基于miR-155/-146α调控p38丝裂原活化蛋白激酶信号通路探讨电针缓解膝骨性关节炎的机制研究

The mechanism of electroacupuncture in relieving knee osteoarthritis based on mir-155/-146αregulating p38 MAPK signaling pathway

目的:基于miR-155/-146α调控p38丝裂原活化蛋白激酶(p38 Mitogen-activated Protein Kinase,p38 MAPK)信号通路探讨电针治疗膝骨性关节炎的作用机制。方法:体内体外模型诱导鉴定后,采用番红O-固绿及苏木精-伊红(Hematoxylineosin,HE)染色观察软骨组织形态学改变,酶联免疫吸附试验(Enzyme-linked Immunosorbent Method,ELISA)检测血清炎性因子白细胞介素(Interleukin,IL)-6、IL-1β、肿瘤坏死因子(Tumor Necrosis Factor,TNF)-α的表达,qPCR及Western blot检测软骨组织和细胞中miR-155、miR-146αmRNA转录水平和p38 MAPK蛋白表达情况,Tunel染色、流式细胞术、激光共聚焦显微镜观察软骨细胞凋亡情况。结果:(1)micro-CT显示:正常组关节面平整,边缘光滑圆润,模型组关节面粗糙,边缘不规则;CollagenⅡ免疫细胞化学染色显示:空白组软骨细胞胞浆呈棕黄色,胞核呈蓝紫色,与空白组比较,模型组软骨细胞胞浆棕黄色颜色变浅。(2)番红O-固绿和HE染色显示:与正常组比较,模型组结构紊乱,软骨表面粗糙,可见部分磨损、缺失,软骨细胞排列杂乱无序;与模型组比较,电针组明显改善,四层结构清晰,软骨表面光滑,软骨细胞排列整齐有序。(3)ELISA结果显示:与正常组比较,模型组IL-6、IL-1β、TNF-α促炎细胞因子显著增加;与模型组比较,电针组IL-6、IL-1β、TNF-α的表达有效减少(P<0.05)。(4)在软骨组织中,与正常组比较,模型组miR-155、miR-146αmRNA和p38 MAPK蛋白表达显著增高,与模型组比较,电针组有效抑制了miR-155、miR-146αmRNA及p38 MAPK蛋白表达(P<0.05);在软骨细胞中,与空白组比较,模型组miR-155、mi R-146αmRNA和p38 MAPK蛋白表达显著增高,与模型组比较,电针组和抑制剂组有效抑制了miR-155、miR-146αmRNA及p38 MAPK蛋白表达(P<0.05)。(5)软骨组织Tunel染色显示:与正常组比较,模型组软骨组织细胞核皱缩数量增多,凋亡细胞显著增多;与模型组比较,电针组细胞核皱缩数量减少,凋亡减少。流式细胞检测及激光共聚焦显微镜显示:与空白组比较,模型组软骨细胞凋亡和细胞色素C蛋白表达显著增多,电针组和抑制剂组均有效抑制了细胞凋亡和细胞色素C蛋白表达(P<0.01)。结论:电针可以改善软骨退变、抑制炎症从而减轻凋亡,其具体机制与介导miR-155/-146α调控p38 MAPK信号通路有关。

Objective:To explore the mechanism of electroacupuncture on knee osteoarthritis based on the regulation of p38 MAPK signaling pathway by miR-155/-146α.Methods:After induction and identification in vitro and in vivo models,the morphological changes of cartilage were observed by saffranine O-solid green and HE staining.Enzyme-linked immunosorbent method was used to detect serum inflammatory factors such as IL-6,IL-1βand TNF-α.The mRNA transcription levels of miR-155 and miR-146αand the protein expression of p38 MAPK in cartilage tissues and cells were detected by qPCR and Western blot.The apoptosis of chondrocytes was observed by Tunel staining,flow cytometry and confocal laser microscopy.Results:micro-CT showed that the joint surface in the normal group was smooth and the edge was smooth and round,while the joint surface in the model group was rough and the edge was irregular.Collagen II immunocytochemical staining showed that the cytoplasm of chondrocytes in the blank group was brown-yellow and the nucleus blue-purple,and the cytoplasm of chondrocytes in the model group was lighter than that in the blank group,etc.Conclusion:Electroacupuncture can improve cartilage degeneration and inhibit inflammation so as to alleviate apoptosis,which is related to the regulation of p38 MAPK signaling pathway by miR-155/-146α.