牙龈卟啉单胞菌在怀孕小鼠中的施用增强了糖酵解和组蛋白乳酸化/ADAM17，导致后代腭裂-动脉网

Administration of Porphyromonas gingivalis in pregnant mice enhances glycolysis and histone lactylation/ADAM17 leading to cleft palate in offspring

Nature 等信源发布 2025-03-13 12:50

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Abstract

摘要

Periodontal disease is a risk factor for many systemic diseases such as Alzheimer’s disease and adverse pregnancy outcomes. Cleft palate (CP), the most common congenital craniofacial defect, has a multifaceted etiology influenced by complex genetic and environmental risk factors such as maternal bacterial or virus infection.

牙周病是许多系统性疾病的风险因素，如阿尔茨海默病和不良妊娠结局。腭裂（CP）是最常见的先天性颅面缺陷，其病因复杂，受复杂遗传和环境风险因素的影响，例如母体细菌或病毒感染。

A prior case-control study revealed a surprisingly strong association between maternal periodontal disease and CP in offspring. However, the precise relationship remains unclear. In this study, the relationship between maternal oral pathogen and CP in offspring was studied by sonicated .

先前的一项病例对照研究揭示了母亲牙周病与子代脑瘫之间存在惊人强烈的关联。然而，确切的关系仍不清楚。本研究通过超声处理研究了母体口腔病原体与子代脑瘫之间的关系。

P. gingivalis

牙龈卟啉单胞菌

injected intravenously and orally into pregnant mice. We investigated an obvious increasing CP (12.5%) in sonicated

静脉注射和口服给怀孕小鼠。我们观察到超声处理后CP明显增加（12.5%）。

P. gingivalis

牙龈卟啉单胞菌

group which had inhibited osteogenesis in mesenchyme and blocked efferocytosis in epithelium. Then glycolysis and H4K12 lactylation (H4K12la) were detected to elevate in both mouse embryonic palatal mesenchyme (MEPM) cells and macrophages under

该组抑制了间充质中的骨生成并阻断了上皮中的胞葬作用。随后检测到糖酵解和H4K12乳酸化（H4K12la）在小鼠胚胎腭间充质（MEPM）细胞和巨噬细胞中均升高。

P. gingivalis

牙龈卟啉单胞菌

exposure which further promoted the transcription of metallopeptidase domain17 (ADAM17), subsequently mediated the shedding of transforming growth factor-beta receptor 1 (TGFBR1) in MEPM cells and mer tyrosine kinase (MerTK) in macrophages and resulted in the suppression of efferocytosis and osteogenesis in palate, eventually caused abnormalities in palate fusion and ossification.

暴露进一步促进了金属肽酶结构域17 (ADAM17) 的转录，随后介导了MEPM细胞中转化生长因子-β受体1 (TGFBR1) 的脱落以及巨噬细胞中Mer酪氨酸激酶 (MerTK) 的脱落，导致腭部胞葬作用和成骨作用的抑制，最终引起腭融合和骨化异常。

The abnormal efferocytosis also led to a predominance of M1 macrophages, which indirectly inhibited palatal osteogenesis via extracellular vesicles. Furthermore, pharmacological ADAM17 inhibition could ameliorate the abnormality of .

异常的胞葬作用还导致了M1型巨噬细胞占主导地位，其通过细胞外囊泡间接抑制腭骨生成。此外，药理学上对ADAM17的抑制可以改善这一异常。

P. gingivalis

牙龈卟啉单胞菌

-induced abnormal palate development. Therefore, our study extends the knowledge of how maternal oral pathogen affects fetal palate development and provides a novel perspective to understand the pathogenesis of CP.

诱导的腭发育异常。因此，我们的研究扩展了关于母体口腔病原体如何影响胎儿腭发育的知识，并为理解CP的发病机制提供了新的视角。

Introduction

介绍

Cleft palate (CP), the most common congenital craniofacial defect, can significantly impact quality of life, even following surgical interventions, and places a considerable strain on families and society.

唇腭裂（CP）是最常见的先天性颅面缺陷，即使在手术干预后，也会显著影响生活质量，并给家庭和社会带来沉重负担。

CP has a multifaceted etiology influenced by complex genetic and environmental risk factors.

CP的病因复杂，受多种遗传和环境风险因素的影响。

Maternal environment is important to embryonic development, and disturbing it may cause deformity. The maternal intrauterine microenvironment, which surrounds the fetus and affects it directly, was once believed to be sterile. However, recent evidences have demonstrated the vertical transmission of maternal oral microbes to the uterus, potentially introducing them into the intrauterine microenvironment..

母体环境对胚胎发育很重要，干扰它可能会导致畸形。母体子宫内围绕胎儿并直接影响胎儿的微环境，曾经被认为无菌。然而，最近的证据表明，母体口腔微生物可垂直传播至子宫，可能将它们引入子宫内的微环境中。

，

Periodontitis, one of the major public health issues often caused by

牙周炎，一种常由多种因素引起的重大公共卫生问题，

Porphyromonas gingivalis

牙龈卟啉单胞菌

(

P. gingivalis

牙龈卟啉单胞菌

) among other periodontal pathogens, is confirmed relating to more and more systemic diseases such as Alzheimer’s disease, atherosclerosis and adverse pregnancy outcomes.

）在内的多种牙周病原体，已被证实与越来越多的全身性疾病相关，如阿尔茨海默病、动脉粥样硬化和不良妊娠结局。

An unexpectedly strong association between maternal periodontal disease and CP in children was observed in a previous case-control study.

之前的一项病例对照研究观察到母亲牙周病与儿童脑瘫之间存在意外强烈的相关性。

However, the exact mechanism of this relationship remains unclear.

然而，这种关系的确切机制尚不清楚。

Mice is the most common animal model in exploring etiology of CP. The secondary palatal shelves in mice, between embryonic days (E) 12.5 and 15.5, undergo a sequence of vertical growth, elevation, horizontal growth, fusion, and osteogenesis.

小鼠是研究CP病因最常见的动物模型。小鼠的次级腭架在胚胎第12.5天至第15.5天期间，会经历垂直生长、抬升、水平生长、融合和骨化等一系列过程。

This process mirrors human palate development to a large extent and any disturbance during the process will result in CP.

这一过程在很大程度上反映了人类腭部的发育过程，任何过程中出现的异常都会导致腭裂。

Recently, metabolic shifts stemming from environmental and dietary changes has been revealed to influence cell fate in both physiological and pathological states via epigenetic effect.

最近，由于环境和饮食变化引起的代谢转变已被揭示通过表观遗传效应影响生理和病理状态下的细胞命运。

Studies have observed dramatic shifts in glucose metabolism during the migration and differentiation of neural crest cells, which share homology with palatine mesenchymal cells.

研究表明，在神经嵴细胞迁移和分化过程中，葡萄糖代谢发生了显著变化，这些细胞与腭间充质细胞具有同源性。

，

Our previous study also demonstrated that mouse embryonic palatal mesenchyme (MEPM) cells at E13.5 undergo significant growth and primarily depend on glycolysis, then shift to oxidative phosphorylation (OXPHOS) as they adapt to osteogenesis in response to functional demands and environmental cues at E15.5..

我们之前的研究还表明，E13.5 的小鼠胚胎腭间充质 (MEPM) 细胞经历显著的生长，主要依赖糖酵解，随后在适应成骨过程中，随着 E15.5 时功能需求和环境信号的变化，转向氧化磷酸化 (OXPHOS)。

However, little is known about whether aberrated shifts in glucose metabolism involve in the increasing risk of CP. On the other hand, numerous adverse outcomes in offspring, including metabolic disorders, have been associated with maternal

然而，目前尚不清楚葡萄糖代谢的异常变化是否与脑瘫风险的增加有关。另一方面，许多不良的后代结果，包括代谢紊乱，都与母体有关。

P. gingivalis

牙龈卟啉单胞菌

infection.

感染。

，

Moreover, prior studies suggested that

此外，先前的研究表明

P. gingivalis

牙龈卟啉单胞菌

infection can trigger a metabolic shift from OXPHOS to glycolysis as an adaptation to stressful conditions,

感染可以触发从氧化磷酸化到糖酵解的代谢转变，以适应压力条件，

，

which is opposite to the normal metabolic progression of palate development in our previous study.

这与我们之前研究中腭发育的正常代谢进程相反。

We then wonder if

我们然后想知道是否

P. gingivalis

牙龈卟啉单胞菌

infection disrupts glucose metabolism will affect palatogenesis.

感染会破坏葡萄糖代谢，从而影响腭形成。

Historically, studies on palate development have focused predominantly on functional changes in MEPM cells, while potential contributions of immune cells have been largely overlooked. Emerging evidences indicate that macrophages (Mφs) play an active role during the key stage of palatal fusion by phagocytosing apoptotic epithelial fragments in the decomposed midpalate epithelial suture (MES)..

历史上，关于腭发育的研究主要集中在MEPM细胞的功能变化上，而免疫细胞的潜在贡献在很大程度上被忽视。新兴证据表明，巨噬细胞 (Mφs) 在腭融合的关键阶段通过吞噬分解的中腭上皮缝 (MES) 中的凋亡上皮碎片发挥了积极作用。

，

This activity fine-tunes palatal fusion, suggesting a significant role for Mφ-mediated efferocytosis in palatal fusion regulation. It has also been demonstrated that sustained glycolysis can negatively impact on Mφ functions. For example, the functions such as phagocytosis and chemotaxis of microglia, the resident Mφs in brain, can be compromised when glycolytic metabolism is activated..

该活动微调了腭融合，表明巨噬细胞介导的胞葬作用在腭融合调控中具有重要作用。同时也有研究表明，持续的糖酵解可能对巨噬细胞功能产生负面影响。例如，当糖酵解代谢被激活时，大脑中的常驻巨噬细胞——小胶质细胞的吞噬作用和趋化功能可能会受到损害。

Nevertheless, the relationship between Mφ and metabolism in palate development has not been reported.

然而，Mφ与腭发育过程中代谢的关系尚未见报道。

While the regulatory role of metabolic pathways in cell function has garnered considerable attention, the specific mechanisms involved remain largely elusive. Recently, a variety of histone modification marks derived from cellular metabolites have been discovered.

虽然代谢途径在细胞功能中的调控作用已引起相当多的关注，但所涉及的具体机制仍然大多不明朗。最近，发现了多种由细胞代谢物衍生的组蛋白修饰标记。

Among these, lactate, a product of glycolysis, has been identified as a substrate for histone lactylation. Histone lactylation has been shown to regulate cell functions, such as Mφ polarization and somatic cell reprogramming.

其中，乳酸作为糖酵解的产物，已被确定为组蛋白乳酸化的一种底物。组蛋白乳酸化已被证明能够调节细胞功能，例如巨噬细胞极化和体细胞重编程。

，

Additionally, our previous study suggested that changes in the maternal gut microbiome could affect fetal palatal development, potentially due to metabolic disorders and lactate accumulation,

此外，我们之前的研究表明，母体肠道微生物组的变化可能影响胎儿腭部发育，这可能是由于代谢紊乱和乳酸积累所致。

which meanest histone lactylation might participate in the process.

这可能意味着组蛋白乳酸化可能参与了这一过程。

In this study, we demonstrated that

在这项研究中，我们证明了

P. gingivalis

牙龈卟啉单胞菌

exposure caused CP in mice which elevates glycolysis and subsequent histone lactylation, esp. H4K12 lactylation in both Mφs and MEPM cells. This increased lactylation promoted the transcription of metallopeptidase domain17 (ADAM17) which mediated the shedding of Mer tyrosine kinase (MerTK) in Mφs and transforming growth factor-beta receptor 1 (TGFBR1) in MEPM cells, inducing the suppression of Mφ efferocytosis in epithelium and MEPM cells osteogenesis, respectively, and ultimately resulting in abnormalities in palate shelves fusion and ossification.

暴露导致小鼠的CP升高，这提升了糖酵解及随后的组蛋白乳酸化，尤其是Mφs和MEPM细胞中的H4K12乳酸化。这种增加的乳酸化促进了金属肽酶结构域17（ADAM17）的转录，其介导了Mφs中Mer酪氨酸激酶（MerTK）以及MEPM细胞中转化生长因子-β受体1（TGFBR1）的脱落，分别抑制了上皮中Mφ的胞葬作用和MEPM细胞的成骨作用，最终导致腭板融合和骨化异常。

Blocking ADAM17 or histone lactylation could rescue the occurrence of CP. Furthermore, the abnormal efferocytosis also led to a predominance of M1 Mφs, which indirectly inhibited palatal osteogenesis via small extracellular vesicles (sEVs). Therefore, our study describes a cascade process from an initial perturbation of how maternal oral pathogen impact on fetal palate development, highlights novel aspects on etiology of congenital malformations and its potential interceptive therapy..

阻断ADAM17或组蛋白乳酸化可挽救CP的发生。此外，异常的胞葬作用还导致M1型巨噬细胞占优势，其通过小细胞外囊泡（sEVs）间接抑制腭骨生成。因此，我们的研究描述了一个从母体口腔病原体如何影响胎儿腭发育的初始扰动开始的级联过程，揭示了先天性畸形病因的新方面及其潜在的干预疗法。

Results

结果

P. gingivalis

牙龈卟啉单胞菌

induced CP with abnormal palate shelves fusion and osteogenesis in palate development in vivo

诱导的CP伴有腭架融合异常及腭发育过程中的骨生成异常。

Sonicated

超声处理

P. gingivalis

牙龈卟啉单胞菌

is a common method in investigating effects of

是研究效应的常用方法

P. gingivalis

牙龈卟啉单胞菌

on the body.

在身体上。

To examine the potential translocation of sonicated

为了检查超声处理后可能的移位

P. gingivalis

牙龈卟啉单胞菌

to the fetal palate and to evaluate the temporal variation in its concentration during the critical period of palatal development, we firstly constructed a mouse model of experimental

针对胎儿腭部，并评估其在腭部发育关键时期浓度的时序变化，我们首先构建了一个实验性的小鼠模型。

P. gingivalis

牙龈卟啉单胞菌

exposure during pregnant (Fig.

怀孕期间的暴露（图。

and assessed the presence of

并评估了是否存在

P. gingivalis

牙龈卟啉单胞菌

gingipain R1 (RgpA) in the amniotic fluid and fetal palatal process utilizing indirect Enzyme-Linked ImmunoSorbent Assay (ELISA). As depicted in Supplementary Fig.

利用间接酶联免疫吸附试验（ELISA）检测羊水和胎儿腭突中的牙龈蛋白酶 R1 (RgpA)。如补充图所示。

S1a, b

, the injection of sonicated

，超声处理的注射

P. gingivalis

牙龈卟啉单胞菌

resulted in a significant elevation of RgpA levels in the amniotic fluid and fetal palate tissue, with a progressive increase observed between E13.5 and E15.5. We then evaluated the impact of sonicated

导致羊水和胎儿腭组织中RgpA水平显著升高，并在E13.5至E15.5之间观察到逐步增加。随后我们评估了超声处理的影响。

P. gingivalis

牙龈卟啉单胞菌

exposure on palate development. In the sonicated

腭部发育暴露。在超声处理的

P. gingivalis

牙龈卟啉单胞菌

-treated group, CP (12.1%, 4 out of 33 embryos) was evident at E16.5, revealed by direct observation under a stereoscope and in histological sections stained with hematoxylin and eosin (HE) (Fig.

-处理组，在E16.5天通过体视镜直接观察以及苏木精和伊红（HE）染色的组织学切片中发现CP（12.1％，33个胚胎中有4个）（图。

1b–d

, Supplementary Table

补充表格

, Supplementary Table

，补充表格

）。

Fig. 1

图1

In vivo evaluation of palatal phenotypes under sonicated

超声处理下腭部表型的体内评估

P. gingivalis

牙龈卟啉单胞菌

treatment.

治疗。

Experimental design and time schedule of the study.

研究的实验设计和时间安排。

Lateral and occlusal views and histological observation of mice palates after treatment with or without sonicated

有或没有超声处理的小鼠腭部的侧面和咬合面视图及组织学观察

P. gingivalis

牙龈卟啉单胞菌

(P. g). The arrowheads indicate the cleft. Bar, 200 μm. PS represents palatal shelves and T represents togue.

(P. g)。箭头指示裂缝。比例尺，200微米。PS代表腭架，T代表舌头。

，

Frequency of cleft palate in P. g group compared with Control group,

P. g 组与对照组相比，腭裂的发生频率，

value was calculated by Fisher’s exact test.

该值通过Fisher精确检验计算得出。

Sequential histological sections from E13.5 to E15.5, black arrows indicate the MES, red arrows indicate osteogenic center. Bar, 200 μm. The frequency of no mesenchymal confluence at E15 (

从E13.5到E15.5的连续组织学切片，黑色箭头指示MES，红色箭头指示成骨中心。比例尺，200微米。在E15时未见间充质汇合的频率（

) and the frequency of cleft in control and P. g mice at E15.5 (

) 以及在 E15.5 时对照组和 P. g 小鼠中裂缝的频率 (

）。

，

Masson staining and statistical analysis (

Masson染色和统计分析(

= 3). red arrows indicate osteogenic center. Bar, 200 μm. The data are shown as the mean ± SD and were statistically analyzed by two-tailed Student’s

= 3)。红色箭头指示成骨中心。比例尺，200 μm。数据以均值±标准差表示，并通过双尾Student’s进行统计分析。

-test (

)

Full size image

全尺寸图像

To better understand the moment of cleft origin and the persistence of the phenotypes, we further analyzed sequential histological sections from E13.5 to E15.5 (Fig.

为了更好地了解裂隙起源的时刻和表型的持续性，我们进一步分析了从E13.5到E15.5的连续组织学切片（图。

). At E13.5, the palatal shelves grew vertically adjacent to the tongue. By E14.5, the secondary palatal shelves started to meet at the middle of the palate. At this point, no abnormal palatal phenotypes were observed under

). 在E13.5时，腭突垂直生长在舌头旁边。到E14.5时，次级腭突开始在腭中部相遇。此时，在以下情况下未观察到异常的腭表型：

P. gingivalis

牙龈卟啉单胞菌

exposure. However, at E15.0, unlike in the control palates when the midline epithelial remnants started to degrade and partial mesenchymal confluence was achieved, the epithelial remnants in the

暴露。然而，在E15.0时，与对照组腭部中线表皮残留物开始降解并实现部分间充质融合不同，表皮残留物在

P. gingivalis

牙龈卟啉单胞菌

-treated group were still present at the midline without any mesenchymal confluence (Fig.

-处理组在中线处仍然存在，没有任何间充质汇合（图。

1e, f

). By E15.5, while complete fusion of the secondary palatal shelves had occurred in the control palate, the fusion failed in the

). 到E15.5时，虽然在对照组的腭中，次级腭架已完全融合，但在实验组中，融合失败了。

P. gingivalis

牙龈卟啉单胞菌

-treated group with scattered epithelial cell fragments at the interface (Fig.

- 治疗组在界面处有散在的上皮细胞碎片（图。

1e, g

). It appeared that the palatal shelves, which made contact at E15.0, became detached by E15.5.

). 似乎在E15.0时接触的腭突在E15.5时脱离了。

Moreover, HE staining results indicated a distinct osteogenic center in the E15.5 control palate, which was absent in the

此外，HE染色结果显示，在E15.5对照组腭中有一个明显的成骨中心，而在实验组中缺失。

P. gingivalis

牙龈卟啉单胞菌

-treated palate (Fig.

- 处理过的上颚（图。

). To investigate whether

）。为了调查是否

P. gingivalis

牙龈卟啉单胞菌

exposure affected palate bone formation later in development, we compared the bone area of the palate using Masson staining. Both Masson staining and statistical analysis revealed a significant decrease in bone areas in the E15.5

暴露影响了后期发育中的腭骨形成，我们使用Masson染色比较了腭的骨面积。Masson染色和统计分析均显示E15.5时骨面积显著减少。

P. gingivalis

牙龈卟啉单胞菌

-treated palate compared with the control (Fig.

-与对照组相比的处理过的上颚（图。

1h, i

1小时，我

）。

In summary, no morphological differences were observed in the

总之，没有观察到形态学差异。

P. gingivalis

牙龈卟啉单胞菌

-treated mice by E14.5. However, disruption of the epithelial fusion began around E15.0, and the palatal phenotypes became evident from E15.5 onwards with disturbed palate shelves fusion and osteogenesis.

在E14.5时处理小鼠。然而，上皮融合的破坏大约从E15.0开始，腭部表型从E15.5开始显现，伴有腭架融合和骨化过程的异常。

MEPM cells migration and osteogenesis were inhibited under

MEPM细胞的迁移和成骨作用受到抑制

P. gingivalis

牙龈卟啉单胞菌

exposure

曝光

MEPM cells migrate to the osteogenic region and undergo proliferation and differentiation to form the bone structures of the palate. To explore the mechanism of the abnormal osteogenesis induced by sonicated

MEPM细胞迁移到成骨区域并进行增殖和分化，以形成腭骨结构。为了探索超声处理诱导的异常成骨机制

P. gingivalis

牙龈卟啉单胞菌

, we further investigated its impact on the biological behavior of MEPM cells in vitro and in vivo. Using a Cell Counting Kit-8 (CCK-8) assay, we observed that sonicated

，我们进一步研究了它对MEPM细胞体外和体内生物学行为的影响。通过细胞计数试剂盒-8（CCK-8）实验，我们观察到超声处理后的

P. gingivalis

牙龈卟啉单胞菌

(at concentrations of 0, 0.25, 0.5, and 1 μg/mL) stimulated MEPM cell proliferation, with the magnitude of the effect corresponding to the concentration and a significant effect was achieved at a concentration of 0.5 μg/mL (Fig.

（在 0、0.25、0.5 和 1 μg/mL 的浓度下）刺激了 MEPM 细胞增殖，其效果的大小与浓度相对应，并且在 0.5 μg/mL 的浓度下达到了显著效果（图。

). Flow cytometry analyses further showed that sonicated

）。流式细胞术分析进一步表明，超声处理的

P. gingivalis

牙龈卟啉单胞菌

inhibited MEPM cell apoptosis (Fig.

抑制了MEPM细胞凋亡（图。

2b, c

2b，c

). Next, proliferating cell nuclear antigen (PCNA) immunohistochemical staining was utilized to evaluate the proliferation of MEPM cells in vivo. Consistent with in vitro findings, our results revealed an upregulation of PCNA expression in the palatal mesenchyme following treatment with sonicated

). 随后，使用增殖细胞核抗原（PCNA）免疫组织化学染色来评估MEPM细胞在体内的增殖情况。与体外研究结果一致，我们的结果显示，经超声处理后，腭间充质中PCNA表达上调。

P. gingivalis

牙龈卟啉单胞菌

(Fig.

（图。

2d, e

2d，e

). Additionally, the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also employed to detect apoptotic cells. We observed a reduction in apoptotic cells within the

此外，还采用了末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）测定法来检测凋亡细胞。我们观察到在

P. gingivalis

牙龈卟啉单胞菌

-exposed mesenchyme (Fig.

暴露的间充质（图。

2f, g

), whereas an increase in epithelial apoptosis was observed around the MES (Fig.

)，而围绕MES观察到上皮细胞凋亡增加（图。

2f, h

2f，h

). These results suggest that osteogenic disorders in mesenchyme caused by sonicated

这些结果表明，由声波引起的间充质骨生成障碍

P. gingivalis

牙龈卟啉单胞菌

were not due to a deficiency in osteoprogenitors.

不是由于骨前体细胞的缺乏。

Fig. 2

图2

Exposure to

暴露于

P. gingivalis

牙龈卟啉单胞菌

(P. g) facilitated the proliferation of MEPM cells and inhibited their apoptosis.

(P. g) 促进了 MEPM 细胞的增殖并抑制了它们的凋亡。

The growth curve of MEPM cells in P. g of different concentrations (

不同浓度的P.g中MEPM细胞的生长曲线(

= 6, **

< 0.01).

< 0.01)。

AnnexinV-FITC/PI flow cytometric analysis and

AnnexinV-FITC/PI流式细胞术分析和

statistical data of MEPM cell apoptosis (

MEPM细胞凋亡的统计学数据 (

= 3).

= 3)。

，

PCNA IHC in E15.5 fetal palate from mice treated with or without P. g, with quantification of positive cells (right panel,

PCNA免疫组化检测E15.5胎鼠腭部，使用或不使用P. g处理，并对阳性细胞进行量化（右图），

= 5). Bar, 100 μm.

= 5）。条形图，100 μm。

，

TUNEL assay in E15.5 fetal palate from mice treated with or without P. g, with quantification of positive cells (right panel,

TUNEL 检测 E15.5 小鼠胎儿腭部（经 P. g 处理或未处理），并对阳性细胞进行量化（右侧面板，

= 3). Bar, 100 μm.

= 3)。标尺，100 μm。

我

Scratch assay for cell migration and cell migration rate was quantified by calculating the wound area (

通过计算伤口面积量化细胞迁移和细胞迁移率的划痕实验

) (

= 9). The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 9）。数据显示为平均值 ± 标准差，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

) or two-tailed Student’s

）或双尾Student’s

-test (

，

). All the

). 所有的

values were two-sided and adjustments were made for multiple comparisons

值是双侧的，并对多重比较进行了调整。

Full size image

全尺寸图像

We then used the concentration of 0.5 μg/mL in subsequent in vitro experiments. Scratch assay results revealed that

我们随后在后续的体外实验中使用了0.5 μg/mL的浓度。划痕实验结果显示，

P. gingivalis

牙龈卟啉单胞菌

inhibited MEPM cells migration (Fig.

抑制了MEPM细胞的迁移（图。

2I, j

2我，j

). Both Alkaline phosphatase (ALP) staining (Fig.

）。碱性磷酸酶（ALP）染色（图。

3a, b

), an early marker of osteogenesis and alizarin red staining (ARS) (Fig.

），骨生成的早期标志物和茜素红染色（ARS）（图。

3c, d

3c，d

), the late osteogenic indicator displayed blocked osteogenic differentiation under sonicated

），晚期成骨指标在超声处理下显示出成骨分化受阻

P. gingivalis

牙龈卟啉单胞菌

exposure after MEPM cells osteogenic induction for 7 and 28 days, along with a decreased osteogenesis indicators such as osteocalcin (

MEPM细胞成骨诱导7天和28天后暴露，同时成骨指标如骨钙素减少 (

Ocn

海洋

), osterix (

Osx

Alp

阿尔卑斯山

, runt related transcription factor 2 (

，runt相关转录因子2 (

Runx2

) (Supplementary Fig.

) （补充图。

). These data suggest that

）。这些数据表明

P. gingivalis

牙龈卟啉单胞菌

inhibited the osteogenic capacity of MEPM cells, consistent with in vivo phenotypes.

抑制了MEPM细胞的成骨能力，与体内表型一致。

Fig. 3

图 3

Sonicated

超声处理

P. gingivalis

牙龈卟啉单胞菌

(P. g) induced osteogenic inhibition is related to decreased TGFBR1 in MEPM cells.

(P. g) 诱导的成骨抑制与 MEPM 细胞中 TGFBR1 的减少有关。

，

The expression levels of ALP were detected using ALP staining and ALP assay (

使用ALP染色和ALP测定法检测ALP的表达水平 (

= 5).

= 5）。

Alizarin red S staining on 28 days and

28天的茜素红S染色和

the calcium concentration was determined by measuring the absorbance at 562 nm on a multiplate reader as shown in the bottom panel (

通过在多孔板读数仪上测量562 nm处的吸光度来确定钙浓度，如底部面板所示（

= 3). Bar, 500 μm.

= 3). 条形图，500 微米。

Western blot assay of the protein in classical TGFβ pathway.

经典TGFβ通路中蛋白质的Western blot检测。

，

TGFBR1 IHC in fetal palate from mice treated with or without P. g, with quantification of positive cells (right panel,

用或不用P. g处理的小鼠胎儿腭部的TGFBR1免疫组化，以及阳性细胞的定量分析（右图），

= 6). Bar, 100 μm.

= 6)。标尺，100 μm。

，

我

The expression levels of ALP were detected using ALP staining and ALP assay (

使用ALP染色和ALP测定法检测ALP的表达水平 (

= 5). Bar, 500 μm.

= 5)。标尺，500 μm。

Alizarin red S staining on 28 days and

28天时的茜素红S染色和

the calcium concentration was determined by measuring the absorbance at 562 nm on a multiplate reader (

通过在多孔板读数仪上测量562 nm处的吸光度来确定钙浓度 (

= 3). Bar, 500 μm. The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 3)。标尺，500 μm。数据以均值±标准差表示，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

我

，

) or two-tailed Student’s

）或双尾Student’s

-test (

，

). All the

`).` 所有的

values were two-sided and adjustments were made for multiple comparisons

这些值是双侧的，并且对多重比较进行了调整。

Full size image

全尺寸图像

Osteogenic inhibition induced by

骨生成抑制诱导

P. gingivalis

牙龈卟啉单胞菌

is related to decreased TGFBR1

与TGFBR1减少有关

Given our prior confirmation of TGFBR1’s crucial role in palatal osteogenic differentiation,

鉴于我们之前已确认TGFBR1在腭部成骨分化中的关键作用，

，

we focused on mediators closely associated with the classical TGFβ pathway. We found that the protein levels of TGFBR1, p- Suppressor of mother against decapentaplegic 2 (p-SMAD2), and p-SMAD3 were significantly downregulated upon MEPM cells exposure to sonicated

我们重点关注与经典TGFβ通路密切相关的介质。我们发现，在MEPM细胞暴露于超声处理后，TGFBR1、p-SMAD2（对抗decapentaplegic 2的抑制子的磷酸化形式）和p-SMAD3的蛋白水平显著下调。

P. gingivalis

牙龈卟啉单胞菌

(Fig.

（图。

, Supplementary Fig.

，补充图。

S3a

). This downregulation of TGFBR1, particularly in regions of palatal osteogenesis, was further confirmed in vivo using immunohistochemistry (Fig.

). 免疫组织化学进一步证实了TGFBR1的下调，尤其是在腭骨发生区域（图。

3f, g

3楼，g

）。

To further illustrate TGFBR1’s association with the abnormal osteogenesis caused by sonicated

为了进一步说明TGFBR1与超声处理引起的异常骨生成之间的关联

P. gingivalis

牙龈卟啉单胞菌

, we introduced TGFβ2 in vitro to activate TGFBR1 because TGFβ2 is mainly expressed in the palatal mesenchyme.

，我们在体外引入了TGFβ2来激活TGFBR1，因为TGFβ2主要在腭中胚层中表达。

ALP assay and ARS showed that the addition of 10 ng/mL TGFβ2 could partially reverse the inhibition of osteogenesis induced by sonicated

ALP检测和ARS显示，添加10 ng/mL的TGFβ2可以部分逆转由超声处理引起的成骨抑制。

P. gingivalis

牙龈卟啉单胞菌

(Fig.

（图。

3h–k

3小时-k

). This indicates that the abnormal osteogenic differentiation induced by sonicated

这表明由超声波引起的异常成骨分化

P. gingivalis

牙龈卟啉单胞菌

is associated with the TGF-β pathway through TGFBR1.

与TGF-β通路通过TGFBR1相关联。

Sonicated

超声处理

P. gingivalis

牙龈卟啉单胞菌

induced TGFBR1 cleavage in MEPM cells by upregulating H4K12la/ADAM17

通过上调H4K12la/ADAM17诱导MEPM细胞中TGFBR1的裂解

Recently glucose metabolic reprogramming was reported important during embryonic development esp. in neural crest.

最近有报道称，葡萄糖代谢重编程在胚胎发育过程中尤为重要，尤其是在神经嵴中。

，

To investigate if the reduction in TGFBR1 was related to glucose metabolic reprogramming, we first evaluated the key mediators of glycolysis and OXPHOS. Western blot analysis revealed that hexokinase-2 (HK2) and lactate dehydrogenase A (LDHA), markers of glycolysis, were upregulated after treatment with sonicated .

为了研究TGFBR1的减少是否与葡萄糖代谢重编程有关，我们首先评估了糖酵解和氧化磷酸化（OXPHOS）的关键介质。蛋白质印迹分析显示，经超声处理后，糖酵解标志物己糖激酶-2（HK2）和乳酸脱氢酶A（LDHA）的表达上调。

P. gingivalis

牙龈卟啉单胞菌

. In contrast, cytochrome c (CytC), a marker of OXPHOS, was downregulated following sonicated

相比之下，作为OXPHOS标志物的细胞色素c（CytC）在超声处理后被下调。

P. gingivalis

牙龈卟啉单胞菌

treatment (Fig.

治疗（图。

, Supplementary Fig.

，补充图。

S3b

). Additionally, the lactate level was significantly increased in the sonicated

此外，超声处理后乳酸水平显著增加。

P. gingivalis

牙龈卟啉单胞菌

group (Fig.

组（图。

). These results demonstrated that

）。这些结果表明

P. gingivalis

牙龈卟啉单胞菌

reversed metabolic reprogramming from OXPHOS to glycolysis in MEPM cells.

在MEPM细胞中，代谢重编程从氧化磷酸化逆转为糖酵解。

Fig. 4

图4

Sonicated

超声处理

P. gingivalis

牙龈卟啉单胞菌

(P. g) induced TGFBR1 cleavage in MEPM cells by upregulating H4K12la/ADAM17.

(P. g) 通过上调 H4K12la/ADAM17 诱导 MEPM 细胞中 TGFBR1 的切割。

Western blot assay of the markers in glycolysis and OXPHOs in MEPM cells treated with or without P. g.

用或不用P. g.处理的MEPM细胞中糖酵解和OXPHOs标志物的Western blot分析。

The production of lactate (

乳酸的产生 (

= 4).

= 4）。

Western blot analysis of Pan- and site-specific histone lactylation, with quantification of protein levels as shown in (

西方墨点法分析泛素和位点特异性组蛋白乳酸化，并对蛋白质水平进行量化，如图所示 (

) (

= 3).

= 3)。

Western blot analysis of H4K12la and H3K18la after added P. g and (or) FX-11, with quantification of protein levels as shown in (

添加P. g和（或）FX-11后，对H4K12la和H3K18la进行蛋白质印迹分析，并对蛋白水平进行量化，如图所示（

) (

= 3).

= 3)。

Western blot analysis of TGFBR1, with quantification of protein levels as shown in (

TGFBR1的蛋白质印迹分析，蛋白质水平的定量如图所示 (

) (

= 3).

= 3)。

我

ChIP-qPCR analysis of the ADAM17 promoters was performed using antibodies against H4K12la in MEPM cells (

使用针对H4K12la的抗体在MEPM细胞中进行了ADAM17启动子的ChIP-qPCR分析 (

= 3).

= 3)。

Western blot analysis of ADAM17 after added P. g and (or) FX-11, with quantification of protein levels as shown in (

添加P. g和（或）FX-11后对ADAM17进行Western blot分析，并对蛋白质水平进行量化，如图所示（

) (

= 3). The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 3）。数据显示为平均值 ± 标准差，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

) or two-tailed Student’s

）或双尾Student’s

-test (

，

我

). All the

）。所有

values were two-sided and adjustments were made for multiple comparisons

这些值是双侧的，并且对多重比较进行了调整。

Full size image

全尺寸图像

Currently histone lactylation induced by lactate is a key epigenetic regulation,

目前，乳酸诱导的组蛋白乳酸化是一种关键的表观遗传调控，

we hypothesized that histone lactylation might be altered under sonicated

我们假设组蛋白乳酸化可能在超声处理下发生改变。

P. gingivalis

牙龈卟啉单胞菌

exposure which was confirmed by western blot analysis with an increase of histone lactylation, specifically H4K12la and H3K18la (Fig.

通过蛋白质印迹分析证实了暴露增加，组蛋白乳酸化增加，特别是 H4K12la 和 H3K18la（图。

4c, d

4c，d

）。

Next, we explored the biological function of histone lactylation induced by sonicated

接下来，我们探讨了由超声诱导的组蛋白乳酸化修饰的生物学功能。

P. gingivalis

牙龈卟啉单胞菌

in MEPM cells. We adjusted the level of histone lactylation in MEPM cells by treating them with sonicated

在MEPM细胞中，我们通过用超声处理调整了MEPM细胞中组蛋白乳酸化水平。

P. gingivalis

牙龈卟啉单胞菌

and/or the lactate dehydrogenase inhibitor, FX-11. As expected, the elevated level of H4K12la caused by sonicated

和/或乳酸脱氢酶抑制剂FX-11。正如预期的那样，由超声处理引起的H4K12la水平升高

P. gingivalis

牙龈卟啉单胞菌

was effectively inhibited by 9 μmol/L FX-11, while changes in H3K18la were not significant (Fig.

被9 μmol/L的FX-11有效抑制，而H3K18la的变化不显著（图。

4e, f

). We then focused on H4K12la in subsequent experiments. Importantly, the inhibition of H4K12la could rescue the downregulation of TGFBR1 induced by sonicated

）。随后我们在后续实验中重点关注了H4K12la。重要的是，抑制H4K12la可以挽救由超声处理引起的TGFBR1下调。

P. gingivalis

牙龈卟啉单胞菌

, suggesting a potential causal link between H4K12la and TGFBR1 (Fig.

，提示H4K12la和TGFBR1之间可能存在潜在的因果关系（图。

4g, h

4克，小时

）。

To determine whether a direct relationship exists between these factors, we examined MEPM cells with ChIP-qPCR using an antibody against H4K12la. The promoter of

为了确定这些因素之间是否存在直接关系，我们使用针对H4K12la的抗体通过ChIP-qPCR检测了MEPM细胞。启动子区域

Tgfbr1

was not detected after treatment with the anti-H4K12la antibody, indicating there might not be a direct interaction between them (Supplementary Fig.

在使用抗H4K12la抗体处理后未被检测到，表明它们之间可能不存在直接相互作用（补充图）。

）

Given that ADAM17 has been previously reported as a protease that cleaves the extracellular region of TGFBR1, and it might be activated in inflammatory or high-glucose environments.

鉴于ADAM17之前已被报道为一种能够切割TGFBR1细胞外区域的蛋白酶，并且它可能在炎症或高糖环境中被激活。

，

Consequently, we aimed to examine the link between histone lactylation and ADAM17 to determine if ADAM17 mediated the indirect regulation of TGFBR1 by H4K12la. Indeed, ChIP-qPCR demonstrated an increase in H4K12la enrichment in the promoter region of

因此，我们旨在研究组蛋白乳酸化与ADAM17之间的联系，以确定ADAM17是否介导了H4K12la对TGFBR1的间接调控。事实上，ChIP-qPCR显示H4K12la在启动子区域的富集增加。

Adam17

(Fig.

（图。

). Consistently, western blot confirmed the upregulation of ADAM17 expression after sonicated

`). 一致地，蛋白质印迹证实了超声处理后 ADAM17 表达的上调。`

P. gingivalis

牙龈卟啉单胞菌

treatment (Fig.

治疗（图。

4j, k

4j，k

). Collectively, these results indicate that H4K12la modification activates the transcription of

`).总的来说，这些结果表明H4K12la修饰激活了`

Adam17

in MEPM cells.

在MEPM细胞中。

Lastly, we further investigated the distribution and intensity of H4K12la, ADAM17, and TGFBR1 during palate development in vivo. Consistent with our in vitro results, in vivo immunofluorescence findings showed that H4K12la and ADAM17 had similar distributions, with a substantial amount of co-localization (Fig.

最后，我们进一步研究了H4K12la、ADAM17和TGFBR1在腭发育过程中的分布和强度。与我们的体外结果一致，体内免疫荧光结果显示H4K12la和ADAM17具有相似的分布，并且存在大量的共定位（图。

。

). The signal intensity of H4K12la and ADAM17 was significantly increased in mice treated with sonicated

）。在经过超声处理的小鼠中，H4K12la 和 ADAM17 的信号强度显著增加，

P. gingivalis

牙龈卟啉单胞菌

(Fig.

（图。

, Supplementary Fig.

，补充图。

S5a, b

S5a，b

). Furthermore, a time-course analysis revealed that the signal intensities of H4K12la and ADAM17 gradually decreased with embryonic development in normal mice, whereas these markers remained at a consistently high level in mice treated with sonicated

此外，时间进程分析显示，在正常小鼠中，H4K12la 和 ADAM17 的信号强度随着胚胎发育逐渐降低，而在经过超声处理的小鼠中，这些标志物始终保持在较高水平。

P. gingivalis

牙龈卟啉单胞菌

(Supplementary Fig.

（补充图。

S5a, b

S5a，b

）。

Fig. 5

图5

Co-staining of H4K12la/ADAM17/TGFBR1 in palate tissue.

腭组织中H4K12la/ADAM17/TGFBR1的共染色。

The distribution and intensity of H4K12la and ADAM17 during palate development. Bar, 250 μm.

腭发育过程中H4K12la和ADAM17的分布与强度。标尺，250微米。

Co-staining of H4K12la/ADAM17/TGFBR1 at E15.5. P. g represents

E15.5时H4K12la/ADAM17/TGFBR1的共染色。P. g 代表

P. gingivalis.

牙龈卟啉单胞菌。

Bar, 100 μm

条形，100微米

Full size image

全尺寸图像

Specifically, at the primary osteogenic site during the osteogenic stage (E15.5), we observed that TGFBR1-positive cells expressed lower levels of H4K12la and ADAM17 in the normal palate. In contrast, in the sonicated

具体来说，在成骨阶段（E15.5）的初级成骨部位，我们观察到在正常腭中，TGFBR1阳性细胞表达较低水平的H4K12la和ADAM17。相反，在超声处理后的腭中，

P. gingivalis

牙龈卟啉单胞菌

-treated CP, the number of TGFBR1-positive cells significantly decreased due to the continuous expression of H4K12la and ADAM17 (Fig.

-处理后的CP，由于H4K12la和ADAM17的持续表达，TGFBR1阳性细胞的数量显著减少（图。

）。

These findings suggest that the increase in H4K12la induced by sonicated

这些发现表明，由超声诱导的 H4K12la 增加

P. gingivalis

牙龈卟啉单胞菌

promotes the expression of ADAM17 which further cleaves TGFBR1, thereby inhibiting osteogenic differentiation in MEPM cells and inducing abnormal palate development.

促进ADAM17的表达，ADAM17进一步切割TGFBR1，从而抑制MEPM细胞的成骨分化并诱导腭发育异常。

Decreased MerTK in Mφs under sonicated

超声处理下 Mφs 中 MerTK 减少

P.gingivalis

牙龈卟啉单胞菌

exposure caused abnormal palate shelves fusion

曝露导致腭板融合异常

In addition to osteogenic abnormalities, as previously mentioned, fetal mice treated with sonicated

除了之前提到的成骨异常之外，经过超声处理的胎鼠

P. gingivalis

牙龈卟啉单胞菌

also displayed persistent epithelial remnants at the fusion stage which is usually medial edge epithelial (MEE) cells, leading to abnormal palatal fusion (Fig.

在融合阶段还显示出持续的上皮残留，这通常是中缘上皮（MEE）细胞，导致腭部融合异常（图。

). To investigate the reason of the abnormity, we then evaluated the effect of sonicated

）。为了调查异常的原因，我们随后评估了超声处理的效果。

P. gingivalis

牙龈卟啉单胞菌

on the apoptosis of MEE. Using flow cytometry analysis, we observed that sonicated

关于MEE的凋亡。通过流式细胞术分析，我们观察到超声处理后的

P. gingivalis

牙龈卟啉单胞菌

promoted apoptosis in MEE cells (Fig.

促进MEE细胞凋亡（图。

6a, b

6a，b

). This suggests that the abnormal fusion induced by sonicated

）。这表明超声处理诱导的异常融合

P. gingivalis

牙龈卟啉单胞菌

may not be due to a reduction in MEE apoptosis.

可能不是由于MEE凋亡减少所致。

Fig. 6

图6

Sonicated

超声处理

P. gingivalis

牙龈卟啉单胞菌

(P. g) induced abnormal fusion is related to decreased MerTK in macrophages.

(P. g) 诱导的异常融合与巨噬细胞中 MerTK 的减少有关。

，

AnnexinV-FITC/PI flow cytometric analysis and statistical data of MEE cell apoptosis (

AnnexinV-FITC/PI流式细胞术分析和MEE细胞凋亡的统计数据（

= 3).

= 3)。

Co-staining of MerTK positive macrophage (green) in TUNEL-positive regions (red), with

在TUNEL阳性区域（红色）中对MerTK阳性巨噬细胞（绿色）进行共染色，

quantification of Free/MerTK-associated apoptotic cells (

游离/MerTK相关凋亡细胞的定量分析 (

= 3). Free apoptotic cells, white arrows. MerTK associated apoptotic cells, white arrowheads. Bar, 100 μm.

= 3）。游离凋亡细胞，白色箭头。MerTK 相关凋亡细胞，白色箭头头。标尺，100 μm。

Western blot analysis of MerTK in Raw 264.7 macrophage, with quantification of protein levels in (

用Western blot分析Raw 264.7巨噬细胞中的MerTK，并对蛋白质水平进行定量(

) (

= 3).

= 3)。

，

Representative fluorescent images showing engulfing of apoptotic MEE cells by Raw 264.7 macrophages in vitro and phagocytic index based on the fluorescent image (

显示Raw 264.7巨噬细胞在体外吞噬凋亡的MEE细胞的代表性荧光图像，以及基于荧光图像的吞噬指数（

= 6). The data are shown as the mean ± SD and were statistically analyzed by two-tailed Student’s

= 6）。数据显示为平均值 ± 标准差，并通过双尾 Student's t 检验进行统计分析。

-test

-测试

Full size image

全尺寸图像

Subsequently, we performed immunofluorescence staining of tissue sections at E15 to examine apoptotic MEE in vivo. TUNEL assay revealed clear apoptosis signals in the midline epithelium of palate shelves in both the control and sonicated

随后，我们对 E15 时期的组织切片进行了免疫荧光染色，以在体内检查 MEE 的凋亡情况。TUNEL 检测显示，在对照组和超声处理组的腭架中线表皮中均观察到明显的凋亡信号。

P. gingivalis

牙龈卟啉单胞菌

-treated groups. However, in the control group, apoptotic cells were predominantly located in MerTK-positive areas, whereas the ratio of free to MerTK-associated apoptotic cells significantly increased in the sonicated

-处理组。然而，在对照组中，凋亡细胞主要位于MerTK阳性区域，而自由凋亡细胞与MerTK相关凋亡细胞的比例在超声处理组中显著增加。

P. gingivalis

牙龈卟啉单胞菌

-treated group (Fig.

-处理组（图。

6c, d

6c，d

). As MerTK plays a crucial role in binding molecules on the apoptotic cell surface or binding bridging molecules that interact with the apoptotic cell surface during efferocytosis, these results imply that sonicated

）。由于MerTK在结合凋亡细胞表面的分子或在胞葬作用过程中与凋亡细胞表面相互作用的桥接分子结合方面起着关键作用，这些结果表明超声处理过的

P. gingivalis

牙龈卟啉单胞菌

may induce inefficient efferocytosis by decreasing MerTK expression in Mφs.

可能通过降低巨噬细胞中MerTK的表达来诱导低效的胞葬作用。

We further investigated whether the expression of the MerTK in Raw 264.7 and isolated mice Bone marrow derived-macrophages (BMDM) was affected by sonicated

我们进一步研究了Raw 264.7细胞和分离的小鼠骨髓来源巨噬细胞（BMDM）中MerTK的表达是否受到超声处理的影响。

P. gingivalis

牙龈卟啉单胞菌

in vitro. As shown in Fig.

体外。如图所示。

6e, f

and Supplementary Fig.

和补充图。

S6a

, MerTK expression decreased when exposed to sonicated

，当暴露于超声处理时，MerTK 表达下降

P. gingivalis

牙龈卟啉单胞菌

. Given that BMDM cells are not amenable to passage and are unsuitable for extended culture periods, we predominantly selected Raw264.7 macrophages for subsequent experimental procedures.

鉴于BMDM细胞不易传代且不适合长时间培养，我们主要选择Raw264.7巨噬细胞用于后续实验步骤。

We then explored the potential impact of sonicated

我们接着探讨了超声处理的潜在影响

P. gingivalis

牙龈卟啉单胞菌

on Mφ efferocytosis in vitro. After the addition of apoptotic cells, Raw 264.7 cells in the control group displayed an enlarged cellular surface area along with the formation of additional filopodia-like structures, which might act as phagocytic tentacles to facilitate phagocytosis. Conversely, in the sonicated .

在体外对Mφ吞噬凋亡细胞的影响。在加入凋亡细胞后，对照组中的Raw 264.7细胞显示出细胞表面区域扩大，并伴随形成额外的丝状伪足样结构，这些结构可能充当吞噬触手以促进吞噬作用。相反，在超声处理组中。

P. gingivalis

牙龈卟啉单胞菌

group, Raw 264.7 cells exhibited a dystrophic morphology, and the phagocytic index, indicating Mφ efferocytosis efficiency, was significantly decreased (Fig.

组中，Raw 264.7 细胞表现出营养不良的形态，且表明 Mφ 清除效率的吞噬指数显著降低（图。

6g, h

6克，h

). Therefore, our data indicate that the abnormal palatal fusion induced by sonicated

因此，我们的数据表明，由超声波处理引起的腭融合异常。

P. gingivalis

牙龈卟啉单胞菌

may be related to decreasing MerTK which causes inefficient efferocytosis of Mφs.

可能与MerTK减少有关，这会导致巨噬细胞（Mφs）的胞葬作用效率低下。

Sonicated

超声处理的

P. gingivalis

牙龈卟啉单胞菌

induced MerTK cleavage in Mφs through upregulating H4K12la/ADAM17

通过上调 H4K12la/ADAM17 诱导 Mφs 中的 MerTK 裂解

To investigate whether the decrease of MerTK was related to glucose metabolic reprogramming and histone lactylation, we identified the changes of glucose metabolism, lactate levels, and histone lactylation in Raw 264.7 cells after sonicated

为了研究MerTK的减少是否与葡萄糖代谢重编程和组蛋白乳酸化有关，我们检测了声波处理后Raw 264.7细胞中葡萄糖代谢、乳酸水平和组蛋白乳酸化的变化。

P. gingivalis

牙龈卟啉单胞菌

treatment similar to the MEPM cells. Our results indicated an increase in the expressions of glycolytic mediators, HK2 and LDHA, a decrease in the expression of Cytc (OXPHOS mediator), and higher levels of lactate and histone lactylation in Raw 264.7 cells treated with sonicated

与MEPM细胞相似的处理。我们的结果显示，糖酵解介质HK2和LDHA的表达增加，Cytc（OXPHOS介质）的表达减少，并且在经过超声处理的Raw 264.7细胞中乳酸和组蛋白乳酸化水平更高。

P. gingivalis

牙龈卟啉单胞菌

, especially H4K5la and H4K12la (Fig.

，特别是 H4K5la 和 H4K12la（图。

7a–e

）。

Fig. 7

图7

In macrophages, glycolysis and H4K12la were also observed to increase upon exposure to

在巨噬细胞中，暴露于外界刺激后，也观察到糖酵解和H4K12la增加。

P. gingivalis

牙龈卟啉单胞菌

(P. g).

（P. g）。

Western blot assay of the markers in glycolysis and OXPHOs in macrophages treated with or without P. g, with quantification of protein levels as shown in (

用或不用P. g处理的巨噬细胞中糖酵解和OXPHOs标志物的Western blot分析，蛋白质水平的定量如图所示 (

) (

= 3).

= 3)。

The production of lactate (

乳酸的产生 (

= 4).

= 4)。

Western blot analysis of Pan- and site-specific histone lactylation, with quantification of protein levels as shown in (

Western blot分析泛素和位点特异性组蛋白乳酸化，蛋白质水平的定量如图所示 (

) (

= 3).

= 3)。

Western blot analysis of H4K12la and H4K5la after added P. g and (or) FX-11, with (

添加P. g和（或）FX-11后，对H4K12la和H4K5la进行Western blot分析，（

) quantification of protein levels (

蛋白质水平的量化 (

= 3). The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 3）。数据显示为平均值 ± 标准差，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

) or two-tailed Student’s

）或双尾Student’s

-test (

). All the

）。所有

values were two-sided and adjustments were made for multiple comparisons

这些值是双侧的，并且对多重比较进行了调整。

Full size image

全尺寸图像

Subsequently, we introduced FX-11 to manipulate histone lactylation levels and examine the relationship between changes in histone lactylation and MerTK expression. The elevated level of H4K12la induced by sonicated

随后，我们引入了FX-11来调控组蛋白乳酸化水平，并研究组蛋白乳酸化变化与MerTK表达之间的关系。超声处理诱导的H4K12la水平升高

P. gingivalis

牙龈卟啉单胞菌

was effectively inhibited by 9 μmol/L FX-11 (Fig.

被9 μmol/L FX-11有效抑制（图。

7f, g

7f，g

). Moreover, the suppression of H4K12la was able to rescue the downregulation of MerTK and efferocytosis instigated by sonicated

). 此外，抑制H4K12la能够挽救由超声处理引发的MerTK和胞葬作用的下调。

P. gingivalis

牙龈卟啉单胞菌

(Fig.

（图。

8a–d

). This suggests that the decrease of MerTK in Raw 264.7 cells may also be related to H4K12la. Interestingly, previous report has indicated that MerTK is also susceptible to ADAM17 cleavage,

这表明Raw 264.7细胞中MerTK的减少也可能与H4K12la有关。有趣的是，之前的报告指出MerTK也容易受到ADAM17切割的影响，

which helps us speculate that ADAM17 may also mediate the indirect regulation of H4K12la on MerTK. Indeed, our ChIP-qPCR and western blot results confirmed our speculation, suggesting that the elevation of H4K12la induced by sonicated

这帮助我们推测ADAM17也可能介导H4K12la对MerTK的间接调控。确实，我们的ChIP-qPCR和western blot结果证实了我们的推测，表明由超声处理诱导的H4K12la升高

P. gingivalis

牙龈卟啉单胞菌

promoted the expression of ADAM17 (Fig.

促进了ADAM17的表达（图。

8e–g

). This may further inhibit MerTK expression and thus suppress efferocytosis in Mφs. Additionally, treatment of isolated mice BMDM with sonicated

）。这可能会进一步抑制MerTK的表达，从而抑制巨噬细胞中的胞葬作用。此外，用超声处理孤立的小鼠BMDM进行治疗时，

P. gingivalis

牙龈卟啉单胞菌

also resulted in increases in HAK12la and ADAM17 (Supplementary Fig.

同时导致HAK12la和ADAM17的增加（补充图）。

S6b, c

S6b，c

）。

Fig. 8

图8

Sonicated

超声处理

P. gingivalis

牙龈卟啉单胞菌

(P. g) induced MerTK cleavage in Raw 264.7 macrophages by upregulating H4K12la/ADAM17.

(P. g) 通过上调 H4K12la/ADAM17 诱导 Raw 264.7 巨噬细胞中的 MerTK 裂解。

Representative fluorescent images showing engulfing of apoptotic MEE cells by macrophages and (

显示巨噬细胞吞噬凋亡的MEE细胞的代表性荧光图像和(

) phagocytic index based on the fluorescent image (

) 基于荧光图像的吞噬指数 (

= 6).

= 6)。

Western blot analysis of MerTK, with (

Western blot分析MerTK，与(

) quantification of protein levels (n = 3).

蛋白质水平的定量分析（n = 3）。

ChIP-qPCR analysis of the ADAM17 promoters was performed using antibodies against H4K12la in macrophages (

使用针对巨噬细胞中H4K12la的抗体进行了ADAM17启动子的ChIP-qPCR分析（

= 3).

= 3)。

Western blot analysis of ADAM17 after added P. g and (or) FX-11, with (

添加P. g和（或）FX-11后对ADAM17进行Western blot分析，（

) quantification of protein levels (

蛋白质水平的量化

= 3).

= 3)。

Co-staining of H4K12la/ADAM17/MerTK. The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

H4K12la/ADAM17/MerTK的共染色。数据以平均值±标准差表示，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

) or two-tailed Student’s

）或双尾Student’s

-test (

). All the

`).` 所有的

values were two-sided and adjustments were made for multiple comparisons

这些值是双侧的，并且对多重比较进行了调整。

Full size image

全尺寸图像

Moreover, we analyzed the distribution and intensity of H4K12la/ADAM17/MerTK at the palate fusion stage (E15) in vivo. Immunofluorescence results showed that in the normal palate, despite a relatively high level of basal glycolysis at E15, a significant number of cells expressed H4K12la and ADAM17 while MerTK-positive Mφs recruited around the MES expressed lower levels of H4K12la and ADAM17.

此外，我们还分析了 H4K12la/ADAM17/MerTK 在腭融合阶段（E15）的分布和强度。免疫荧光结果显示，在正常腭中，尽管在 E15 时基础糖酵解水平相对较高，但大量细胞表达 H4K12la 和 ADAM17，而围绕 MES 招募的 MerTK 阳性 Mφs 表达较低水平的 H4K12la 和 ADAM17。

In contrast, in the sonicated .

相比之下，在超声处理中。

P. gingivalis

牙龈卟啉单胞菌

group, MerTK-positive cells were rarely distributed around the MES due to the high expression of H4K12la and ADAM17 (Fig.

由于H4K12la和ADAM17的高表达，MerTK阳性细胞很少分布在MES周围（图。

8小时

）

Mφs exposed to sonicated

暴露于超声处理的Mφs

P. gingivalis

牙龈卟啉单胞菌

cannot undergo a phenotypic switch upon efferocytosis during palate fusion

在腭融合过程中，通过胞葬作用无法进行表型转换

Previous studies have demonstrated that successful efferocytosis initiates signals for the resolution of inflammation, leading to a phenotypic switch from the inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

以往的研究表明，成功的胞葬作用会启动炎症消退的信号，导致从促炎的M1表型转换为抗炎的M2表型。

As such, we hypothesized that during palate shelves fusion, Mφs would undergo a phenotypic switch after phagocytizing apoptotic MEE, while inefficient efferocytosis induced by sonicated

因此，我们假设在腭架融合过程中，Mφs在吞噬凋亡的MEE后会经历表型转换，而由超声处理诱导的无效胞葬作用。

P. gingivalis

牙龈卟啉单胞菌

could result in a failed phenotypic switch. To test this hypothesis, we began with in vitro experiments. Before being co-cultured with DiI-labeled apoptotic MEE cells, the control Raw 264.7 cells were in an unpolarized state, while the sonicated

可能导致表型转换失败。为了验证这一假设，我们从体外实验开始。在与DiI标记的凋亡MEE细胞共培养之前，对照组Raw 264.7细胞处于非极化状态，而经超声处理的

P. gingivalis

牙龈卟啉单胞菌

-pretreated Raw 264.7 cells showed M1 polarization. After a 30-min co-culture, the M1 phenotype was dominant in both groups. Two hours post-co-culture, Raw 264.7 cells in the control group switched to M2 as most of the apoptotic MEE cells were cleared. However, in the sonicated

预处理的Raw 264.7细胞显示出M1极化。在共培养30分钟后，两组中M1表型均占主导地位。共培养2小时后，对照组中的Raw 264.7细胞转变为M2，因为大部分凋亡的MEE细胞已被清除。然而，在超声处理组中，

P. gingivalis

牙龈卟啉单胞菌

group, only a few Raw 264.7 cells that recognized and phagocytized apoptotic MEE cells switched to M2 (Supplementary Fig.

群体中，只有少数识别并吞噬了凋亡MEE细胞的Raw 264.7细胞转向M2型（补充图）。

）。

Subsequently, we observed Mφ polarization in vivo. At E15, there was no significant difference between the two groups, possibly due to the continuous stimulation by apoptotic MEE cells (Fig.

随后，我们在体内观察了Mφ极化。在E15时，两组之间没有显著差异，可能是由于凋亡的MEE细胞持续刺激（图。

9a, b

). By E15.5, however, Mφs in the control palate were primarily M2, whereas those in the sonicated

). 然而，到E15.5时，对照组腭部的Mφs主要是M2型，而超声处理组的则是

P. gingivalis

牙龈卟啉单胞菌

group were predominantly M1 (Fig.

该组主要为M1（图。

9a, c

) corroborating our in vitro findings (Supplementary Fig.

`) 证实了我们的体外研究结果（补充图。`

). We hypothesized that the different distributions of M1/M2 Mφs in the palate shelves of the two groups could also differentially influence surrounding MEPM cells through intercellular communication except MEE cells. Given that sEVs are the primary mediators of intercellular communication, we next examined the effects of M1 or M2 Mφs-derived sEVs on osteogenesis, the most distinct phenotype observed at E15.5..

）。我们假设两组腭架中M1/M2巨噬细胞的不同分布也可能通过细胞间通讯差异性地影响周围的MEPM细胞，而非MEE细胞。鉴于sEV是细胞间通讯的主要介质，我们接下来检查了M1或M2巨噬细胞衍生的sEV对骨形成的影响，这是在E15.5观察到的最显著表型。

Fig. 9

图 9

Macrophages exposed to sonicated

暴露于超声处理的巨噬细胞

P. gingivalis

牙龈卟啉单胞菌

(P. g) cannot undergo a phenotypic switch upon efferocytosis during palate fusion, and M2-sEVs promoted MEPM osteogenesis while M1-sEVs inhibited it.

(P. g) 在腭融合过程中无法通过胞葬作用进行表型转换，M2-sEVs 促进 MEPM 成骨，而 M1-sEVs 抑制成骨。

Co-staining of CD86 /CD206 and quantification of CD86/CD206 at

CD86/CD206 的共染色及 CD86/CD206 的定量分析

E15 or

E15 或者

E15.5. Bar, 200 μm (

E15.5。条形图，200微米（

= 3).

= 3)。

Macrophages were polarized into M1 or M2 types in vitro

巨噬细胞在体外被极化为M1或M2型

nd western blot analysis of CD86 and CD206 for identification.

通过CD86和CD206的蛋白质印迹分析进行鉴定。

Western blot analysis of osteogenic protein in MEPM cells treated with M1-sEVs or M2-sEVs, with (

用M1-sEVs或M2-sEVs处理的MEPM细胞中成骨蛋白的Western blot分析，具有 (

) quantification of protein levels (

蛋白质水平的量化 (

= 3).

= 3)。

Alizarin red S staining on 21 days for MEPM cells treated with M1-sEVs or M2-sEVs and

用M1-sEVs或M2-sEVs处理的MEPM细胞在21天进行茜素红S染色

the calcium concentration was determined by measuring the absorbance at 562 nm on a multiplate reader (

通过在多孔板读数仪上测量562 nm处的吸光度来确定钙浓度 (

= 3). The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 3）。数据显示为平均值 ± 标准差，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

) or two-tailed Student’s

）或双尾Student’s

-test (

，

). All the

`).` 所有的

values were two-sided and adjustments were made for multiple comparisons

这些值是双侧的，并且对多重比较进行了调整。

Full size image

全尺寸图像

M2-sEVs promoted MEPM osteogenesis while M1-sEVs inhibited it in vitro

M2-sEVs促进MEPM成骨，而M1-sEVs在体外抑制其成骨。

To investigate whether M1-sEVs or M2-sEVs play a role in MEPM osteogenesis, Raw 264.7 cells were polarized into M1 or M2 types by adding 40 ng/mL of IFN-γ and 20 ng/mL of IL-4, respectively. This significantly increased the expression of the M1 marker, CD86, or the M2 marker, CD206 (Fig.

为了研究M1-sEVs或M2-sEVs是否在MEPM成骨过程中发挥作用，通过分别添加40 ng/mL的IFN-γ和20 ng/mL的IL-4，将Raw 264.7细胞极化为M1型或M2型。这显著增加了M1标志物CD86或M2标志物CD206的表达（图。

9天

). SEVs were then isolated from these M1 or M2 Mφs via ultracentrifugation and identified using transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. As depicted in Supplementary Fig.

通过超速离心从这些M1或M2巨噬细胞中分离SEVs，并使用透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和蛋白质印迹分析进行鉴定。如补充图所示。

S8a

, the vesicles exhibited the typical cup-shaped morphology, with particle diameters ranging from 80 to 170 nm (Supplementary Fig.

，囊泡呈现出典型的杯状形态，颗粒直径范围为80至170纳米（补充图。

S8b

). The inclusive markers of sEVs, TSG101 and CD63, were expressed, whereas the exclusive marker, Calnexin, was not expressed (Supplementary Fig.

）。sEVs的包容性标志物TSG101和CD63有表达，而排他性标志物Calnexin没有表达（补充图。

S8c

）。

To examine the impact of M1-sEVs and M2-sEVs on osteogenesis, MEPM cells were cultured in osteogenic induction medium containing either M1-sEVs or M2-sEVs. The protein levels of osteogenic markers, including bone morphogenetic protein 2 (BMP2), osteopontin (OPN), and Runx2, were measured 14 days post osteogenic induction.

为了研究M1-sEVs和M2-sEVs对成骨作用的影响，将MEPM细胞培养在含有M1-sEVs或M2-sEVs的成骨诱导培养基中。成骨诱导14天后，测量了包括骨形态发生蛋白2（BMP2）、骨桥蛋白（OPN）和Runx2在内的成骨标志物的蛋白质水平。

Among these markers, Runx2 expression was significantly inhibited by M1-sEVs but promoted by M2-sEVs in comparison to the control (Fig. .

在这些标志物中，与对照组相比，M1-sEVs显著抑制了Runx2的表达，而M2-sEVs则促进了其表达（图。

9e, f

). ARS and quantitative calcium measurements displayed that mineralization was reduced in MEPM cells cultured with M1-sEVs compared to the control group at 21 days post osteogenic induction, 21 days following osteogenic induction. In contrast, cells cultured with M2-sEVs showed a significant increase in mineralization (Fig.

). ARS和定量钙测量显示，与对照组相比，在成骨诱导后21天，用M1-sEVs培养的MEPM细胞矿化减少，成骨诱导后21天。相反，用M2-sEVs培养的细胞显示出显著增加的矿化（图。

。

9g, h

9克，h

). These findings demonstrate that M2-sEVs enhance MEPM osteogenesis, while M1-sEVs inhibit it in vitro.

这些发现表明，M2-sEVs促进MEPM成骨，而M1-sEVs在体外抑制它。

Pharmacological ADAM17 inhibition rescued efferocytosis disorder, fusion failure, and osteogenic abnormities in the sonicated

药理学ADAM17抑制挽救了超声处理后的胞葬作用障碍、融合失败和成骨异常。

P. gingivalis

牙龈卟啉单胞菌

-treated condition

-治疗条件

To evaluate whether the excessive expression of ADAM17 was causally related to the abnormal palatal fusion and osteogenesis induced by sonicated

为了评估ADAM17的过度表达是否与超声处理引起的腭融合和骨生成异常存在因果关系，

P. gingivalis

牙龈卟啉单胞菌

, we administered GW280264X, an ADAM17 antagonist, to mice exposed to sonicated

，我们给暴露于超声处理的 mice 施用了 GW280264X，一种 ADAM17 拮抗剂，

P. gingivalis

牙龈卟啉单胞菌

from E10.5 to E14.5. In a limited sample size, the use of GW280264X reduced the incidence of CP from 12.5% to 0% under treatment with sonicated

从 E10.5 到 E14.5。在有限的样本量中，使用 GW280264X 在超声处理下将脑瘫（CP）的发生率从 12.5% 降低到 0%。

P. gingivalis

牙龈卟啉单胞菌

(Fig.

（图。

10a–c

, Supplementary Table

，补充表格

, Supplementary Table

补充表格

). We also observed that the palatal morphology was relatively thin in the ADAM17 inhibitor group (Fig.

). 我们还观察到，在ADAM17抑制剂组中，腭部形态相对较薄（图。

10a, d

). In MEPM cells, the reduced expression of TGFBR1 was partially reversed (Fig.

在MEPM细胞中，TGFBR1的表达减少得到了部分逆转（图。

10e, f

). ARS and Masson staining further confirmed that the decreased osteogenesis in mice treated with sonicated

). ARS和Masson染色进一步证实，经超声处理的小鼠骨形成减少。

P. gingivalis

牙龈卟啉单胞菌

was significantly improved following ADAM17 inhibition, both in vitro (Fig.

在ADAM17抑制后显著改善，无论是在体外（图。

10g, h

10克，小时

) and in vivo (Fig.

）和体内（图。

10I, j

10I，j

). For Mφs, the diminished expression of MerTK (Supplementary Fig.

). 对于Mφs，MerTK的表达减少（补充图。

S9a, b

S9a，b

) and efferocytosis was partially restored both in vitro (Fig.

`) 和胞葬作用在体外均部分恢复（图。`

11a, b

) and in vivo (Fig.

）和体内（图。

11c, d

). However, the restorative effect of GW280264X on the average percentages of M1 Mφs and M2 Mφs per high-power field and their ratio in the palate were not significant (Fig.

然而，GW280264X对每高倍视野下M1巨噬细胞和M2巨噬细胞平均百分比及其在腭部中的比例的恢复效果并不显著（图。

11e, f

, Supplementary Fig.

，补充图。

S9c, d

S9c，d

). Taken together, our findings indicate that the upregulation of ADAM17 due to glycolysis enhanced H4K12la, and the subsequent cleavage of TGFBR1 and MerTK, govern the CP with abnormal palate shelves fusion and osteogenesis caused by sonicated

). 综上所述，我们的研究结果表明，糖酵解增强的H4K12la导致ADAM17上调，以及随后TGFBR1和MerTK的切割，控制了因超声处理引起的腭架融合异常和成骨作用的CP。

P. gingivalis

牙龈卟啉单胞菌

during palate development.

在腭发育期间。

Fig. 10

图10

Pharmacological ADAM17 inhibition ameliorated osteogenic abnormities in the sonicated

药理学ADAM17抑制改善了声波处理后的成骨异常。

P. gingivalis

牙龈卟啉单胞菌

(P. g)-treated condition.

（P. g）处理条件。

Lateral and occlusal views and histological observation of mice palates after treated with P. g and (or) GW280264X. Bar, 200 μm, PS represents palatal shelves and T represents togue.

用P. g和（或）GW280264X处理后的小鼠腭部的侧面与咬合面视图及组织学观察。标尺：200微米，PS代表腭板，T代表舌头。

The frequency of cleft.

裂隙的频率。

Frequency of cleft palate in P. g group compared with other groups,

P.g组与其他组相比，腭裂的发生频率，

value was calculated by Fisher’s exact test.

该值通过Fisher精确检验计算得出。

Quantification of the sectional thickness of embryonic palate in the control and GW280264X group.

对照组和GW280264X组胚胎腭部切片厚度的量化。

Western blot analysis of TGFBR1, with (

转化生长因子β受体1的蛋白质印迹分析，与 (

) quantification of protein levels (

蛋白质水平的量化 (

= 3).

= 3)。

Alizarin red S staining on 28 days and

28天时的茜素红S染色和

the calcium concentration was determined by measuring the absorbance at 562 nm on a multiplate reader (

钙浓度通过在多孔板读数仪上测量562 nm处的吸光度来确定 (

= 3). Bar, 500 μm.

= 3）。标尺，500 μm。

，

Masson staining and statistical analysis (

Masson染色和统计分析(

= 4). Bar, 200 μm. The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 4)。标尺，200 μm。数据以均值 ± 标准差表示，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

) or two-tailed Student’s

）或双尾Student’s

-test (

). All the

). 所有的

values were two-sided and adjustments were made for multiple comparisons

这些值是双侧的，并且对多重比较进行了调整。

Full size image

全尺寸图像

Fig. 11

图11

Pharmacological ADAM17 inhibition ameliorated efferocytosis disorder and fusion failure.

药理学ADAM17抑制改善了胞葬作用障碍和融合失败。

Representative fluorescent images showing engulfing of apoptotic MEE cells by macrophages and

显示巨噬细胞吞噬凋亡的MEE细胞的代表性荧光图像和

phagocytic index based on the fluorescent image (

基于荧光图像的吞噬指数 (

= 6).

= 6）。

Co-staining of MerTK positive macrophage (green) in TUNEL-positive regions (red), with

在TUNEL阳性区域（红色）中，MerTK阳性巨噬细胞（绿色）的共染色，

quantification of Free/MerTK-associated apoptotic cells (

游离/MerTK相关凋亡细胞的定量分析 (

= 4). Bar, 200 μm.

= 4）。标尺，200 μm。

Co-staining of CD86 /CD206 and

CD86/CD206 的共染色和

quantification of CD86/CD206 at E15.5 (

E15.5时CD86/CD206的量化 (

= 4). Bar, 200 μm. The data are shown as the mean ± SD and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test (

= 4)。标尺，200 μm。数据以均值 ± 标准差表示，并通过单因素方差分析结合Tukey多重比较检验进行统计分析 (

，

). All the

). 所有的

values were two-sided and adjustments were made for multiple comparisons. P. g represents

值是双侧的，并且对多重比较进行了调整。P. g 代表

P. gingivalis

牙龈卟啉单胞菌

Full size image

全尺寸图像

Discussion

讨论

Maternal periodontal disease has been reported to correlate with adverse pregnancy outcomes.

母体牙周病据报道与不良妊娠结局相关。

，

A prior case-control study even revealed a surprisingly strong association between maternal periodontal disease and CP in offspring.

既往一项病例对照研究甚至揭示了母体牙周病与子代脑瘫之间存在惊人强烈的关联。

However, the precise relationship has remained unclear for over a decade. Although a variety of periodontal pathogens are prevalent in the dental plaque of patients with periodontitis, it is noteworthy that the majority of periodontal pathogens identified in the placenta and amniotic fluid tissues of pregnant women experiencing adverse pregnancy outcomes, such as eclampsia and premature delivery, are predominantly .

然而，十多年来，这种精确的关系一直不清楚。尽管在牙周炎患者的牙菌斑中普遍存在多种牙周病原体，但值得注意的是，在经历不良妊娠结局（如子痫和早产）的孕妇的胎盘和羊水组织中发现的大多数牙周病原体主要是。

P. gingivalis

牙龈卟啉单胞菌

。

Consequently, we selected

因此，我们选择了

P. gingivalis

牙龈卟啉单胞菌

as the focal point of our research.

作为我们研究的焦点。

In our study, we exposed pregnant mice to sonicated

在我们的研究中，我们让怀孕的小鼠接触了超声波处理的环境。

P. gingivalis

牙龈卟啉单胞菌

via both intravenous injection and oral administration starting from E2.5, as periodontal disease is characterized by both endotoxemia and the ingestion of periodontal bacteria.

通过静脉注射和口服给药，从E2.5开始，因为牙周病的特征在于内毒素血症和牙周细菌的摄入。

，

We found that treatment with sonicated

我们发现用超声处理后

P. gingivalis

牙龈卟啉单胞菌

resulted in CP with cleft between the palate shelves and reduced osteogenesis. At E15, the incidence of residual epithelial remnants at the midline without any mesenchymal confluency in the sonicated

导致腭裂发生在腭板之间，并减少了骨生成。在E15，声波处理后中线处残留上皮残余物的发生率没有任何间充质融合。

P. gingivalis

牙龈卟啉单胞菌

group was 21.4%. However, CP was seen in 12.1% of the litters when evaluated at E16.5.

该组为21.4%。然而，在E16.5评估时，12.1%的窝中观察到CP。

We posit two possible explanations for this frequency disparity. Firstly, given that previous studies reported an association between

我们提出了这种频率差异的两种可能解释。首先，鉴于之前的研究报告了之间的关联

P. gingivalis

牙龈卟啉单胞菌

infection and spontaneous abortion, we hypothesize that severely affected fetal mice at E15 might have ultimately undergone abortion or embryonic absorption by E16.5 as it might influence not only palate development, but also the whole body.

感染和自然流产，我们推测在E15严重受影响的胎鼠可能最终在E16.5时经历了流产或胚胎吸收，因为它可能不仅影响腭的发育，还影响整个身体。

During the course of the experiment, it was additionally observed that embryos exposed to

在实验过程中，另外观察到暴露于

P. gingivalis

牙龈卟啉单胞菌

exhibited various craniofacial, ocular and dermatological abnormalities. Secondly, the mating time might vary between different female mice by several hours, and offspring within the same pregnant mice could even develop at different rates. Consequently, at E15, some late-mating or slower-developing offspring might not have initiated epithelial apoptosis at the midline, but they were still included in the abnormal phenotype count.

表现出各种颅面、眼部和皮肤异常。其次，不同雌性小鼠之间的交配时间可能相差数小时，甚至同一怀孕小鼠的后代也可能以不同的速度发育。因此，在E15时，一些晚交配或发育较慢的后代可能尚未在中线启动上皮细胞凋亡，但它们仍被计入异常表型数量中。

To eliminate the influence of variable developmental periods, we selected offspring with no signs of developmental delay for our subsequent study based on dental germ morphology..

为了消除可变发育期的影响，我们根据牙胚形态选择了没有发育迟缓迹象的后代进行后续研究。

To investigate the mechanisms of CP underlying the abnormal palate shelves fusion and osteogenesis caused by sonicated

超声作用下腭架融合异常及成骨作用的CP机制研究

P. gingivalis

牙龈卟啉单胞菌

, we further examined the impact of sonicated

，我们进一步检查了超声处理的影响

P. gingivalis

牙龈卟啉单胞菌

on MEPM cells and Mφs, respectively. A crucial aspect of cellular physiology that has recently garnered attention in embryonic development is the regulation of glucose metabolism. We discovered that both MEPM cells and Mφs underwent a metabolic shift from OXPHOS to glycolysis to cope with

在MEPM细胞和Mφs上，分别。细胞生理学中一个关键方面，最近在胚胎发育中引起关注的是葡萄糖代谢的调控。我们发现，MEPM细胞和Mφs都经历了从氧化磷酸化到糖酵解的代谢转变，以应对

P. gingivalis

牙龈卟啉单胞菌

exposure. This shift is contrary to the typical metabolic changes seen during palate development, leading to metabolic disorder.

暴露。这种转变与腭发育过程中常见的代谢变化相反，导致代谢紊乱。

Considering previous studies that have identified a link between glycolysis and inflammation,

考虑到先前已确定糖酵解和炎症之间存在联系的研究，

，

we hypothesize that metabolic alterations may be instigated by inflammation (see Supplementary Fig.

我们假设代谢改变可能是由炎症引发的（见补充图）。

S10

). This hypothesis warrants further exploration.

）。这个假设需要进一步探讨。

Most metabolic changes can indirectly determine cell identities by epigenetic regulation.

大多数代谢变化可以通过表观遗传调控间接决定细胞身份。

，

More recently, glycolysis-derived lactate was identified as a substrate for histone lactylation and lactate-derived histone lactylation was shown to stimulate gene expressions.

最近，糖酵解衍生的乳酸被确定为组蛋白乳酸化的底物，并且已证明乳酸衍生的组蛋白乳酸化可以刺激基因表达。

Zhang et al. discovered that

张等人发现

P. gingivalis

牙龈卟啉单胞菌

msRNAP.G_45033 could induce Aβ production by enhancing glycolysis and histone lactylation in Mφs.

msRNAP.G_45033能够通过增强巨噬细胞中的糖酵解和组蛋白乳酸化来诱导Aβ的产生。

Consist with this finding, in this research, we demonstrated that an increase in the lactate pool due to sonicated

与此发现一致，在这项研究中，我们证明了由于超声作用导致乳酸池的增加。

P. gingivalis

牙龈卟啉单胞菌

facilitates glycolysis, then enhanced histone lactylation which played a significant role in defining ADAM17 binding, as indicated by H4K12la and ADAM17 ChIP-qPCR analysis of MEPM cells and Mφs.

促进糖酵解，然后增强的组蛋白乳酸化在定义ADAM17结合中发挥了重要作用，正如通过H4K12la和ADAM17 ChIP-qPCR分析MEPM细胞和Mφs所表明的那样。

Previous research has indicated that H4K12la specifically upregulates multiple genes related to cell proliferation.

以往的研究表明，H4K12la 特异性上调多个与细胞增殖相关的基因。

Consistent with these findings, ADAM17 has been reported to play a crucial role in cell proliferation during development. Earlier studies on the developing cerebellum and spinal cord found that the expression level of ADAM17 was higher during early stages of development—when cell proliferation is predominant—and gradually declines in response to environmental and functional changes..

与这些发现一致，据报道，ADAM17在发育过程中的细胞增殖中起着关键作用。早期关于小脑和脊髓发育的研究发现，ADAM17的表达水平在发育早期（细胞增殖占主导时）较高，并随着环境和功能变化逐渐下降。

，

In our study, we observed that the palatal morphology was relatively thin in the ADAM17 inhibitor group, suggesting that ADAM17 might also play a significant role in cell proliferation during palate development. Furthermore, we noted that the expression of ADAM17 gradually decreased with maturation during normal palate development.

在我们的研究中，我们观察到ADAM17抑制剂组的腭部形态相对较薄，这表明ADAM17在腭部发育过程中可能也对细胞增殖起着重要作用。此外，我们注意到在正常的腭部发育过程中，ADAM17的表达随着成熟逐渐减少。

However, in the sonicated .

然而，在超声处理中。

P. gingivalis

牙龈卟啉单胞菌

-treated group, H4K12la facilitated the transcription of ADAM17, disrupting its usual temporal expression pattern (Supplementary Fig.

-处理组中，H4K12la促进了ADAM17的转录，破坏了其通常的时间表达模式（补充图）。

). The sustained high expression of ADAM17 consequently mediated the shedding of MerTK in Mφs and TGFBR1 in MEPM cells, leading to abnormal efferocytosis and osteogenesis in palate development. Our findings, along with previous studies, suggest that glucose metabolism and its derivative histone lactylation fine-tune the temporal expression of ADAM17, underlining the pivotal role of metabolism-associated epitranscriptomic machinery in coordinating organ growth and functional maturation timing during palate development..

）。ADAM17 的持续高表达随后介导了 Mφ 细胞中 MerTK 和 MEPM 细胞中 TGFBR1 的脱落，导致腭发育过程中异常的胞葬作用和成骨作用。我们的研究结果结合先前的研究表明，葡萄糖代谢及其衍生的组蛋白乳酸化精细调控了 ADAM17 的时间表达，突显了与代谢相关的表观转录组机制在腭发育过程中协调器官生长和功能成熟时机的关键作用。

Palate shelves fusion and wound repair are regenerative processes sharing common signaling pathways and gene regulatory networks.

腭架融合和伤口修复是共享共同信号通路和基因调控网络的再生过程。

Previous studies demonstrated Mφ-mediated efferocytosis played a prominent role in wound healing by removing apoptotic cells and then dampening inflammation and secreting factors that regulate the proliferation.

以往的研究表明，Mφ介导的胞葬作用通过清除凋亡细胞从而在伤口愈合中发挥了重要作用，进而减轻炎症并分泌调节增殖的因素。

The normal exercise of efferocytosis can induce the Mφ reprogramming, so that Mφ transition from pro-inflammatory M1 to reparative M2 for tissue repair.

正常的胞葬作用可以诱导巨噬细胞重编程，使巨噬细胞从促炎的M1型转变为修复性的M2型以促进组织修复。

To determine whether Mφs play a similar role in palatal fusion, Christiaan et al. investigated whether palatal fusion was disrupted in heme oxygenase-2 knockout (HO-2 KO) mice due to altered epithelial cell and Mφ interactions within the MES and they found HO-2 KO Mφs were still functional in phagocytosing apoptotic fragments, with no disturbance was observed in palatal fusion in HO-2 KO foetuses.

为了确定Mφs在腭融合中是否发挥类似作用，Christiaan等人研究了血红素加氧酶-2基因敲除（HO-2 KO）小鼠的腭融合是否因MES内上皮细胞和Mφ相互作用改变而受到破坏，他们发现HO-2 KO Mφs在吞噬凋亡碎片方面仍然功能正常，并且在HO-2 KO胎儿中未观察到腭融合异常。

This may be due to their examination of Mφ function in a non-pathological environment, with the heightened expression of HO-1 possibly compensating for the deletion of HO-2 in Mφs recruited to the fusing palate shelves..

这可能是由于他们在非病理环境中对Mφ功能的检查，HO-1表达的增强可能补偿了募集到融合腭架的Mφ中HO-2的缺失。

In contrast, our study provides additional evidence that under pathological conditions, disrupted efferocytosis due to MerTK shedding hinders the clearance of apoptotic epithelium in the MES, resulting in a cleft between the palate shelves. These observations emphasize the significant influence of the pathological environment on Mφ function, a situation analogous to inflammatory stresses in the wound healing context.

相比之下，我们的研究提供了更多证据表明，在病理条件下，由于MerTK脱落导致的胞吐作用受阻会阻碍MES中凋亡上皮的清除，从而导致腭架之间出现裂缝。这些观察结果强调了病理环境对Mφ功能的重大影响，这种情况类似于伤口愈合过程中炎症应激的状况。

Given that palatogenesis and wound healing share many key cellular behaviors, deepening our understanding of one can inform that of the other, leading to the development of novel therapeutic strategies..

鉴于腭发生和伤口愈合有许多关键的细胞行为相同，加深对其中一个的理解可以为另一个提供信息，从而开发出新的治疗策略。

Moreover, we observed that the weakened efferocytosis inhibited the phenotypic transformation of Mφs, led to a predominant distribution of M1 Mφs at E15.5, which might further impact MEPM osteogenesis through sEV-mediated cross-talk. Interestingly, among the detected osteogenic markers, the expression of Runx2 but not BMP2 and OPN was significantly regulated by M1-sEVs or M2-sEVs.

此外，我们观察到吞噬作用的减弱抑制了Mφs的表型转化，导致在E15.5时M1 Mφs占主导分布，这可能通过sEV介导的串扰进一步影响MEPM成骨。有趣的是，在检测的成骨标志物中，Runx2而非BMP2和OPN的表达显著受到M1-sEVs或M2-sEVs的调控。

Since previous studies demonstrated BMPs functioned by activating SMAD proteins to stimulate expression of Runx2 which induced expression of osteoblast markers such as collagen type I, ALP and OCN,.

由于之前的研究表明，BMPs通过激活SMAD蛋白来刺激Runx2的表达，从而诱导成骨细胞标志物如I型胶原、碱性磷酸酶和骨钙素的表达。

，

we speculated that M1-sEVs or M2-sEVs might act downstream of BMP2, while the insignificant variation of OPN might be due to its not being the main target gene for direct binding of Runx2 in MEPM cells. Therefore, understanding the sEV-mediated interaction among different type of cells especially investigating the Runx2 function in more detail will facilitate the cognition of palatogenesis and development of targeted drugs for CP in the future..

我们推测M1-sEVs或M2-sEVs可能作用于BMP2的下游，而OPN的微小变化可能是由于它不是MEPM细胞中Runx2直接结合的主要目标基因。因此，深入了解sEV介导的不同类型细胞间的相互作用，特别是更详细地研究Runx2的功能，将有助于理解腭发育过程，并推动未来针对唇腭裂（CP）的靶向药物开发。

Additionally, given our findings that pharmacological inhibition of ADAM17 using GW280264X ameliorated the aberrant efferocytosis induced by

此外，鉴于我们发现使用GW280264X对ADAM17进行药理学抑制改善了由以下因素引起的异常胞葬作用：

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牙龈卟啉单胞菌

, we subsequently investigated whether such inhibition could also rescue the abnormal polarization of Mφs and enhance osteogenic differentiation. However, the restorative effect of GW280264X on the Mφ phenotypic shift was not significant, possibly due to the challenge of phenotypic switching in the chronic inflammatory environment induced by sonicated .

，我们随后调查了这种抑制是否也能挽救Mφs的异常极化并增强成骨分化。然而，GW280264X对Mφ表型转变的恢复效果并不显著，可能是由于在超声诱导的慢性炎症环境中表型转换的挑战所致。

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. These results suggested that future research should undertake a more detailed investigation at the molecular level to identify specific substances within

这些结果表明，未来的研究应进行更详细的分子水平调查，以确定其中的具体物质。

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, as well as components in other pathogens, that may activate the embryonic inflammatory environment and induce Mφ phenotype transformation.

，以及其它病原体中的成分，可能会激活胚胎炎症环境并诱导巨噬细胞表型转化。

Although Mφ polarization remained unchanged, GW280264X successfully ameliorated

尽管Mφ极化保持不变，GW280264X成功改善了

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-induced osteogenesis abnormalities. This finding confirmed that the elevated expression of ADAM17 induced by

诱导的成骨异常。这一发现证实了由诱导的ADAM17表达升高导致的

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could also influence osteogenesis through alternative pathways. Supporting evidence for this assertion is provided by our findings, which illustrate that, beyond the indirect osteogenic regulation facilitated by sEVs, direct regulation induced by sonicated

也可能通过其他途径影响骨生成。支持这一说法的证据由我们的研究结果提供，这些结果表明，除了由sEVs促进的间接成骨调节外，超声处理诱导的直接调节也发挥了作用。

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also results in aberrant osteogenesis. This phenomenon is attributed to the shedding of TGFBR1 in MEPM cells.

还导致异常的骨生成。这种现象归因于MEPM细胞中TGFBR1的脱落。

TGFBR1 plays important roles in morphogenesis of many craniofacial tissues. Dudas et al. reported missing and dysplastic craniofacial skeletal structures in

TGFBR1在许多颅面组织的形态发生中起着重要作用。Dudas等人报道了缺失和发育不良的颅面骨骼结构。

Tgfbr1/Wnt1

-Cre mutants, which deleted Tgfbr1 only in cells that express

-Cre突变体，仅在表达Tgfbr1的细胞中将其删除

Wnt1

Wnt1基因

-Cre, i.e., in neural crest cells, and in the neural plate. They suggested that the observed phenotypes in

-Cre，即在神经嵴细胞和神经板中。他们认为观察到的表型在

Tgfbr1/Wnt1

-Cre mutants could be caused by defective cell survival.

-Cre突变体可能是由于细胞存活缺陷引起的。

However, in our study, we did not observe the negative regulation of sonicated

然而，在我们的研究中，我们没有观察到超声处理的负调节作用。

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on cell survival. On the contrary, both in vitro and in vivo results showed that sonicated

超声处理对细胞存活的影响。相反，体外和体内结果都显示

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promoted the proliferation of MEPM cells and inhibited the apoptosis. Since early studies have shown that bacteria-induced metabolic reprogramming to glycolysis promotes cell proliferation as a strategy for bacterial initiation of disseminated infection,

促进了MEPM细胞的增殖并抑制了细胞凋亡。由于早期研究表明细菌诱导的代谢重编程为糖酵解促进细胞增殖是细菌引发播散性感染的一种策略，

，

and given the association between ADAM17 and cell proliferation,

鉴于ADAM17与细胞增殖之间的关联，

，

we suggested that the effects of metabolic reprogramming and subsequent ADAM17 upregulation might counteract the negative regulation of TGFBR1 on cell survival. We found that although the density of MEPM cells was not reduced, the shedding of TGFBR1 led to an obstruction of MEPM cells migration and osteogenic differentiation, ultimately leading to osteogenic abnormalities..

我们推测代谢重编程和随后的ADAM17上调可能抵消了TGFBR1对细胞存活的负调控作用。我们发现，虽然MEPM细胞的密度没有减少，但TGFBR1的脱落导致了MEPM细胞迁移和成骨分化的障碍，最终导致成骨异常。

In conclusion, we demonstrate the etiology of CP caused by sonicated

总之，我们证明了由声波引起的脑瘫的病因学

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by upregulation of ADAM17 due to glycolysis enhanced H4K12la, and the resulting cleavage of TGFBR1 and MerTK, underlie the abnormal palatal fusion and osteogenesis during palate development (Fig.

通过糖酵解增强的H4K12la上调ADAM17，以及由此产生的TGFBR1和MerTK的裂解，是腭发育过程中异常腭融合和成骨的原因（图。

). This may explain the strong association between maternal periodontal disease and CP in their children. Since the molecular processes and signaling pathways associated with palatal ontogeny are mirrored in the embryogenesis of multiple other systems,

）。这可能解释了母亲牙周病和孩子CP之间的强关联。由于与腭部发育相关的分子过程和信号通路在多个其他系统的胚胎发生中也有体现，

，

understanding the pathological mechanisms underlying palate development during maternal periodontal disease will also allow us to explore potential targets for preventing or treating infection-induced congenital birth defects.

了解母体牙周病期间腭发育的病理机制，也将使我们能够探索预防或治疗感染引起的先天性出生缺陷的潜在靶点。

Fig. 12

图12

Schematic diagram of the etiology of CP caused by sonicated

超声波引起的CP病因示意图

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. Glycolysis and H4K12la were enhanced in both macrophages and MEPM cells under

. 在巨噬细胞和MEPM细胞中，糖酵解和H4K12la均得到增强

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exposure which further promoted the transcription of ADAM17, subsequently mediated the shedding of MerTK in macrophages and TGFBR1 in MEPM cells and resulted in the suppression of efferocytosis and osteogenesis in fetal palate, eventually caused abnormalities in palate fusion and ossification. The abnormal efferocytosis also led to a predominance of M1 macrophages, which indirectly inhibited palatal osteogenesis via sEVs.

暴露进一步促进了ADAM17的转录，随后介导了巨噬细胞中MerTK和MEPM细胞中TGFBR1的脱落，导致胎鼠腭部胞葬作用和成骨作用的抑制，最终引起腭融合与骨化异常。异常的胞葬作用还导致M1型巨噬细胞占主导地位，其通过sEVs间接抑制了腭部成骨。

Full size image

全尺寸图像

Materials and methods

材料与方法

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culture

文化

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(ATCC 33277) was cultured following a previously described method.

(ATCC 33277) 按照先前描述的方法进行培养。

A bacterial suspension of 10

细菌悬浮液为10

CFU/mL was sonicated at 300 W for 90 cycles (10 s on, 10 s off) on ice using an ultrasonic cell disruptor (JYD-150, Zhixin Instrument Co., China), ensuring thorough bacterial lysis. The sonicated extract was then sterilized by filtration through 0.22-μm-pore-size filter. The total protein concentration was measured, and the solution was stored at −80 °C..

每毫升菌落形成单位 (CFU/mL) 在冰上使用超声细胞破碎仪 (JYD-150，智信仪器公司，中国) 以300瓦功率进行90个循环（10秒开，10秒关）的超声处理，以确保细菌充分裂解。随后通过0.22微米孔径的滤膜过滤对超声提取物进行灭菌。测量总蛋白浓度后，将溶液保存在-80℃。

Cell culture

细胞培养

MEPM cells were obtained from E15.5 ICR pregnant mice as previously described.

MEPM细胞是从E15.5 ICR孕鼠中获得的，如前所述。

The cells were cultured in the Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12; SV30023.01, Hyclone, USA) containing 10% fetal bovine serum (FBS; 10099-141, Gibco, USA) and 1% penicillin/streptomycin (C100C5, NCM Biotech, China).

细胞在含10%胎牛血清（FBS；10099-141，Gibco，美国）和1%青霉素/链霉素（C100C5，NCM Biotech，中国）的Dulbecco改良Eagle培养基/F12（DMEM/F12；SV30023.01，Hyclone，美国）中培养。

The RAW264.7 murine macrophage cell line (CL-0190, Procell, China) was purchased from Procell company and cultured in Raw264.7 specialized medium (CM-0190, Procell, China).

RAW264.7小鼠巨噬细胞系（CL-0190，普罗塞尔，中国）购自普罗塞尔公司，并在Raw264.7专用培养基（CM-0190，普罗塞尔，中国）中培养。

BMDM were prepared as previously described

BMDM的制备如前所述。

and cultured for 7 d in RPMI 1640 medium (SH30809.01, HyClone, USA) with 10% FBS and 1% penicillin/streptomycin and 10 ng/mL M-CSF (81627-83-0, Sigma-Aldrich, USA).

在含10%胎牛血清、1%青霉素/链霉素和10 ng/mL M-CSF（81627-83-0，Sigma-Aldrich，美国）的RPMI 1640培养基（SH30809.01，HyClone，美国）中培养7天。

The MEE cell line (BFN607200453, DSMZ Cell Bank, China) were obtained from the cell bank of Chinese Academy of Sciences, and cultured in DMEM medium (SH30022.01, HyClone, USA), supplemented with 15% FBS and 1% penicillin–streptomycin.

MEE细胞系（BFN607200453，DSMZ细胞库，中国）购自中国科学院细胞库，并在DMEM培养基（SH30022.01，HyClone，美国）中培养，添加15%胎牛血清和1%青霉素-链霉素。

Preparation and characterization of sEVs

sEVs的制备与表征

M1-sEVs and M2-sEVs were extracted and isolated and characterized as described previously.

M1-sEVs和M2-sEVs按照之前描述的方法进行提取、分离和表征。

The morphology of sEVs was identified using TEM. The concentration and particle size distribution of sEVs were analyzed by NTA, and the surface sEV-markers were tested by western blot. 100 μg/mL sEVs were used to treat MEPM cells.

使用透射电子显微镜（TEM）鉴定sEVs的形态。通过纳米颗粒跟踪分析（NTA）分析sEVs的浓度和粒径分布，并通过蛋白质印迹（western blot）检测表面sEV标记物。使用100 μg/mL sEVs处理MEPM细胞。

Mice experiments

小鼠实验

Healthy female C57BL/6 mice (8–10 weeks old; weight, 22–25 g) were paired with healthy males (12–14 weeks old) in a 2:1 ratio overnight. The presence of vaginal plugs was checked the next morning and marked as day 0.5 of gestation (E0.5). The mice were randomly divided into two groups: those administered sonicated .

健康的雌性C57BL/6小鼠（8-10周龄；体重22-25克）与健康的雄性小鼠（12-14周龄）以2:1的比例过夜配对。次日早晨检查阴道栓的存在，并标记为妊娠第0.5天（E0.5）。小鼠被随机分为两组：一组接受超声处理。

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suspension in saline and those receiving saline only. Experimental periodontal disease was induced as previously described.

盐水悬浮液和仅接受盐水的组别。实验性牙周病按照先前描述的方法诱导。

Administration was carried out through both oral gavage and intravenous injection in each group. A sonicated

每组均通过口服灌胃和静脉注射进行给药。超声处理的

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suspension (10

悬挂（10

CFU in 100 µL saline) and saline only (100 µL) were administered by the two methods, respectively. Administration was performed every other day from E2.5. Pregnant mice were dissected at E13.5, E14.5, E15, E15.5, and E16.5 and euthanized by cervical dislocation (

分别通过两种方法给予CFU（100 µL盐水）和仅盐水（100 µL）。从E2.5开始每隔一天进行给药。怀孕小鼠在E13.5、E14.5、E15、E15.5和E16.5时被解剖，并通过颈椎脱位处死。

= 6 at each time point per group), and fetal litters were removed via dissection to observe palate development. The embryo samples were embedded for histological sections. For GW280264X (HY-115670, MCE, USA) treatment, pregnant mice were injected with the working solution (0.1 μg/g body weight) from E10.5 to E14.5..

每组每个时间点=6只），通过解剖取出胎仔以观察腭部发育情况。胚胎样本被包埋用于组织学切片。对于GW280264X（HY-115670，MCE，美国）处理，怀孕小鼠从E10.5到E14.5被注射工作溶液（0.1 μg/g体重）。

ELISA assays for RgpA

RgpA的ELISA检测

Amniotic fluid and fetal palate tissue were collected from four pregnant mice at each designated time point. These samples were subsequently analyzed and quantified for the presence of

在每个指定时间点，从四只怀孕小鼠收集羊水和胎儿腭组织。随后对这些样本进行分析并量化以确定

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gingipain R1 (RgpA) using an RgpA-specific antibody (orb243611, biorbyt, UK) and Ancillary Reagent Kit (E-ELIR-K001, Elabscience, China) following the manufacturer’s protocol.

使用针对RgpA的特异性抗体（orb243611，biorbyt，英国）和辅助试剂盒（E-ELIR-K001，Elabscience，中国）按照制造商的方案检测牙龈蛋白酶R1（RgpA）。

Histological analysis

组织学分析

Palate tissue histological sections were sliced to a thickness of 5 µm using a rotary microtome, from the nasal cavity to the ear. The sections were stained with HE and Masson following methods described previously.

腭组织的组织学切片使用旋转式切片机从鼻腔到耳部切成5微米厚。切片按照先前描述的方法用HE和Masson染色。

Microphotographs were captured using a digital camera attached to a microscope (BX61, Olympus, Japan). The collagen volume was measured using ImageJ.

使用连接到显微镜（BX61，奥林巴斯，日本）的数码相机拍摄显微照片。使用ImageJ测量胶原蛋白体积。

Immunohistochemistry

免疫组织化学

All sections underwent antigen activation. Post washing and blocking, the slides were incubated overnight at 4 °C with the primary antibody (TGFBR1, ab288303, Abcam, UK; PCNA, 10205-2-AP, Proteintech, China), diluted 1:100 in PBS. The sections were rinse, then incubated with secondary antibodies (goat anti-rabbit IgG-HRP; PV9001, ZSGB-BIO, China) and diluted 1:400 in PBS for 1 h.

所有切片均进行了抗原激活。清洗和封闭后，将切片与一抗（TGFBR1，ab288303，Abcam，英国；PCNA，10205-2-AP，Proteintech，中国）在4°C下孵育过夜，一抗用PBS稀释至1:100。切片经过冲洗后，与二抗（山羊抗兔IgG-HRP；PV9001，ZSGB-BIO，中国）在PBS中以1:400稀释并孵育1小时。

Following another round of washing, the sections were detected using a standard DAB detection system (ZLI-9019, ZSGB-BIO, China). The quantification of positive cells was measured using ImageJ..

经过另一轮洗涤后，使用标准的DAB检测系统（ZLI-9019，中杉金桥生物公司，中国）对切片进行检测。阳性细胞的定量通过ImageJ进行测量。

Assessment of apoptosis and efferocytosis in the medial edge seam

内侧边缘接缝中细胞凋亡和胞葬作用的评估

Apoptotic cells in the medial edge seam were examined using an in situ TUNEL kit. MerTK, a member of the TAM family (Tyro3, Axl, and MerTK), is known for its contribution to the regulation of Mφ efferocytosis. Mφ efferocytosis in this study was assessed by co-staining of apoptotic cells and MerTK+ Mφs, following a previous protocol.

使用原位TUNEL试剂盒检测了中间边缘缝中的凋亡细胞。MerTK是TAM家族（Tyro3、Axl和MerTK）的一员，因其在调控巨噬细胞胞葬作用中的贡献而闻名。本研究按照先前的方案，通过凋亡细胞与MerTK+巨噬细胞的共染色来评估巨噬细胞的胞葬作用。

Mφs were stained with a rabbit anti-MerTK antibody (ab52968, Abcam, UK, final dilution, 1:50) and then a FITC-labeled goat anti-rabbit IgG (35552, Thermo Fisher, USA, final dilution, 1:200). The ratio of free to MerTK-associated apoptotic cells was calculated. Images were captured using identical exposure settings and quantified using ImageJ..

Mφs用兔抗MerTK抗体（ab52968，Abcam，英国，最终稀释度1:50）染色，然后用FITC标记的山羊抗兔IgG（35552，Thermo Fisher，美国，最终稀释度1:200）染色。计算了游离与MerTK相关凋亡细胞的比例。使用相同的曝光设置捕获图像，并使用ImageJ进行量化。

Fluorescence multiplex immunohistochemical analysis

荧光多重免疫组织化学分析

Fluorescence multiplex immunohistochemical analysis was performed according to the manufacturer’s instructions. The sections underwent three rounds of staining in the order of H4K12la (PTM-1411, PTM BIO, China, 1:150), ADAM17 (ab39162, Abcam, UK, 1:150), MerTK (ab52968, Abcam, UK, 1:150), or TGFBR1 (ab288303, Abcam, UK, 1:150) each employing a separate fluorescent tyramide signal amplification system (Quadruple-Fluorescence Immunohistochemical Mouse/Rabbit Kit [RS0037]; ImmunoWay, USA).

根据制造商的说明进行荧光多重免疫组织化学分析。切片按照 H4K12la（PTM-1411，PTM BIO，中国，1:150）、ADAM17（ab39162，Abcam，英国，1:150）、MerTK（ab52968，Abcam，英国，1:150）或 TGFBR1（ab288303，Abcam，英国，1:150）的顺序进行了三轮染色，每种染色均使用独立的荧光酪胺信号放大系统（四重荧光免疫组化小鼠/兔试剂盒 [RS0037]；ImmunoWay，美国）。

Images were captured by confocal laser scanning microscopy (TSC SP5, Leica, Germany) using identical exposure settings and quantified using ImageJ..

图像通过共聚焦激光扫描显微镜（TSC SP5，徕卡，德国）使用相同的曝光设置捕获，并使用ImageJ进行量化。

In vitro efferocytosis analysis

体外胞葬作用分析

MEE cells were resuspended in DMEM medium supplemented with 15% FBS and 0.5 μmol/L actinomycin D and incubated for 12 h. At this time, 85% of the MEPM cells were apoptotic. Apoptotic MEE cells were stained with DiI fluorescent dye (D8700, Solarbio, China), following the manufacturer’s instructions. Raw 264.7 cells (1 × 10.

MEE细胞重新悬浮于添加了15%胎牛血清（FBS）和0.5 μmol/L放线菌素D的DMEM培养基中，并孵育12小时。此时，85%的MEPM细胞已发生凋亡。按照制造商的说明，用DiI荧光染料（D8700，Solarbio，中国）对凋亡的MEE细胞进行染色。Raw 264.7细胞（1 × 10...

cells per well) were plated in 12-well plates. After a 24-h incubation, the cells were exposed to DiI-labeled apoptotic for 1 h. The ratio of Raw 264.7 cells to MEE cells was 10:1. Undigested cells were removed by washing three times with ice-cold PBS, and Raw 264.7 cells were stained with Phalloidin.

每孔细胞数）接种于12孔板中。经过24小时孵育后，将细胞暴露于DiI标记的凋亡细胞1小时。Raw 264.7细胞与MEE细胞的比例为10:1。通过用冰冷的PBS洗涤三次去除未消化的细胞，并用鬼笔环肽对Raw 264.7细胞进行染色。

Efferocytosis of apoptotic cells was visualized using fluorescence microscopy..

使用荧光显微镜观察了凋亡细胞的胞葬作用。

CCK-8 assay

CCK-8 检测

Third-generation palate cells were plated into 96-well plates at a density of 7 × 10

第三代腭细胞以7×10的密度接种到96孔板中

cells per well with six independent replicates. After the cells adhered to the wall, 10 μL of CCK-8 reagent (CK04; Dojindo Laboratories) was added separately to each well at 0, 24, 48, and 72 h, followed by incubation at 37 °C for 2 h. Each sample was analyzed at 450 nm with a SpectraMax Paradigm microplate reader (10822-512; Molecular Devices, USA)..

每孔细胞数，设置六个独立复孔。细胞贴壁后，分别在 0、24、48 和 72 小时时向每孔中加入 10 μL 的 CCK-8 试剂 (CK04；Dojindo Laboratories)，并在 37°C 下孵育 2 小时。使用 SpectraMax Paradigm 微孔板读板机 (10822-512；Molecular Devices，美国) 在 450 nm 处对每个样品进行分析。

Apoptosis assay

凋亡测定

About 2.5 × 10

大约 2.5 × 10

cells per well were seeded in 6-well plates and incubated at 37 °C. The following day, cells were harvested using trypsin digestion without EDTA, and then stained with annexin V-fluorescein isothiocyanate (FITC) and PI following the instructions of the FITC-Annexin V Apoptosis Detection Kit (C1062L; Beyotime, China).

每孔接种的细胞置于6孔板中，并在37°C下孵育。次日，使用不含EDTA的胰酶消化收集细胞，然后按照FITC-Annexin V凋亡检测试剂盒（C1062L；碧云天，中国）说明书，用 Annexin V-异硫氰酸荧光素（FITC）和PI进行染色。

Cell analysis was carried out via flow cytometry (Accuri C6; BD, USA)..

通过流式细胞术（Accuri C6；BD，美国）进行细胞分析。

Scratch test

刮痕测试

About 3 × 10

大约 3×10

cells per well were seeded into 6-well plates. A 1-ml pipette tip was used to horizontally scratch the plate on the next day. Images were obtained using an inverted phase-contrast microscope after 24 and 48 h. The migration areas were detected and calculated using Image-Pro Plus 6.0

每孔的细胞被接种到6孔板中。第二天使用1毫升的移液器吸头水平划伤培养板。在24和48小时后，使用倒置相差显微镜获取图像。迁移区域通过Image-Pro Plus 6.0检测并计算。

Western blot

蛋白质印迹法

Cells were washed thrice with ice-cold PBS and lysed in RIPA (C1053, Applygen, China) buffer containing protease inhibitor cocktail (PIC; P8340, Sigma-Aldrich, USA) and phenylmethylsulfonyl fluoride (PMSF; 93482, Sigma-Aldrich, USA) for 10 min on ice before being centrifuged at 14 000 ×

细胞用冰冷的PBS洗涤三次，并在含蛋白酶抑制剂混合物（PIC；P8340，Sigma-Aldrich，美国）和苯甲基磺酰氟（PMSF；93482，Sigma-Aldrich，美国）的RIPA（C1053，Applygen，中国）缓冲液中冰上裂解10分钟，然后以14,000×g离心。

for 15 min at 4 °C. Protein samples were resolved on 10% SDS–PAGE gels and subsequently transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline-Tween for 1 h at room temperature, and then incubated with primary antibodies (listed in Supplementary Table .

在4°C下进行15分钟。蛋白质样品在10% SDS-PAGE凝胶上分离，随后转移到聚偏二氟乙烯膜上。膜在室温下用含5%（w/v）牛血清白蛋白（BSA）的Tris缓冲盐水-Tween中封闭1小时，然后与一抗（列于补充表中）孵育。

, final dilution 1:1 000) followed by the appropriate secondary antibody. Proteins were detected by enhanced chemiluminescence detection reagents (P10300, NCM 377 Biotech, China). The quantification of protein level was measured using ImageJ.

，最终稀释度为 1:1 000），然后加入适当的二抗。蛋白质通过增强化学发光检测试剂（P10300，NCM 377 Biotech，中国）进行检测。蛋白质水平的定量使用 ImageJ 进行测量。

Quantitative real-time polymerase chain reaction (qRT-PCR)

定量实时聚合酶链反应 (qRT-PCR)

Total RNA was extracted using TRIzol (CW0580; ComWin Biotech, China) and converted to cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, R233-01, China). Expressions was analyzed in triplicate using qRT-PCR instrument (CFX96 Touch Real-Time PCR Detection System; Bio-Rad Laboratories, USA) and MagicSYBR Mixture (CW2601H; ComWin Biotech, China).

使用TRIzol（CW0580；康为世纪生物，中国）提取总RNA，并使用HiScript II 第一链cDNA合成试剂盒（Vazyme，R233-01，中国）将其转化为cDNA。使用qRT-PCR仪器（CFX96 Touch实时荧光定量PCR检测系统；Bio-Rad Laboratories，美国）和MagicSYBR Mixture（CW2601H；康为世纪生物，中国）进行三重复分析表达。

The final results were normalized to β-actin levels and analyzed by calculating the comparative cycle threshold values (2.

最终结果被归一化为β-actin水平，并通过计算相对循环阈值（2来分析。

−ΔΔCt

). All primer sequences are listed in Supplementary Table

`). 所有引物序列均列于补充表中`

。

Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR)

染色质免疫沉淀后接定量PCR（ChIP-qPCR）

ChIP-qPCR was measured using the ChIP assay Kit (P2078, Beyotime, China) and performed according to the manufacturer’s instructions. For immunoprecipitation, the diluted chromatin was incubated with H4K12la antibodies (listed in Supplementary Table

使用ChIP检测试剂盒（P2078，Beyotime，中国）测量ChIP-qPCR，并按照制造商的说明进行操作。对于免疫沉淀，将稀释的染色质与H4K12la抗体（列于补充表中）孵育。

, final dilution 1:50) overnight at 4 °C with continuous rotation, followed by a 2-h incubation with 50 ml of 50% (v/v) protein A/G and washed with TSE I, TSE II, and TSE III buffers. The pulled-down chromatin complex was eluted by TE, and both eluted and input samples were de-crosslinked at 55 °C for 12 h in elution buffer.

，最终稀释比例为 1:50）在 4°C 下连续旋转孵育过夜，随后与 50 ml 50% (v/v) 的蛋白 A/G 孵育 2 小时，并用 TSE I、TSE II 和 TSE III 缓冲液洗涤。拉下的染色质复合物用 TE 洗脱，洗脱样品和输入样品均在洗脱缓冲液中于 55°C 下进行 12 小时的去交联。

DNA was purified with the QIAquick PCR Purification Kit. qPCR was performed as previously described, and the primer sequences for ChIP-qPCR are listed in Supplementary Table .

使用QIAquick PCR纯化试剂盒纯化DNA。qPCR按照之前描述的方法进行，ChIP-qPCR的引物序列列在补充表中。

。

Alkaline phosphatase (ALP) and Alizarin red staining (ARS)

碱性磷酸酶（ALP）和茜素红染色（ARS）

ALP staining was measured using the BCIP/NBT Alkaline Phosphatase Color Development Kit according to the instruction from manufacturer’s protocol (C3206, Beyotime, China). ALP activity assay was performed using an ALP activity kit according to the instruction from manufacturer’s protocol (245-325-0, Sigma-Aldrich, USA).

使用BCIP/NBT碱性磷酸酶显色试剂盒（C3206，碧云天，中国）按照制造商的方案测量ALP染色。使用ALP活性检测试剂盒（245-325-0，Sigma-Aldrich，美国）按照制造商的方案进行ALP活性测定。

Signal strength was normalized based on protein concentration. For ARS, MEPM cells were fixed for 30 min and then stained with 1% alizarin red (A5533, Sigma-Aldrich, USA) at room temperature. For quantification, calcium mineralization was measured as described in prior literature..

信号强度根据蛋白质浓度进行了标准化。对于ARS，MEPM细胞固定30分钟后，在室温下用1%茜素红（A5533，Sigma-Aldrich，美国）染色。对于定量分析，钙矿化程度按照先前文献中描述的方法进行测量。

Statistical analyses

统计分析

Statistical analyses were performed using Prism 8 (GraphPad Software) and data are presented as mean ± SD. An unpaired two-tailed Student’s t-test was used to determine significance between two groups of normally distributed data. For comparisons between multiple groups, an ordinary one-way or two-way ANOVA was used, followed by Tukey’s test.

使用 Prism 8（GraphPad Software）进行统计分析，数据以均值 ± 标准差表示。对于两组正态分布数据之间的显著性差异，采用未配对双尾 Student's t 检验。对于多组之间的比较，使用单因素或双因素方差分析（ANOVA），随后进行 Tukey 检验。

Comparisons of CP frequency were performed using Fisher’s exact test. The statistical tests used for each experiment are indicated in the figure legends. A value of .

使用Fisher精确检验进行CP频率的比较。每个实验所使用的统计检验方法在图例中注明。值为。

< 0.05 was considered statistically significant. All in vitro experiments were performed in triplicate. Animal feeding, treatments and histological analyses were performed in a single-blinded fashion.

< 0.05 被认为具有统计学意义。所有体外实验均进行三次重复。动物饲养、处理及组织学分析均以单盲方式进行。

Ethics approval and consent to participate

伦理批准与知情同意

All animal experimentation was approved by the Animal Care and Use Committee at Beijing Stomatological Hospital, affiliated with Capital Medical University (permit number: KQYY-202208-003, Beijing, China).

所有动物实验均经首都医科大学附属北京口腔医院动物护理与使用委员会批准（批准号：KQYY-202208-003，中国北京）。

Data availability

数据可用性

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

本研究结果的数据可在合理要求下由通讯作者提供。

References

参考文献

Lewis, C. W., Jacob, L. S. & Lehmann, C. U. The primary care pediatrician and the care of children with cleft lip and/or cleft palate.

刘易斯，C. W.，雅各布，L. S.，莱曼，C. U. 基层儿科医生与唇裂和/或腭裂儿童的护理。

Pediatrics

儿科

139

, e20170628 (2017).

，e20170628（2017）。

Dixon, M. J., Marazita, M. L., Beaty, T. H. & Murray, J. C. Cleft lip and palate: understanding genetic and environmental influences.

达克森，M. J.，马拉齐塔，M. L.，贝蒂，T. H.，穆雷，J. C. 唇腭裂：了解遗传和环境影响。

Nat. Rev. Genet.

自然遗传学评论

, 167–178 (2011).

，167-178页（2011年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术搜索

Aagaard, K. et al. The placenta harbors a unique microbiome.

Aagaard, K. 等。胎盘拥有独特的微生物组。

Sci. Transl. Med.

科学转化医学

, 237ra65 (2014).

，237ra65（2014）。

PubMed

PubMed Central

Google Scholar

谷歌学术

Wu, S. et al. Do maternal microbes shape newborn oral microbes?

吴珊等。母体微生物是否会影响新生儿口腔微生物？

Indian J. Microbiol.

印度微生物学杂志

, 16–23 (2021).

，16-23页（2021年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术索

Bobetsis, Y. A., Graziani, F., Gürsoy, M. & Madianos, P. N. Periodontal disease and adverse pregnancy outcomes.

Bobetsis, Y. A., Graziani, F., Gürsoy, M. & Madianos, P. N. 牙周病与不良妊娠结局。

Periodontology

牙周病学

, 154–174 (2020).

，154-174页（2020年）。

Google Scholar

谷歌学术

Bánhidy, F., Acs, N., Puhó, E. H. & Czeizel, A. E. A possible association of periodontal infectious diseases in pregnant women with isolated orofacial clefts in their children: a population-based case-control study.

Bánhidy, F., Acs, N., Puhó, E. H. & Czeizel, A. E. 孕妇牙周感染疾病与子女孤立性口腔面裂的可能关联：一项基于人群的病例对照研究。

Birth Defects Res A Clin. Mol. Teratol.

出生缺陷研究A：临床与分子致畸学

, 466–473 (2010).

，466-473页（2010年）。

PubMed

Google Scholar

谷歌学术

Hammond, N. L. & Dixon, M. J. Revisiting the embryogenesis of lip and palate development.

Hammond, N. L. & Dixon, M. J. 重新审视唇腭发育的胚胎发生。

Oral. Dis.

口腔科。

, 1306–1326 (2022).

，1306-1326（2022）。

PubMed

PubMed Central

Google Scholar

谷歌学术

Figlia, G., Willnow, P. & Teleman, A. A. Metabolites regulate cell signaling and growth via covalent modification of proteins.

菲利亚，G.，威尔诺，P.，特莱曼，A. A. 代谢物通过蛋白质的共价修饰调控细胞信号传导和生长。

Dev. Cell

开发。细胞

, 156–170 (2020).

，156-170页（2020年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Bhattacharya, D., Khan, B. & Simoes-Costa, M. Neural crest metabolism: at the crossroads of development and disease.

Bhattacharya, D., Khan, B. & Simoes-Costa, M. 神经嵴代谢：发育与疾病的十字路口。

Dev. Biol.

发育生物学

475

, 245–255 (2021).

，245-255（2021）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Mehrotra, P. et al. Wnt/BMP mediated metabolic reprogramming preserves multipotency of neural crest-like stem cells.

Mehrotra, P. 等。Wnt/BMP 介导的代谢重编程保持神经嵴样干细胞的多能性。

Stem Cells

干细胞

, 287–305 (2023).

，287-305页（2023年）。

PubMed

PubMed Central

Google Scholar

谷歌学术索

Peng, X. et al. Osteogenic microenvironment affects palatal development through glycolysis.

彭，X. 等。成骨微环境通过糖酵解影响腭部发育。

Differentiation

分化

133

, 1–11 (2023).

，1-11页（2023年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Chopra, A., Radhakrishnan, R. & Sharma, M. Porphyromonas gingivalis and adverse pregnancy outcomes: a review on its intricate pathogenic mechanisms.

乔普拉，A.，拉达克里希南，R.，夏尔马，M. 牙龈卟啉单胞菌与不良妊娠结局：其复杂致病机制的综述。

Crit. Rev. Microbiol.

微生物学评论

, 213–236 (2020).

，213-236页（2020年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Starzyńska, A. et al. Association between maternal periodontitis and development of systematic diseases in offspring.

Starzyńska, A. 等。母亲牙周炎与后代系统性疾病发展之间的关联。

Int. J. Mol. Sci.

国际分子科学杂志

, 2473 (2022).

，2473（2022）。

PubMed

PubMed Central

Google Scholar

谷歌学术搜索

Fleetwood, A. J. et al. Metabolic remodeling, inflammasome activation, and pyroptosis in macrophages stimulated by porphyromonas gingivalis and its outer membrane vesicles.

Fleetwood, A. J. 等。牙龈卟啉单胞菌及其外膜囊泡刺激巨噬细胞中的代谢重塑、炎症小体激活和细胞焦亡。

Front. Cell. Infect. Microbiol.

前言。细胞。感染。微生物。

, 351 (2017).

，351（2017）。

PubMed

PubMed Central

Google Scholar

谷歌学术

Yu, Y., Jiang, L., Li, J., Lei, L. & Li, H. Hexokinase 2-mediated glycolysis promotes receptor activator of NF-κB ligand expression in Porphyromonas gingivalis lipopolysaccharide-treated osteoblasts.

余洋、江莉、李杰、雷蕾、李红。己糖激酶2介导的糖酵解促进牙龈卟啉单胞菌脂多糖处理的成骨细胞中NF-κB受体活化因子配体的表达。

J. Periodontol.

牙周病学杂志

, 1036–1047 (2022).

，1036–1047（2022）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术搜索

Suttorp, C. M. et al. Chemokine signaling during midline epithelial seam disintegration facilitates palatal fusion.

Suttorp, C. M. 等。趋化因子信号在中线表皮缝分解过程中促进腭融合。

Front. Cell Dev. Biol.

细胞与发育生物学前沿

, 94 (2017).

，94（2017）。

PubMed

PubMed Central

Google Scholar

谷歌学术索

Xu, Y., Yuan, D., Fan, Z., Wang, S. & Du, J. Identification and profiles of microRNAs in different development stages of miniature pig secondary palate.

徐，Y.，袁，D.，范，Z.，王，S.，杜，J. 不同发育阶段小型猪次级腭中微小RNA的鉴定与表达谱。

Genomics

基因组学

113

, 2634–2644 (2021).

，2634–2644（2021）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

McIntosh, A. et al. Iron accumulation in microglia triggers a cascade of events that leads to altered metabolism and compromised function in APP/PS1 mice.

麦金托什，A. 等。小胶质细胞中铁的积累引发了一系列事件，导致APP/PS1小鼠代谢改变和功能受损。

Brain Pathol.

脑病理学。

, 606–621 (2019).

，606-621页（2019年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Sun, L., Zhang, H. & Gao, P. Metabolic reprogramming and epigenetic modifications on the path to cancer.

孙，L.，张，H.，高，P. 代谢重编程和表观遗传修饰在癌症路径上的变化。

Protein Cell

蛋白质细胞

, 877–919 (2022).

，877-919（2022）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术索

Zhang, D. et al. Metabolic regulation of gene expression by histone lactylation.

张，D. 等。组蛋白乳酸化对基因表达的代谢调控。

Nature

自然

574

, 575–580 (2019).

，575-580页（2019年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Chen, J. et al. Histone lactylation driven by mROS-mediated glycolytic shift promotes hypoxic pulmonary hypertension.

陈杰等。组蛋白乳酸化通过活性氧介导的糖酵解转变促进低氧性肺动脉高压。

J. Mol. Cell Biol.

分子细胞生物学杂志

, mjac073 (2023).

，mjac073（2023）。

PubMed

Google Scholar

谷歌学术

Wang, Y. et al. Novel investigations in retinoic-acid-induced cleft palate about the gut microbiome of pregnant mice.

王，Y. 等。关于孕鼠肠道微生物组的视黄酸诱导腭裂的新研究。

Front. Cell. Infect. Microbiol.

前言。细胞。感染。微生物学。

, 1042779 (2022).

，1042779（2022）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Hayashi, K. et al. Continuous oral administration of sonicated

林，K. 等。超声波连续口服给药

P. gingivalis

牙龈卟啉单胞菌

delays rat skeletal muscle healing post-treadmill training.

跑步机训练后延迟大鼠骨骼肌愈合。

Int J. Environ. Res. Public Health

国际环境研究与公共卫生杂志

, 13046 (2022).

，13046（2022）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Yoshida, S. et al. Porphyromonas gingivalis administration induces gestational obesity, alters gene expression in the liver and brown adipose tissue in pregnant mice, and causes underweight in fetuses.

吉田，S. 等。牙龈卟啉单胞菌给药诱导妊娠肥胖，改变怀孕小鼠肝脏和棕色脂肪组织中的基因表达，并导致胎儿体重不足。

Front. Cell. Infect. Microbiol.

前言。细胞。感染。微生物学。

, 745117 (2021).

，745117（2021）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Guo, W., Fan, Z., Wang, S. & Du, J. ALK5 is essential for tooth germ differentiation during tooth development.

郭，W.，范，Z.，王，S.，杜，J. ALK5在牙齿发育过程中对牙胚分化至关重要。

Biotech. Histochem.

生物技术。组织化学。

, 481–490 (2019).

，481-490页（2019年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术搜索

Liu, H. & Du, J. Effect of transforming growth factor β2 on chondrogenic differentiation of mouse embryonic palatal cells(MEPC).

刘, H. & 杜, J. 转化生长因子β2对小鼠胚胎腭细胞(MEPC)软骨分化的影响。

Beijing J. Stom

北京口腔医学期刊

(2), 61–85 (2017).

Google Scholar

谷歌学术

Dudas, M. et al. Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion.

Dudas, M. 等。I型TGF-β受体ALK5在面部形态发生和腭融合过程中的上皮和外中胚层作用。

Dev. Biol.

发育生物学

296

, 298–314 (2006).

，298-314页（2006年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Liu, C., Xu, P., Lamouille, S., Xu, J. & Derynck, R. TACE-mediated ectodomain shedding of the type I TGF-beta receptor downregulates TGF-beta signaling.

刘, C., 徐, P., 拉穆伊, S., 徐, J. & 德林克, R. TACE介导的I型TGF-β受体胞外域脱落下调TGF-β信号传导。

Mol. Cell

分子细胞

, 26–36 (2009).

，26-36页（2009年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Düsterhöft, S., Lokau, J. & Garbers, C. The metalloprotease ADAM17 in inflammation and cancer.

杜斯特霍夫特，S.，洛考，J. & 加伯斯，C. 金属蛋白酶ADAM17在炎症和癌症中的作用。

Pathol. Res Pract.

病理学研究与实践

215

, 152410 (2019).

，152410（2019）。

PubMed

Google Scholar

谷歌学术索

Toda, G., Yamauchi, T., Kadowaki, T. & Ueki, K. Preparation and culture of bone marrow-derived macrophages from mice for functional analysis.

户田，G.，山内，T.，影崎，T.，上树，K. 从小鼠制备和培养骨髓来源的巨噬细胞用于功能分析。

STAR Protoc.

星形协议。

, 100246 (2021).

，100246（2021）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Zhang, Y. et al. Angiotensin II deteriorates advanced atherosclerosis by promoting MerTK cleavage and impairing efferocytosis through the AT(1)R/ROS/p38 MAPK/ADAM17 pathway.

张, Y. 等。血管紧张素 II 通过 AT(1)R/ROS/p38 MAPK/ADAM17 通路促进 MerTK 裂解并损害胞葬作用，从而加剧晚期动脉粥样硬化。

Am. J. Physiol. Cell Physiol.

美国生理学杂志：细胞生理学

317

, C776–c787 (2019).

，C776–c787（2019）。

PubMed

Google Scholar

谷歌学术索

Zhang, S. et al. Efferocytosis fuels requirements of fatty acid oxidation and the electron transport chain to polarize macrophages for tissue repair.

张，S. 等。尸体清除作用满足脂肪酸氧化和电子传递链的需求，以极化巨噬细胞进行组织修复。

Cell Metab.

细胞代谢

, 443–456.e5 (2019).

，443-456.e5（2019）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Hajishengallis, G. Periodontitis: from microbial immune subversion to systemic inflammation.

Hajishengallis, G. 牙周炎：从微生物免疫颠覆到系统性炎症。

Nat. Rev. Immunol.

自然免疫学评论

, 30–44 (2015).

，30-44页（2015年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Brandes, R. P. & Rezende, F. Glycolysis and inflammation: partners in crime!

布兰德斯，R. P. & 雷津德，F. 糖酵解与炎症：犯罪伙伴！

Circ. Res

循环研究

。

129

, 30–32 (2021).

，30-32页（2021年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Ying, J. et al. Inflammation-mediated aberrant glucose metabolism in subchondral bone induces osteoarthritis.

英, J. 等。炎症介导的软骨下骨葡萄糖代谢异常诱发骨关节炎。

Stem Cells

干细胞

, 482–492 (2023).

，482-492页（2023年）。

PubMed

Google Scholar

谷歌学术索

Zhang, Y. et al. Porphyromonas gingivalis msRNA P.G_45033 induces amyloid-β production by enhancing glycolysis and histone lactylation in macrophages.

张, Y. 等。牙龈卟啉单胞菌 msRNA P.G_45033 通过增强巨噬细胞中的糖酵解和组蛋白乳酸化诱导淀粉样蛋白-β 的产生。

Int. Immunopharmacol.

国际免疫药理学。

121

, 110468 (2023).

，110468（2023）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Wang, X. et al. BRAFV600E restructures cellular lactylation to promote anaplastic thyroid cancer proliferation.

王，X. 等。BRAFV600E 重构细胞乳酸化以促进间变性甲状腺癌增殖。

Endocr. Relat. Cancer

内分泌相关癌症

, e220344 (2023).

，e220344（2023）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Markus, A., Yan, X., Rolfs, A. & Luo, J. Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development.

Markus, A., Yan, X., Rolfs, A. & Luo, J. 鸡脑发育过程中区域ADAM蛋白的前体和活性形式的定量和动态表达谱。

Cell Mol. Biol. Lett.

细胞与分子生物学快报

, 431–451 (2011).

，431-451页（2011年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术索

Lin, J. et al. Expression of seven members of the ADAM family in developing chicken spinal cord.

林，J. 等。鸡胚脊髓发育过程中ADAM家族七个成员的表达。

Dev. Dyn.

发育动态

239

, 1246–1254 (2010).

，1246-1254（2010）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Yan, X., Lin, J., Rolfs, A. & Luo, J. Differential expression of the ADAMs in developing chicken retina.

颜晓，林静，罗尔夫斯，罗军。ADAMs在发育中的鸡视网膜中的差异表达。

Dev. Growth Differ.

发育、生长和分化。

, 726–739 (2011).

，726-739页（2011年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Biggs, L. C., Goudy, S. L. & Dunnwald, M. Palatogenesis and cutaneous repair: a two-headed coin.

Biggs, L. C., Goudy, S. L. & Dunnwald, M. 腭发生与皮肤修复：一枚双面硬币。

Dev. Dyn.

发育动态

244

, 289–310 (2015).

，289-310页（2015年）。

PubMed

Google Scholar

谷歌学术

Aitcheson, S. M., Frentiu, F. D., Hurn, S. E., Edwards, K. & Murray, R. Z. Skin wound healing: normal macrophage function and macrophage dysfunction in diabetic wounds.

艾奇逊，S. M.，弗伦蒂乌，F. D.，赫恩，S. E.，爱德华兹，K.，穆雷，R. Z. 皮肤伤口愈合：正常巨噬细胞功能与糖尿病伤口中的巨噬细胞功能障碍。

Molecules

分子

, 4917 (2021).

，4917（2021）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术搜索

Zhao, Z., Zhao, M., Xiao, G. & Franceschi, R. T. Gene transfer of the Runx2 transcription factor enhances osteogenic activity of bone marrow stromal cells in vitro and in vivo.

赵，Z.，赵，M.，肖，G.，& 弗朗西斯奇，R. T. Runx2转录因子的基因转移增强了骨髓基质细胞在体外和体内的成骨活性。

Mol. Ther.

分子治疗学

, 247–253 (2005).

，247-253页（2005年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Chen, G., Deng, C. & Li, Y. P. TGF-β and BMP signaling in osteoblast differentiation and bone formation.

陈, G., 邓, C. & 李, Y. P. TGF-β 和 BMP 信号在成骨细胞分化和骨形成中的作用。

Int. J. Biol. Sci.

国际生物科学杂志

, 272–288 (2012).

，272-288页（2012年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Escoll, P. et al. Legionella pneumophila modulates mitochondrial dynamics to trigger metabolic repurposing of infected macrophages.

埃斯科尔，P. 等。嗜肺军团菌调节线粒体动力学以触发受感染巨噬细胞的代谢重编程。

Cell Host Microbe

细胞宿主微生物

, 302–316.e7 (2017).

，302-316.e7（2017）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Kuboniwa, M. et al. P. gingivalis accelerates gingival epithelial cell progression through the cell cycle.

久保庭，M. 等。牙龈卟啉单胞菌通过细胞周期加速牙龈上皮细胞的进程。

Microbes Infect.

微生物感染。

, 122–128 (2008).

，122-128页（2008年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Liu, J. et al. Dynamic mRNA expression analysis of the secondary palatal morphogenesis in miniature pigs.

刘, J. 等。微型猪次级腭形态发生中的动态mRNA表达分析。

Int J. Mol. Sci.

国际分子科学杂志

, 4284 (2019).

，4284（2019）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术

Jia, L. et al.

贾，L. 等。

Porphyromonas gingivalis

牙龈卟啉单胞菌

and

和

Lactobacillus rhamnosus

鼠李糖乳杆菌

GG regulate the Th17/Treg balance in colitis via TLR4 and TLR2.

GG通过TLR4和TLR2调节结肠炎中的Th17/Treg平衡。

Clin. Transl. Immunol.

临床与转化免疫学

, e1213 (2020).

，e1213（2020）。

CAS

中国科学院

Google Scholar

谷歌学术

Zhang, X. et al. Inflammatory macrophages exacerbate neutrophil-driven joint damage through ADP/P2Y(1) signaling in rheumatoid arthritis.

张, X. 等。炎症性巨噬细胞通过类风湿性关节炎中的ADP/P2Y(1)信号通路加剧中性粒细胞驱动的关节损伤。

Sci. China Life Sci.

中国科学：生命科学

, 953–968 (2022).

，953-968页（2022年）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Wang, Y. et al. M2 macrophage-derived exosomes promote diabetic fracture healing by acting as an immunomodulator.

王宇等。M2巨噬细胞衍生的外泌体通过作为免疫调节剂促进糖尿病骨折愈合。

Bioact. Mater.

生物活性材料

, 273–283 (2023).

，273-283页（2023年）。

CAS

中国科学院

PubMed

PubMed Central

Google Scholar

谷歌学术索

Chen, J. et al. Autophagy triggered by the ROS/ERK signaling pathway protects mouse embryonic palatal cells from apoptosis induced by nicotine.

陈杰等。ROS/ERK信号通路触发的自噬保护小鼠胚胎腭细胞免受尼古丁诱导的凋亡。

Environ. Sci. Pollut. Res. Int.

环境科学与污染研究国际版

, 81909–81922 (2022).

，81909–81922（2022）。

CAS

中国科学院

PubMed

Google Scholar

谷歌学术

Download references

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Acknowledgements

致谢

This research was funded by grants from the National Natural Science Foundation of China (grant numbers 82170912 and 82370910), and the Beijing Stomatological Hospital, Capital Medical University Young Scientist Program (No. YSP202404).

本研究由中国国家自然科学基金（批准号：82170912 和 82370910）以及首都医科大学附属北京口腔医院青年科学家计划（编号：YSP202404）资助。

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Laboratory of Orofacial Development, Laboratory of Molecular Signaling and Stem Cells Therapy, Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China

中国北京，首都医科大学口腔医学院，口腔颌面发育实验室，分子信号与干细胞治疗实验室，基因治疗与牙齿再生分子实验室，北京牙齿再生与功能重建重点实验室

Xige Zhao, Xiaoyu Zheng, Yijia Wang, Jing Chen, Xiaotong Wang, Xia Peng, Ying Liu, Zhiwei Wang & Juan Du

赵锡革，郑晓宇，王一佳，陈静，王晓彤，彭霞，刘颖，王志伟，杜娟

Department of geriatric dentistry, Capital Medical University School of Stomatology, Beijing, China

中国北京首都医科大学口腔医学院老年牙科系

Dong Yuan & Juan Du

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Contributions

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Conceptualization, J.D.; Methodology, X.-G.Z., X.-Y.Z., Y.-J.W., J.C., and X.-T.W.; Software, X.P., Z.-W.W.; Validation, D.Y., Y.L.; Writing—Original Draft Preparation, X.-G.Z.; Writing—Review & Editing, J.D.; Funding Acquisition, J.D., X.-G.Z. All authors gave final approval and agreed to be accountable for all aspects of the work..

概念化，J.D.；方法论，X.-G.Z.，X.-Y.Z.，Y.-J.W.，J.C.，和 X.-T.W.；软件，X.P.，Z.-W.W.；验证，D.Y.，Y.L.；撰写—原稿准备，X.-G.Z.；撰写—审阅与编辑，J.D.；资金获取，J.D.，X.-G.Z.。所有作者均给予最终批准并同意对工作的各个方面负责。

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Juan Du

杜安

。

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Zhao, X., Zheng, X., Wang, Y.