b

2a、b

and Supplementary Fig.

和补充图。

). This finding was further supported by immunofluorescence staining, which revealed fewer MMP13-positive and 8-oxo-dG-positive chondrocytes in

)。免疫荧光染色进一步支持了这一发现，该染色显示MMP13阳性和8-氧代-dG阳性软骨细胞较少

Pdk2

KO mice than in control mice, indicating a decrease in the levels of ECM-degrading proteases and oxidative stress-induced DNA damage, respectively (Fig.

KO小鼠比对照小鼠，表明ECM降解蛋白酶和氧化应激诱导的DNA损伤水平分别降低（图）。

2a, c

2a，c

). This effect was accompanied by decreased pain-related behaviors in

)。这种效应伴随着疼痛相关行为的减少

Pdk2

KO mice compared with WT mice beginning at 6 weeks post-DMM surgery, as observed in tests such as static weight bearing over the hind limbs (incapacitance test), paw withdrawal time on a hot plate, and the von Frey test (Fig.

KO小鼠与DMM手术后6周开始的WT小鼠相比，如在后肢静态负重（失能试验），热板上的缩爪时间和von Frey试验等试验中观察到的。

二维

Fig. 2: PDK2 deficiency reduces the severity of cartilage degradation, oxidative stress-related DNA damage and pain-related behaviors in DMM-induced OA mice.

图2:PDK2缺陷降低了DMM诱导的OA小鼠软骨降解，氧化应激相关DNA损伤和疼痛相关行为的严重程度。

一

Safranin-O staining and immunostaining for MMP13 and 8-oxo-dG were performed on WT and

对野生型和野生型MMP13和8-氧代-dG进行番红-O染色和免疫染色

Pdk2

KO mice 8 weeks after DMM and sham surgery, and representative images are displayed. Scale bars, 100 μm.

在DMM和假手术后8周，KO小鼠显示代表性图像。比例尺，100微米。

b类

OARSI grade, subchondral bone plate thickness and osteophyte size were quantified and are expressed as mean ± s.e.m. *

量化OARSI等级，软骨下骨板厚度和骨赘大小，并表示为平均值±s.e.m*

< 0.05, Mann–Whitney

< 0.05，曼-惠特尼

test.

测试。

= 7.

= 7。

c级

Quantification of the percentage of MMP13- and 8-oxo-dG-positive chondrocytes above the tidemark. ***

潮标上方MMP13和8-氧代-dG阳性软骨细胞百分比的定量***

< 0.001; mean ± s.e.m.; Mann–Whitney

< 0.001;平均值±标准误差。，曼-惠特尼

test;

试验；

= 7.

= 7。

Pain behavior tests were conducted once a week on DMM and sham-operated mice. Weight bearing on the hind paw was assessed by an incapacitance test, which represents the ratio of weight bearing between the ipsilateral and contralateral hind paws; thus, any percentage less than 100% indicates hind limb unweighting.

对DMM和假手术小鼠每周进行一次疼痛行为测试。通过失能测试评估后爪的负重，该测试代表同侧和对侧后爪之间的负重比率；因此，任何小于100%的百分比都表示后肢不加权。

Thermal and mechanical pain sensations were assessed with the Hargreaves test via a hot plate and the von Frey test, respectively. *.

通过Hargreaves测试分别通过热板和von Frey测试评估热和机械疼痛感。*。

< 0.05, compared between WT and

<0.05，比较WT和

Pdk2

KO mice in the DMM group, Mann–Whitney

DMM组的KO小鼠，Mann-Whitney

test.

测试。

= 7.

= 7。

Full size image

全尺寸图像

PDK2 deficiency partially restores the IL-1β-mediated metabolic shift toward glycolysis in chondrocytes

PDK2缺乏部分恢复了IL-1β介导的软骨细胞向糖酵解的代谢转变

To determine whether PDK2 deficiency impacts chondrocyte metabolism, we assessed OxPhos and glycolysis under IL-1β-induced catabolic conditions using the Seahorse XF96 Extracellular Flux Analyzer. IL-1β treatment for 24 h markedly suppressed OxPhos, as reflected by a decrease in the OCR of cultured primary chondrocytes, whereas it increased glycolysis, as indicated by an increased ECAR.

为了确定PDK2缺乏是否影响软骨细胞代谢，我们使用Seahorse XF96细胞外通量分析仪评估了IL-1β诱导的分解代谢条件下的OxPhos和糖酵解。IL-1β处理24小时显着抑制OxPhos，如培养的原代软骨细胞的OCR降低所反映的，而它增加了糖酵解，如ECAR增加所示。

PDK2 deficiency partially restored the OCR by increasing basal respiration and ATP production in chondrocytes under IL-1β-treated conditions (Fig. .

在IL-1β处理的条件下，PDK2缺陷通过增加软骨细胞的基础呼吸和ATP产生来部分恢复OCR（图）。

3a级

). Meanwhile, the IL-1β-mediated increase in the ECAR was significantly reduced in

)。同时，IL-1β介导的ECAR增加在

Pdk2-

deficient chondrocytes, resulting from decreased glycolysis and glycolytic capacity (Fig.

缺乏软骨细胞，由糖酵解和糖酵解能力下降引起（图）。

3b级

). This PDK2-mediated metabolic reprogramming also led to a significant increase in ATP and the NAD

)。这种PDK2介导的代谢重编程也导致ATP和NAD的显着增加

/NADH ratio in the culture supernatant from

/培养上清液中NADH的比例

Pdk2-

deficient chondrocytes (Fig.

软骨细胞缺陷（图）。

3c, d

3c，d

). These findings suggest that the inhibition of PDK2 enhances OxPhos, potentially restoring the balance of energy homeostasis in chondrocytes under catabolic conditions such as those in OA.

)。这些发现表明，PDK2的抑制增强了OxPhos，可能在分解代谢条件下（例如OA）恢复软骨细胞能量稳态的平衡。

Fig. 3: PDK2 deficiency partially restores IL-1β-induced metabolic alterations, leading to increases in ATP and NAD

图3:PDK2缺乏部分恢复IL-1β诱导的代谢改变，导致ATP和NAD增加

/NADH levels in primary chondrocytes.

/原代软骨细胞中的NADH水平。

一

b类

, OCRs (

，OCRs(

一

) and ECARs (

)和ECAR(

b类

) were measured in primary chondrocytes isolated from WT and

)在从WT和WT分离的原代软骨细胞中进行了测量

Pdk2

KO mice via an XF96 Seahorse analyzer. Basal respiration and ATP production in the OCR analysis and glycolysis and glycolytic capacity in the ECAR analysis at one time point were quantified and are presented as the mean ± s.e.m. *

。OCR分析中的基础呼吸和ATP产生以及ECAR分析中一个时间点的糖酵解和糖酵解能力被量化，并表示为平均值±s.e.m*

< 0.05, Mann–Whitney

<0.05，曼-惠特尼

test.

测试。

= 4.

c级

, The ATP levels (

，ATP水平(

c级

) and NAD

)和NAD

/NADH ratios (

/NADH比率(

) in the culture media were quantified via colorimetric assays at 570 nm and 450 nm, respectively; mean ± s.e.m., *

)通过比色测定分别在570 nm和450 nm处对培养基中的蛋白质进行定量；平均值±s.e.m*

< 0.05, Mann–Whitney

< 0.05，曼-惠特尼

test.

测试。

= 3.

= 3。

Full size image

全尺寸图像

PDK2 deficiency enhances PDH activity and anabolic effects, which lead to reduced oxidative stress and cellular senescence in chondrocytes under IL-1β-treated conditions

PDK2缺乏会增强PDH活性和合成代谢作用，从而在IL-1β处理的条件下降低软骨细胞的氧化应激和细胞衰老

To investigate the impact of PDK2 deficiency on chondrocyte homeostasis under catabolic conditions, we then examined the mRNA expression of chondrogenic markers (

为了研究分解代谢条件下PDK2缺乏对软骨细胞稳态的影响，我们检测了软骨形成标志物的mRNA表达(

Col2

and

和

Aggrecan

聚集蛋白聚糖

), catabolic proteases (

)，分解代谢蛋白酶(

Mmp13

and

和

Adamts5

亚当斯5

) and senescence-associated secretory phenotype (SASP)-related genes (

)和衰老相关分泌表型（SASP）相关基因(

Il-6

and

和

Vegf

血管内皮生长因子

). Under IL-1β-treated conditions, PDK2 deficiency increased

)。在IL-1β处理的条件下，PDK2缺乏症增加

Col2

and

和

Aggrecan

聚集蛋白聚糖

expression and decreased

表达和减少

MMP13

and

和

IL-6

expression but had no effect on

表达，但对

Adamts5

亚当斯5

或

Vegf

血管内皮生长因子

expression (Fig.

表达（图）。

4a级

). PDH enzymatic activity was enhanced in

)。PDH酶活性增强

Pdk2

-deficient chondrocytes, as indicated by lower levels of phosphorylated PDH (p-S

-磷酸化PDH（p-S）水平较低表明软骨细胞缺乏

293

-PDH) under IL-1β-treated conditions. PDK2 deficiency significantly increased the protein levels of PDK1 and PDK4 under IL-1β-treated catabolic conditions but not under untreated control conditions, suggesting compensatory upregulation of other PDK isoforms. Similarly, the protein levels of Col2 increased in PDK2-deficient chondrocytes compared with WT chondrocytes under IL-1β-treated conditions, whereas those of MMP13 decreased.

-PDH）在IL-1β处理的条件下。在IL-1β处理的分解代谢条件下，PDK2缺乏显着增加了PDK1和PDK4的蛋白质水平，但在未处理的对照条件下却没有，这表明其他PDK亚型的补偿性上调。同样，在IL-1β处理的条件下，与WT软骨细胞相比，PDK2缺陷型软骨细胞中Col2的蛋白水平升高，而MMP13的蛋白水平降低。

The protein levels of Sirt1 remained unaffected, but the levels of antioxidant proteins, such as NRF2 and HO-1, were increased in .

Sirt1的蛋白质水平不受影响，但抗氧化蛋白（如NRF2和HO-1）的水平在年增加。

Pdk2

-deficient chondrocytes under IL-1β-treated conditions (Fig.

-在IL-1β处理的条件下缺乏软骨细胞（图）。

4b级

). Next, we assessed the role of PDK2 in oxidative stress under IL-1β-treated catabolic conditions. IL-1β treatment (10 ng/ml) significantly increased the production of ROS as well as 8-oxo-dG, a DNA damage product that occurs as a result of oxidative stress. Under these conditions,

)。接下来，我们评估了在IL-1β处理的分解代谢条件下PDK2在氧化应激中的作用。IL-1β处理（10ng/ml）显着增加了ROS以及8-氧代-dG（一种由于氧化应激而发生的DNA损伤产物）的产生。在这些条件下，

Pdk2

-deficient chondrocytes presented a significant decrease in ROS and 8-oxo-dG production, suggesting that PDK2 can reduce oxidative stress under IL-1β-mediated catabolic conditions (Fig.

-缺乏的软骨细胞表现出ROS和8-氧代-dG产生的显着降低，表明PDK2可以在IL-1β介导的分解代谢条件下降低氧化应激（图）。

4c级

). Consistent with these results, the expression of

)。与这些结果一致，表达

Hmox

Hmx

and

和

Fth

, which are target genes of oxidative stress, was decreased in

是氧化应激的靶基因，在

Pdk2

-deficient chondrocytes (Fig.

-软骨细胞缺陷（图）。

4d级

). We further investigated the effect of PDK2 deficiency on the senescence of chondrocytes, as mitochondrial dysfunction and ROS production are closely related to cellular senescence

)。我们进一步研究了PDK2缺乏对软骨细胞衰老的影响，因为线粒体功能障碍和活性氧的产生与细胞衰老密切相关

. After 48 h of treatment with IL-1β, a marked decrease in the expression of senescence-associated β-galactosidase (SA-β-gal), a senescence marker, was observed in chondrocytes from PDK2 KO mice compared with those from WT mice (Fig.

用IL-1β处理48小时后，与来自WT小鼠的软骨细胞相比，在PDK2 KO小鼠的软骨细胞中观察到衰老相关β-半乳糖苷酶（SA-β-gal）的表达显着降低（图1）。

4e级

). Our results collectively indicate that PDK2 loss of function leads to anabolic effects and attenuates ROS levels as well as the senescence of chondrocytes under catabolic conditions.

)。我们的结果共同表明，PDK2功能丧失会导致合成代谢作用，并减弱ROS水平以及分解代谢条件下软骨细胞的衰老。

Fig. 4: PDK2 deficiency not only increases the active form of PDH and has anabolic effects on IL-1β-treated chondrocytes but also decreases oxidative stress and cellular senescence.

图4:PDK2缺陷不仅增加了PDH的活性形式，并且对IL-1β处理的软骨细胞具有合成代谢作用，而且还降低了氧化应激和细胞衰老。

一

The expression of chondrogenic marker genes such as

软骨形成标记基因的表达，例如

Col2

and

和

Aggrecan

聚集蛋白聚糖

, catabolic proteases such as

，分解代谢蛋白酶，例如

Mmp13

and

和

Adamts5

亚当斯5

, and SASP-related biomarkers such as

，以及与SASP相关的生物标志物，例如

Il-6

and

和

Vegf

血管内皮生长因子

was assessed via qRT‒PCR. The results for mRNA expression are displayed as the fold increase in gene expression normalized to

通过qRT-PCR评估。mRNA表达的结果显示为基因表达的倍数增加归一化为

Gapdh

. Mean ± s.e.m., *

.平均值±s.e.m*

< 0.05, Mann–Whitney

< 0.05，曼-惠特尼

test.

测试。

= 4.

= 4。

b类

The phosphorylated-PDH (p-S

磷酸化PDH（p-S

293

-PDH), PDH-E1, PDK1, PDK2, PDK3, PDK4, Col2, MMP13, Sirt1, NRF2 and HO-1 protein levels in WT and

-PDH），PDH-E1，PDK1，PDK2，PDK3，PDK4，Col2，MMP13，Sirt1，NRF2和HO-1蛋白水平

Pdk2

KO chondrocytes after 24 h of IL-1β were assessed by western blot analysis. Western blot band quantification for p-S

通过蛋白质印迹分析评估IL-1β24小时后的KO软骨细胞。p-S的蛋白质印迹带定量

293

-PDH protein levels was normalized to PDH-E1, and the levels of PDK1, PDK2, PDK3, PDK4, Col2, MMP13, Sirt1, NRF2 and HO-1 were normalized to those of β-actin. *

-将PDH蛋白水平标准化为PDH-E1，并将PDK1，PDK2，PDK3，PDK4，Col2，MMP13，Sirt1，NRF2和HO-1的水平标准化为β-肌动蛋白的水平*

< 0.05, Mann–Whitney

<0.05，曼-惠特尼

test.

测试。

= 3.

= 3。

c级

Staining with DHE (red), a fluorescent probe for ROS, and 8-oxo-dG (green), a marker for oxidative DNA damage, was conducted in primary chondrocytes from WT and

用活性氧荧光探针DHE（红色）和氧化性DNA损伤标记物8-氧代-dG（绿色）对WT和WT的原代软骨细胞进行染色

Pdk2

KO mice treated with IL-1β (10 ng/ml) for 24 h. The nuclei of the cells were counterstained with DAPI (blue). Scale bar, 100 μm. DHE and 8-oxo-dG fluorescence-positive cells were quantified and expressed as ratios to DAPI-positive cells. *

用IL-1β（10ng/ml）处理KO小鼠24小时。细胞核用DAPI（蓝色）复染。比例尺，100微米。*

< 0.05. Mann–Whitney

< 0.05.曼-惠特尼

test.

测试。

= 4.

= 4。

The relative expression of oxidative stress marker genes such as

氧化应激标记基因的相对表达，例如

Hmox

Hmx

and

和

Fth

was evaluated via qRT‒PCR. The mRNA expression results are displayed as the fold increase in gene expression normalized to

通过qRT-PCR评估。mRNA表达结果显示为基因表达的倍数增加，归一化为

Gapdh

. *

< 0.05. Mann–Whitney

< 0.05。曼 - 惠特尼

test.

测试。

= 4.

= 4。

SA-β-gal staining was conducted on primary chondrocytes treated with IL-1β (10 ng/ml) for the indicated times. Scale bar, 100 μm. β-Gal-positive cells were counted from six different fields from three biological replicates, and the percentages of positive cells were determined. *

在用IL-1β（10ng/ml）处理指定时间的原代软骨细胞上进行SA-β-gal染色。比例尺，100微米。从三个生物学重复的六个不同区域计数β-Gal阳性细胞，并确定阳性细胞的百分比*

< 0.05. Mann–Whitney

< 0.05。曼 - 惠特尼

test.

测试。

= 3.

= 3。

Full size image

全尺寸图像

PDK2 deficiency reduces p38 signaling activity and sustains AMPK activation in response to IL-1β stimulation

PDK2缺乏会降低p38信号传导活性，并响应IL-1β刺激维持AMPK激活

We further examined the signaling mechanisms involved in the anabolic effects on PDK2-deficient chondrocytes under catabolic conditions, with a particular focus on metabolism and oxidative stress-related pathways such as the AMPK, mTOR, FoxO3a, AKT, NF-κB and MAPK pathways. Among these signaling pathways, the phosphorylation of p38 MAPK (Thr180/Tyr182) was most prominently suppressed in .

我们进一步研究了分解代谢条件下PDK2缺陷型软骨细胞合成代谢作用中涉及的信号传导机制，特别关注代谢和氧化应激相关途径，如AMPK，mTOR，FoxO3a，AKT，NF-κB和MAPK途径。在这些信号传导途径中，p38 MAPK（Thr180/Tyr182）的磷酸化被最显着地抑制。

Pdk2

-deficient chondrocytes compared with WT chondrocytes. The phosphorylation of AMPK (Thr172), which is typically downregulated by IL-1β stimulation, remained in an activated state in

-与WT软骨细胞相比，软骨细胞缺乏。AMPK（Thr172）的磷酸化通常被IL-1β刺激下调，在

Pdk2

-null chondrocytes. PDK2 deficiency led to a discontinuous increase in FoxO3a phosphorylation (Ser253) at 5 and 60 min after IL-1β stimulation, which suppressed FoxO3a activity through cytoplasmic export and proteasomal degradation

-空软骨细胞。PDK2缺陷导致IL-1β刺激后5分钟和60分钟FoxO3a磷酸化（Ser253）不连续增加，这通过细胞质输出和蛋白酶体降解抑制了FoxO3a活性

. The activation of mTOR, JNK, p65 and AKT signaling was not affected by PDK2 deficiency (Fig.

mTOR、JNK、p65和AKT信号的激活不受PDK2缺陷的影响（图）。

5a, b

5a、b

Fig. 5: PDK2 deficiency enhances FoxO3a signaling and prevents the downregulation of AMPK signaling, while it suppresses p38 MAPK activity under IL-1β-induced catabolic conditions.

。

一

The phosphorylation of FoxO3a, AMPKα, p38, JNK, mTOR, p65 and Akt was assessed in primary chondrocytes from WT and Pdk2 KO mice stimulated with IL-1β (20 ng/ml) for the indicated durations. The experiments were performed in triplicate, and representative blots are shown.

在用IL-1β（20ng/ml）刺激的WT和Pdk2 KO小鼠的原代软骨细胞中，在指定的持续时间内评估FoxO3a，AMPKα，p38，JNK，mTOR，p65和Akt的磷酸化。实验一式三份进行，并显示了代表性的印迹。

b类

The western blot band quantification for phosphorylated proteins was based on normalization to the corresponding nonphosphorylated total protein. *

磷酸化蛋白的蛋白质印迹带定量基于对相应的非磷酸化总蛋白的标准化*

< 0.05. Mann–Whitney

< 0.05.曼-惠特尼

test.

测试。

= 3.

= 3。

Full size image

全尺寸图像

p38 MAPK is essential for generating ROS in chondrocytes with mitochondrial dysfunction, and mitochondrial ROS, in turn, activate p38 MAPK under catabolic conditions

p38 MAPK对于在线粒体功能障碍的软骨细胞中产生ROS至关重要，而线粒体ROS又在分解代谢条件下激活p38 MAPK

To determine whether the decrease in p38 MAPK phosphorylation is simply a result of reduced ROS or if it directly involves a reduction in ROS in PDK2-deficient conditions, we treated WT and

为了确定p38 MAPK磷酸化的降低是否仅仅是ROS降低的结果，或者是否直接涉及PDK2缺陷条件下ROS的降低，我们处理了WT和

Pdk2

-deficient chondrocytes with the chemical p38 inhibitor SB203580 and the specific scavenger of mitochondrial superoxide Mito-TEMPO. The inhibition of p38 significantly suppressed ROS production and cellular senescence in WT chondrocytes, but this effect was not observed in

-化学p38抑制剂SB203580和线粒体超氧化物Mito-TEMPO的特异性清除剂缺乏软骨细胞。p38的抑制显着抑制了WT软骨细胞中ROS的产生和细胞衰老，但在

Pdk2

-deficient chondrocytes, where ROS generation and cellular senescence remained unchanged despite p38 MAPK inhibition (Fig.

-缺乏软骨细胞，尽管p38 MAPK抑制，ROS产生和细胞衰老保持不变（图）。

). This finding implies that, in a metabolic environment in which OxPhos is increased due to PDK2 deficiency, p38 MAPK does not play a significant role in ROS production and subsequent cellular senescence.

)。这一发现意味着，在由于PDK2缺乏而导致OxPhos增加的代谢环境中，p38 MAPK在ROS产生和随后的细胞衰老中不起重要作用。

Fig. 6: PDK2 is involved in a positive feedback loop between p38 MAPK and mitochondrial ROS production.

图6:PDK2参与p38 MAPK和线粒体ROS产生之间的正反馈回路。

一

p38 MAPK inhibition led to a decrease in ROS production and cellular senescence in WT chondrocytes but not in

Pdk2

-deficient chondrocytes. Chondrocytes from WT and

-软骨细胞缺陷。来自WT和

Pdk2

KO plants were pretreated with the p38 MAPK inhibitor SB203580 and the mitochondrial ROS inhibitor Mito-TEMPO for 30 min. The cells were then treated with IL-1β (10 ng/ml) for 24 h and stained with DHE (red) for total cellular ROS, 8-oxo-dG (green) for DNA damage, and MitoSOX (red) for mitochondrial ROS, and their nuclei were counterstained with DAPI (blue).

KO植物用p38 MAPK抑制剂SB203580和线粒体ROS抑制剂Mito-TEMPO预处理30分钟。然后用IL-1β（10 ng/ml）处理细胞24小时，并用DHE（红色）染色总细胞ROS，8-oxo-dG（绿色）用于DNA损伤，MitoSOX（红色）用于线粒体ROS，细胞核用DAPI（蓝色）复染。

Cellular senescence was assessed via SA-β-gal staining. Scale bar, 100 μm. DHE, 8-oxo-dG and MitoSOX fluorescence-positive cells were quantified and expressed as ratios to DAPI-positive cells. The area of SA-β-gal expression was quantified via densitometry with ImageJ software. *.

通过SA-β-gal染色评估细胞衰老。比例尺，100微米。定量DHE，8-氧代-dG和MitoSOX荧光阳性细胞，并表示为与DAPI阳性细胞的比率。使用ImageJ软件通过光密度测定法定量SA-β-gal表达的面积。*。

< 0.05,

< 0.05，

< 0.05. Mann–Whitney

< 0.05。曼 - 惠特尼

test.

测试。

= 4.

b类

p38 MAPK phosphorylation was assessed in the presence of a p38 inhibitor (SB203580) and a mitochondrial ROS inhibitor (Mito-TEMPO). Chondrocytes from WT and

在p38抑制剂（SB203580）和线粒体ROS抑制剂（Mito-TEMPO）存在下评估p38 MAPK磷酸化。来自WT和

Pdk2

KO mice were stimulated with IL-1β (20 ng/ml) for the indicated times and subjected to western blot analysis to assess the phosphorylated and total protein levels of p38 MAPK. *

用IL-1β（20ng/ml）刺激KO小鼠指定的时间，并进行蛋白质印迹分析以评估p38 MAPK的磷酸化和总蛋白水平*

< 0.05 compared with the fold change in p-p38/p38 in WT PBS controls at 15 min after stimulation.

与刺激后15分钟WT PBS对照中p-p38/p38的倍数变化相比，<0.05。

< 0.05 compared with p-p38/p38 in

与p-p38/p38相比<0.05

Pdk2

KO PBS controls at 15 min after stimulation. Mann–Whitney

。曼-惠特尼

test.

测试。

= 4.

= 4。

Full size image

全尺寸图像

Next, we investigated how the inhibition of mitochondrial ROS affects the activation of p38 MAPK. PDK2 deficiency led to a significant reduction in the phosphorylation of p38 MAPK compared with that in chondrocytes from WT littermates under IL-1β stimulation, and treatment with SB203580 substantially delayed the activation of p38 MAPK in .

接下来，我们研究了线粒体ROS的抑制如何影响p38 MAPK的激活。与IL-1β刺激下来自WT同窝仔的软骨细胞相比，PDK2缺乏导致p38 MAPK磷酸化显着降低，并且用SB203580处理显着延迟了p38 MAPK的激活。

Pdk2

-deficient chondrocytes. As expected, scavenging mitochondrial superoxide via Mito-TEMPO significantly reduced the phosphorylation of p38 MAPK at 15 min compared with that of both the WT and

-软骨细胞缺陷。

Pdk2

-deficient controls, indicating that mitochondrial ROS are crucial for the activation of p38 (Fig.

-缺乏对照，表明线粒体ROS对p38的激活至关重要（图）。

6b条

). Collectively, these results suggest the presence of a positive feedback loop between ROS and p38 MAPK and highlight the significant role of the PDK-mediated glycolytic metabolic shift in p38 MAPK-mediated ROS generation.

)。总的来说，这些结果表明ROS和p38 MAPK之间存在正反馈环，并突出了PDK介导的糖酵解代谢转变在p38 MAPK介导的ROS产生中的重要作用。

Discussion

讨论

Increasing evidence suggests that alterations in chondrocyte metabolism toward glycolysis, associated with mitochondrial dysfunction, are critically linked to OA pathogenesis

越来越多的证据表明，与线粒体功能障碍相关的软骨细胞代谢向糖酵解的改变与OA发病机制密切相关

. In this context, PDK-dependent inhibition of PDH activity may be a pivotal mechanism responsible for the glycolytic metabolic shift in catabolic chondrocytes

在这种情况下，PDK依赖性抑制PDH活性可能是导致分解代谢软骨细胞糖酵解代谢转变的关键机制

. Here, we revealed that PDK2 is specifically upregulated in OA chondrocytes and that its loss of function leads to an increase in PDH activity in order to restore the IL-1β-mediated metabolic shift toward glycolysis in chondrocytes. In addition, PDK2 deficiency resulted in a protective effect in a surgically induced OA model, which was accompanied by reduced oxidative stress and cellular senescence.

在这里，我们发现PDK2在OA软骨细胞中特异性上调，其功能丧失导致PDH活性增加，以恢复IL-1β介导的软骨细胞向糖酵解的代谢转变。此外，PDK2缺乏症在手术诱导的OA模型中产生保护作用，伴随着氧化应激和细胞衰老的减少。

Mechanistically, PDK2 deficiency led to decreased activation of p38 MAPK, along with sustained activation of AMPK signaling under IL-1β-treated conditions (Fig. .

从机制上讲，PDK2缺陷导致p38 MAPK的激活减少，以及在IL-1β处理的条件下AMPK信号的持续激活（图）。

). Taken together, our data shed light on the potential of metabolic reprogramming toward OxPhos as a novel therapeutic approach for OA.

)。综上所述，我们的数据揭示了代谢重编程对OxPhos作为OA新型治疗方法的潜力。

Fig. 7: Schematic diagram depicting the anabolic effects of metabolic reprogramming toward oxidative phosphorylation caused by PDK2 deficiency under catabolic conditions.

图7：描绘在分解代谢条件下由PDK2缺陷引起的代谢重编程对氧化磷酸化的合成代谢作用的示意图。

Among PDK isoforms, PDK2 was expressed primarily under catabolic and in vivo OA conditions. The loss of PDK2 function enhanced OxPhos and ATP/NAD

在PDK同种型中，PDK2主要在分解代谢和体内OA条件下表达。PDK2功能的丧失增强了OxPhos和ATP/NAD

production, which led to a reduction in oxidative stress. Mechanistically, PDK2 plays a crucial role in the positive feedback loop between oxidative stress and p38 MAPK under catabolic conditions in chondrocytes.

产生，从而减少了氧化应激。从机制上讲，PDK2在软骨细胞分解代谢条件下氧化应激和p38 MAPK之间的正反馈回路中起着至关重要的作用。

Full size image

全尺寸图像

Several lines of evidence indicate the critical involvement of mitochondrial dysfunction in the pathogenesis of OA

几条证据表明线粒体功能障碍在OA的发病机制中起关键作用

. Specifically, chondrocytes from patients with advanced OA exhibit a decrease in respiring mitochondria, as indicated by decreased rhodamine 123 staining

具体而言，罗丹明123染色减少表明，晚期OA患者的软骨细胞呼吸线粒体减少

. Morphologically, these mitochondria are characterized by increased length relative to width, coupled with an overall reduction in count and disrupted morphology

形态学上，这些线粒体的特征是长度相对于宽度的增加，加上数量的总体减少和形态的破坏

. This elongation is particularly noteworthy, as it implies increased mitochondrial fusion, a phenomenon often observed under conditions such as nutrient withdrawal or increased OxPhos

这种伸长特别值得注意，因为它意味着线粒体融合增加，这种现象通常在营养缺乏或OxPhos增加的情况下观察到

. Chondrocytes from relatively preserved articular cartilage demonstrate significantly greater mitochondrial respiration capacity than those from severely damaged lesions do

.相对保存的关节软骨中的软骨细胞显示出比严重受损病变中的软骨细胞更大的线粒体呼吸能力

. However, as OA progresses, these metabolic adaptations begin to fail. This is indicated by a diminished capacity of the respiratory chain and by a decrease in the number of mitochondria coupled with an increase in mitochondrial fission, leading to mitochondrial dysfunction

然而，随着OA的进展，这些代谢适应开始失败。这表现为呼吸链的能力下降，线粒体数量减少，线粒体裂变增加，导致线粒体功能障碍

. Consequently, impaired mitochondrial function can disrupt ATP production and increase oxidative stress in chondrocytes, both of which are key contributors to the pathogenesis of OA

因此，线粒体功能受损会破坏ATP的产生并增加软骨细胞的氧化应激，这两者都是OA发病机制的关键因素

From this perspective, the central hypothesis of this study was that enhancing OxPhos could inhibit the progression of OA. This study revealed that PDK2 deficiency, along with the subsequent increase in PDH activity, increased OxPhos under catabolic conditions, leading to a reduction in oxidative stress and the inhibition of OA progression.

从这个角度来看，这项研究的中心假设是增强OxPhos可以抑制OA的进展。这项研究表明，PDK2缺乏症以及随后PDH活性的增加，在分解代谢条件下会增加OxPhos，从而导致氧化应激的减少和OA进展的抑制。

These results conflict with the general consensus that increasing OxPhos leads to an increase in ROS.

这些结果与增加OxPhos导致ROS增加的普遍共识相冲突。

. However, a line of evidence has shown that inhibiting mitochondrial respiration can actually increase ROS production

然而，一系列证据表明，抑制线粒体呼吸实际上可以增加活性氧的产生

. Taken together, these findings suggest that both excessive and defective mitochondrial respiration can contribute to increased ROS production, depending on the cellular context. Our in vitro experiments revealed that treatment with dichloroacetate, which is a pan-PDK inhibitor that induces a switch in cellular metabolism toward OxPhos, increased ROS production in nonsenescent chondrocytes but decreased ROS in H2O2- and IL-1β-induced senescent chondrocytes (Supplementary Fig.

综上所述，这些发现表明，线粒体呼吸过度和缺陷均可导致ROS产生增加，具体取决于细胞环境。我们的体外实验表明，用二氯乙酸盐（一种泛PDK抑制剂，可诱导细胞代谢向OxPhos转变）处理可增加非衰老软骨细胞中的ROS产生，但可降低H2O2和IL-1β诱导的衰老软骨细胞中的ROS（补充图）。

). This result suggests a context-dependent role of OxPhos in ROS production; under physiological conditions, an increase in OxPhos increases ROS, but under stress or senescent conditions, an increase in OxPhos may conversely reduce ROS production. Our data suggest that restoring OxPhos in OA chondrocytes is a promising approach to reduce ROS and the catabolic cascade, thereby potentially inhibiting the progression of OA..

)。该结果表明OxPhos在ROS产生中的上下文依赖性作用；在生理条件下，OxPhos的增加会增加ROS，但在压力或衰老条件下，OxPhos的增加可能会反过来减少ROS的产生。我们的数据表明，恢复OA软骨细胞中的OxPhos是减少ROS和分解代谢级联反应的有前途的方法，从而可能抑制OA的进展。。

So far, the molecular mechanism underlying the metabolic shift toward glycolysis in OA chondrocytes has remained largely unclear. In this study, we demonstrated a significant increase in PDK2 among PDK isoforms under IL-1β-mediated catabolic conditions and in OA chondrocytes (Fig.

到目前为止，OA软骨细胞代谢向糖酵解转变的分子机制仍不清楚。在这项研究中，我们证明了在IL-1β介导的分解代谢条件下和OA软骨细胞中PDK亚型中PDK2的显着增加（图）。

). Moreover, PDK2 deficiency led to a decrease in the phosphorylation of PDH under IL-1β-treated catabolic conditions, indicating inactivation of the PDH complex, which converts pyruvate to acetyl-CoA (Fig.

)。此外，在IL-1β处理的分解代谢条件下，PDK2缺乏导致PDH磷酸化降低，表明PDH复合物失活，PDH复合物将丙酮酸转化为乙酰辅酶a（图）。

4b级

). These findings suggest that PDK2 may be a key regulator of chondrocyte metabolism under catabolic conditions. Indeed, our data confirmed that PDK2 deficiency, at least partially, enhanced OxPhos in IL-1β-treated chondrocytes while reducing glycolysis (Fig.

)。这些发现表明，PDK2可能是分解代谢条件下软骨细胞代谢的关键调节剂。事实上，我们的数据证实，PDK2缺陷至少部分增强了IL-1β处理的软骨细胞中的OxPhos，同时减少了糖酵解（图）。

). PDK, which serves as a negative feedback mechanism, is activated by the products of the PDH reaction and the TCA cycle, such as NADH, high energy charge and acetyl-CoA

)。PDK作为一种负反馈机制，被PDH反应和TCA循环的产物激活，如NADH、高能电荷和乙酰辅酶a

. This activation leads to inactivation of the PDH. However, a decreasing energy charge and increasing pyruvate concentration inhibit PDK activity, thereby leading to increased PDH activation

。这种激活会导致PDH失活。然而，降低能量电荷和增加丙酮酸浓度会抑制PDK活性，从而导致PDH活化增加

. Although several mechanisms, including lactate dehydrogenase-A, hypoxia-inducible factor 1A, the AKT–mTOR signaling pathway and pyruvate kinase M2, are known to control the glycolytic shift in chondrocytes

尽管已知几种机制，包括乳酸脱氢酶A，缺氧诱导因子1A，AKT-mTOR信号通路和丙酮酸激酶M2，可以控制软骨细胞的糖酵解转变

, PDK is known to mediate the Warburg effect, which is characterized by increased aerobic glycolysis

，已知PDK介导Warburg效应，其特征是有氧糖酵解增加

. This may directly lead to the glycolytic shift observed in OA. Given these findings, inhibiting PDK2 could be a promising approach for the metabolic reprogramming of chondrocytes.

。这可能直接导致OA中观察到的糖酵解转变。鉴于这些发现，抑制PDK2可能是软骨细胞代谢重编程的有前途的方法。

The expression of PDK isoforms in chondrocytes has not been extensively characterized. Our data revealed an increase in PDK2 and a decrease in other PDK isoforms, such as PDK1, PDK3 and PDK4, in IL-1β-treated catabolic chondrocytes and in vivo OA cartilage (Fig.

PDK亚型在软骨细胞中的表达尚未得到广泛表征。。

). Consistent with our findings, a recent study reported significant downregulation of PDK1 mRNA and protein expression in OA articular cartilage, although it did not specify the expression of other PDK isoforms

)。与我们的发现一致，最近的一项研究报道了OA关节软骨中PDK1 mRNA和蛋白表达的显着下调，尽管它没有指定其他PDK亚型的表达

. The lack of any noticeable phenotype in endochondral bone formation in

。软骨内骨形成缺乏任何明显的表型

Pdk2

-deficient mice also suggests that PDK2 plays a limited role in the physiological maturation of chondrocytes (data not shown). This evidence that PDK2 is specifically expressed in catabolic chondrocytes suggests that targeting PDK2 could minimally affect normal cartilage physiology while effectively addressing OA conditions, thereby offering a potential advantage in the development of OA drugs targeting PDK2..

-缺陷小鼠还表明，PDK2在软骨细胞的生理成熟中起着有限的作用（数据未显示）。PDK2在分解代谢软骨细胞中特异性表达的证据表明，靶向PDK2可以最小程度地影响正常软骨生理，同时有效解决OA病症，从而为开发靶向PDK2的OA药物提供潜在优势。。

Our data revealed that IL-1β-mediated p38 MAPK phosphorylation was significantly reduced in

我们的数据显示，IL-1β介导的p38 MAPK磷酸化在

Pdk2

-deficient chondrocytes (Fig.

-软骨细胞缺陷（图）。

). Apoptosis signal-regulating kinase 1, which is positioned upstream of p38 MAPK, is a well-known redox-sensitive kinase

)。凋亡信号调节激酶1位于p38 MAPK的上游，是一种众所周知的氧化还原敏感激酶

, implying that lower ROS levels in

，这意味着较低的活性氧水平

Pdk2

-deficient chondrocytes may lead to reduced phosphorylation of p38 MAPK. Furthermore, p38 MAPK signaling itself can induce oxidative stress via MAP kinase-activated protein kinase 2, potentially creating a positive feedback loop between p38 MAPK and oxidative stress under catabolic conditions

-缺陷的软骨细胞可能导致p38 MAPK的磷酸化降低。此外，p38 MAPK信号本身可以通过MAP激酶激活的蛋白激酶2诱导氧化应激，在分解代谢条件下可能在p38 MAPK和氧化应激之间产生正反馈回路

. Furthermore, our data revealed that the p38 inhibitor significantly suppressed ROS generation in WT chondrocytes, whereas this inhibition of ROS was not observed in PDK2 KO chondrocytes (Fig.

此外，我们的数据显示p38抑制剂显着抑制WT软骨细胞中ROS的产生，而在PDK2 KO软骨细胞中未观察到ROS的这种抑制（图）。

). This finding implies that p38 MAPK does not influence ROS generation in conditions prone to OxPhos due to PDK2 deficiency. In other words, p38 MAPK may primarily contribute to an increase in ROS in situations of mitochondrial dysfunction, characterized by reduced OxPhos. Although OA is primarily a degenerative disease, omics data from OA articular cartilage have revealed a sustained increase in inflammatory signatures.

)。这一发现意味着p38 MAPK在由于PDK2缺乏而容易发生OxPhos的条件下不影响ROS的产生。换句话说，p38 MAPK可能主要导致线粒体功能障碍情况下ROS的增加，其特征是OxPhos减少。虽然OA主要是一种退行性疾病，但OA关节软骨的组学数据显示炎症特征持续增加。

. Our findings indicate that p38 MAPK could be an essential intermediary linking metabolic alterations to the inflammatory gene signature in OA cartilage.

我们的研究结果表明，p38 MAPK可能是将代谢改变与OA软骨中的炎症基因特征联系起来的重要中介。

Another significant observation regarding signaling changes associated with PDK2 deficiency is the more gradual reduction in AMPK phosphorylation (Thr172) caused by IL-1β stimulation. As implied by its name ‘AMP-activated protein kinase’, AMPK is activated by AMP, which typically increases under metabolic stress conditions.

关于与PDK2缺乏症相关的信号变化的另一个重要观察结果是由IL-1β刺激引起的AMPK磷酸化（Thr172）逐渐减少。正如其名称“AMP激活的蛋白激酶”所暗示的那样，AMPK被AMP激活，AMP通常在代谢应激条件下增加。

. Conversely, when metabolic balance is restored and ATP levels rise, this leads to inactivation of the kinase

相反，当代谢平衡恢复并且ATP水平升高时，这会导致激酶失活

. In addition to metabolic conditions, the regulation of AMPK involves several upstream kinases; LKB1, CaMKKβ and TAK1 are key activators, whereas PKC, AKT, PKA and PP2A contribute to its inactivation

除了代谢条件外，AMPK的调节还涉及几种上游激酶；LKB1、CaMKK和TAK1是关键的激活因子，而PKC、AKT、PKA和PP2A有助于其失活

. With respect to oxidative stress, although AMPK activation helps suppress it, oxidative stress can, in turn, lead to the inactivation of AMPK

.关于氧化应激，尽管AMPK激活有助于抑制它，但氧化应激反过来会导致AMPK失活

. Thus, the reduced levels of ROS observed in PDK2 deficiency could slow the inactivation of AMPK, potentially aiding in the maintenance of metabolic homeostasis in chondrocytes under catabolic conditions.

因此，在PDK2缺乏症中观察到的ROS水平降低可以减缓AMPK的失活，可能有助于在分解代谢条件下维持软骨细胞的代谢稳态。

In conclusion, the loss of function of PDK2, which is upregulated under catabolic conditions in chondrocytes, leads to a metabolic shift toward OxPhos. This shift is associated with a reduction in oxidative stress and cellular senescence, providing protective effects against OA progression. Our findings suggest that metabolic modulation toward OxPhos deserves particular attention as a potential target for OA treatment..

总之，在软骨细胞分解代谢条件下上调的PDK2功能丧失导致代谢向OxPhos转变。这种转变与氧化应激和细胞衰老的减少有关，为OA进展提供保护作用。我们的研究结果表明，对OxPhos的代谢调节值得特别关注，作为OA治疗的潜在靶点。。

Data availability

数据可用性

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

本研究中使用和/或分析的数据集可根据合理要求从通讯作者处获得。

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Acknowledgements

致谢

We thank I.-K. Lee (Endocrinology, Kyungpook National University) for valuable advice on this project and for assisting with the animal experiments. We thank H.-Y. Lee (Kyungpook National University) for helping with the extracellular flux analysis. This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) (grant number RS-2023-00274788 and NRF-2020R1A2C1004517)..

我们感谢I.-K.Lee（庆浦国立大学内分泌学）对该项目的宝贵建议以及对动物实验的协助。我们感谢H.-Y.Lee（庆浦国立大学）帮助进行细胞外通量分析。这项研究得到了韩国政府（科学和信息通信技术部）资助的韩国国家研究基金会（NRF）的资助（资助号RS-2023-00274788和NRF-2020R1A2C1004517）。。

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Laboratory for Arthritis and Cartilage Biology, Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Republic of Korea

大韩民国大邱庆北国立大学衰老与代谢研究所关节炎与软骨生物学实验室

Jin Han, Yoon Hee Kim & Seungwoo Han

Jin Han、Yoon Hee Kim 和 Seungwoo Han

Cell & Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, Republic of Korea

韩国大邱庆浦国立大学医学院细胞与基质研究所

Jin Han & Seungwoo Han

金汉 & 胜宇汉

Division of Rheumatology, Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu, Republic of Korea

大韩民国大邱庆北国立大学医学院内科风湿科

Seungwoo Han

韩成宇

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Jin Han

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Contributions

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Study conception and design: J.H., Y.H.K. and S.H.; acquisition of data: J.H. and Y.H.K.; analysis and/or interpretation of data: J.H. and S.H.; writing of the paper: J.H. and S.H.; critical revision: J.H., Y.H.K. and S.H. All authors read and approved the final manuscript.

研究概念和设计：J.H.，Y.H.K.和S.H。；数据采集：J.H.和Y.H.K。；数据分析和/或解释：J.H.和S.H。；论文撰写：J.H.和S.H。；。

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All experiments were conducted in accordance with approved animal protocols and guidelines established by the Animal Care Committee of Kyungpook National University (approval no. KNU-2018-62/54).

所有实验均按照庆浦国立大学动物护理委员会批准的动物方案和指南进行（批准号KNU-2018-62/54）。

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Han, J., Kim, Y.H. & Han, S. Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

Han，J.，Kim，Y.H。＆Han，S。通过丙酮酸脱氢酶激酶2缺乏增加氧化磷酸化改善了手术诱导的骨关节炎小鼠的软骨降解。

Exp Mol Med

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(2025). https://doi.org/10.1038/s12276-025-01400-9

(2025).https://doi.org/10.1038/s12276-025-01400-9

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2024年2月10日

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