透骨消痛胶囊调节Ras-Raf-MEK1/2-ERK1/2信号通路抑制骨关节炎炎症反应的机制研究  

Mechanism of Inhibition of Inflammation in Osteoarthritis by Tougu Xiaotong Capsule via Regulating Ras-Raf-MEK1/2-ERK1/2 Signaling Pathway

作　　者：林木南 邵翔 许丽梅 张欣 朱在师 韩一旦 段辛威 陈建梅 高松 李西海 LIN Munan;SHAO Xiang;XU Limei;ZHANG Xin;ZHU Zaishi;HAN Yidan;DUAN Xinwei;CHEN Jianmei;GAO song;LI Xihai(The 900th Hospital of the Joint Logistics Support Force,PLA,Fuzhou,Fujian 350025,China;College of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China)

机构地区：[1]中国人民解放军联勤保障部队第九〇〇医院,福建福州350025 [2]福建中医药大学中西医结合学院,福建福州350122

出　　处：《康复学报》2021年第3期228-233,共6页Rehabilitation Medicine

基　　金：国家自然科学基金项目(81573998)。

摘　　要：目的:探讨透骨消痛胶囊调节Ras-Raf-MEK1/2-ERK1/2信号通路抑制骨关节炎炎症反应的作用机制。方法:取4周龄SD雄性大鼠的双侧膝关节软骨,经Ⅱ型胶原酶消化法获得大鼠软骨细胞,并采用Ⅱ型胶原免疫组化法对所得细胞进行鉴定;取第2代软骨细胞分为空白组、模型组(10 ng/mL LPS干预8 h)、抑制剂组(10 ng/mL U0126先干预30 min后再加入10 ng/mL LPS干预8 h)、透骨消痛胶囊组(10 ng/mL LPS+300μg/mL透骨消痛胶囊一同干预8 h)。采用Western blot检测软骨细胞中Ras、Raf、p-ERK1/2、MEK1/2、MMP-3和MMP-13蛋白表达,qPCR检测软骨细胞中MEK1/2、ERK1/2、MMP-3和MMP-13基因表达及miR-27b、miR-34a和miR-146a表达。结果:①软骨细胞经Ⅱ型胶原免疫组化处理后,阳性组细胞胞浆被染为棕黄色,阴性组不显色,表明所提取细胞为软骨细胞。②与空白组相比,模型组中Ras、Raf、MEK1/2、MMP-3和MMP-13蛋白表达明显升高(P<0.01,P<0.05),p-ERK1/2蛋白表达量也升高,但差异无统计学意义(P>0.05);与模型组相比,抑制剂组与透骨消痛胶囊组Ras、Raf、MEK1/2、p-ERK1/2、MMP-3、MMP-13蛋白表达均下降(P<0.01,P<0.05);透骨消痛胶囊组与模型组相比,MEK1/2蛋白表达降低,但差异无统计学意义(P>0.05)。③与空白组相比,模型组中MEK1/2、ERK1/2、MMP-3、MMP-13基因表达明显升高(P<0.01);与模型组相比,透骨消痛胶囊组与抑制剂组上述基因表达均下降(P<0.01,P<0.05)。④模型组miR-27b的表达低于空白组(P<0.01),抑制剂组、透骨消痛胶囊组miR-27b的表达高于模型组(P<0.01);模型组miR-34a、miR-146a的表达均高于空白组(P<0.01,P<0.05),而抑制剂组、透骨消痛胶囊组均低于模型组(P<0.01,P<0.05)。结论:透骨消痛胶囊通过调控Ras-Raf-MEK1/2-ERK1/2信号通路中关键因子及miR-27b、miR-34a和miR-146a的表达,调节软骨细胞炎症反应,从而抑制骨关节炎炎症,保护关节软骨。Objective:To investigate the mechanism of inhibition of inflammation in osteoarthritis by Tougu Xiaotong capsule(TXC)via regulating Ras-Raf-MEK1/2-ERK1/2 signaling pathways.Methods:The chondrocytes from 4-week-old SD male rats obtained by the mechanical typeⅡcollagenase digestion method were cultured in vitro and identified by typeⅡcollagen immunohistochemical staining.Isolated chondrocytes of passage two were divided into blank group,model group(treatment with 10 ng/mL of LPS for 8 h),inhibitor group(treatment with 10 ng/mL of U0126 for 30 min followed by 10 ng/mL of LPS for additional 8 h)and TXC group(treatment with 10 ng/mL of LPS plus 300μg/mL of TXC for 8 h).Western blot was carried out to detect the expression of Ras,Raf,p-ERK1/2,MEK1/2,MMP-3 and MMP-13.qPCR was used to detect the mRNAs expression of MEK1/2,ERK1/2,MMP-3 and MMP-13,as well as the expression of miR-27b,miR-34a and miR-146a.Results:1)The cytoplasm of isolated cells(P2)from the cartilage of knee joint of 4-week-old SD male rats was stained brown by typeⅡcollagen immunohistochemical staining as compared with the negative control cells,which indicated that the extracted cells were chondrocytes.2)The expression of Ras,Raf,MEK1/2,MMP-3,MMP-13 in the model group was significantly higher than that in the blank group,respectively(P<0.01,P<0.05),but the expression of p-ERK1/2 had no significant difference(P>0.05);the expression of Ras,Raf,p-ERK1/2,MEK1/2,MMP-3,MMP-13 in both the inhibitor group and the TXC group was lower than that in the model group,respectively(P<0.01,P<0.05),but the expression of MEK1/2 in the TXC group had no significant difference compared to that in the model group(P>0.05).3)The mRNA expression of MEK1/2,ERK1/2,MMP-3 and MMP-13 in the model group was significantly higher than that in the blank group,respectively(P<0.01);their expression in both the inhibitor group and the TXC group was lower than that in the model group,respectively(P<0.01,P<0.05).4)The expression of miR-27b in the model group was significantly lower than

关 键 词：骨关节炎 透骨消痛胶囊 MIRNAS Ras-Raf-MEK1/2-ERK1/2信号通路 

分 类 号：R285.5[医药卫生—中药学]