牙龈卟啉单胞菌脂多糖通过MEK/ERK/MLCK/MLC2途径破坏口腔上皮屏障功能的机制初探

口腔生物医学 ›› 2025, Vol. 16 ›› Issue (3): 127-134.

牙龈卟啉单胞菌脂多糖通过MEK/ERK/MLCK/MLC2途径破坏口腔上皮屏障功能的机制初探

钱文博,郝新宇,孙颖

南京医科大学附属口腔医院牙周病科，江苏省口腔疾病研究重点实验室，江苏省口腔转化医学工程研究中心

收稿日期:2025-04-21 修回日期:2025-05-22 出版日期:2025-06-25 发布日期:2025-06-30
通讯作者: 孙颖 E-mail:ebolasun@163.com
基金资助:
江苏省自然科学基金;江苏省卫健委重点项目;江苏省科教能力提升工程—江苏省研究型医院;江苏省医学创新中心

Porphyromonas gingivalis lipopolysaccharide disrupts oral epithelial barrier function viaMEK/ERK/MLCK/MLC2 pathway

Received:2025-04-21 Revised:2025-05-22 Online:2025-06-25 Published:2025-06-30

摘要/Abstract

摘要： 目的：探讨牙龈卟啉单胞菌（P. gingivalis）脂多糖（LPS）构建的炎症微环境中口腔上皮紧密连接（TJ）蛋白的表达变化与调控机制以及上述变化对上皮屏障功能的影响。方法：收集16例牙周炎患者和14例牙周健康者的牙龈组织样本，免疫组化检测咬合蛋白（Occludin）、闭锁小带蛋白-1（ZO-1）等TJ蛋白和肌球蛋白轻链激酶（MLCK）、肌球蛋白轻链2（MLC2）等信号分子表达水平，分析其与牙周临床指标牙龈指数（GI）、探诊深度（PD）、菌斑指数（PLI）和临床附着丧失（CAL）的相关性。细胞外信号调节激酶（MEK）抑制剂U0126或MLCK抑制剂ML7预处理后，采用P. gingivalis LPS刺激人口腔角化细胞（HOKs），测量跨上皮电阻（TEER）值和异硫氰酸荧光素葡聚糖4（FD4）的渗透系数，Western blot检测Occludin、ZO-1和信号分子MEK、p-MEK、ERK、p-ERK、MLCK、MLC2、p-MLC2表达水平。结果：与牙周健康者相比，牙周炎患者牙龈上皮细胞中，Occludin和ZO-1的胞膜表达降低，Occludin的胞质表达增高，ZO-1的胞质表达降低，MLCK和MLC2在胞质表达增强（P<0.05）。Occludin、ZO-1在胞膜的表达与GI、PD、CAL呈负相关（P<0.05），MLCK、MLC2与GI、PD、CAL呈正相关（P<0.05）。P. gingivalis LPS刺激6 h后，HOKs的TEER值降低，FD4的渗透系数增加，Occludin和ZO-1蛋白表达水平降低（P<0.05）；P. gingivalis LPS刺激1 h后，p-MEK/MEK、p-ERK/ERK、MLCK和p-MLC2/MLC2水平增高（P<0.05），加入U0126或ML7后，上述信号分子表达降低，TJ蛋白表达升高，TEER值增加，渗透系数减小（P<0.05）。结论：P. gingivalis LPS可能通过MEK/ERK/MLCK/MLC2途径调控口腔上皮TJ蛋白Occludin和ZO-1表达，进而破坏上皮屏障功能。

关键词: 牙龈卟啉单胞菌, 脂多糖, 上皮屏障, 紧密连接, 肌球蛋白轻链2

Abstract: Objective: To investigate the changes in the expression of tight junction (TJ) proteins and the underlying regulatory mechanism in inflammatory microenvironment induced by Porphyromonas gingivalis(P.gingivalis) lipopolysaccharide (LPS), as well as the effect of these changes on epithelial barrier. Methods: Gingivae were collected from 16 periodontitis patients and 14 periodontally healthy individuals. The expression levels of TJ proteins, Occludin and zona occluden-1 (ZO-1), and signaling molecules, myosin light chain kinase (MLCK) and myosin light chain 2 (MLC2), were detected by immunohistochemistry, and the correlations between them and periodontal clinical parameters, gingival index (GI), probing depth (PD), plaque index (PLI) and clinical attachment loss (CAL), were analyzed. Human oral keratinocytes (HOKs) were pretreated with or without MLCK inhibitor ML7 or MEK inhibitor U0126, followed by stimulation with P. gingivalis LPS. Transepithelial electrical resistance (TEER) and permeability coefficient of fluorescein isothiocyanate dextran 4 (FD4) were measured. The expression levels of Occludin, ZO-1, MEK, ERK, MLCK and MLC2 were detected by Western blot. Results: Compared with the healthy controls, there were decreased expressions of Occludin and ZO-1 in cell membrane, increased cytoplasmic expression of Occludin, MLCK and MLC2, and decreased cytoplasmic expression of ZO-1 (P<0.05). Levels of MLCK and MLC2 were positively correlated with GI, PD and CAL (P<0.05), and cytosolic expressions of Occludin and ZO-1 were negatively correlated with GI, PD and CAL (P<0.05). After P. gingivalis LPS stimulation for 6 h, reduced levels of TEER, Occludin and ZO-1, and increased permeability coefficient of FD4 in HOKs were observed (P<0.05). After LPS stimulation for 1 h, the levels of p-MEK/MEK, p-ERK/ERK, MLCK and p-MLC2/MLC2 were increased (P<0.05). Pretreatment with U0126 or ML7 reduced the expressions of the aforementioned signaling molecules, increased TJ protein expressions, elevated TEER and decreased permeability coefficient of FD4 (P<0.05). Conclusions: P. gingivalis LPS may regulate the expressions of TJ proteins (Occludin and ZO-1) in oral epithelia via MEK/ERK/MLCK/MLC2 pathway, thereby disrupting epithelial barrier.

Key words: Porphyromonas gingivalis, lipopolysaccharide, epithelial barrier, tight junction, myosin light chain 2

钱文博郝新宇孙颖. 牙龈卟啉单胞菌脂多糖通过MEK/ERK/MLCK/MLC2途径破坏口腔上皮屏障功能的机制初探[J]. 口腔生物医学, 2025, 16(3): 127-134.

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