骨关节炎大鼠软骨PI3K/Akt-mTOR及Beclin-1自噬通路的表达及相关性分析  

Expression of Autophagy Pathway PI3K/Akt-mTOR and Beclin-1 and Correlation Analysis in Osteoarthritis Rats

作　　者：阮丽萍[1] 刘健[2] 葛瑶[2] 万磊[2] 王亚黎[1] 叶文芳[1] 

机构地区：[1]安徽中医药大学研究生部,合肥230038 [2]安徽中医药大学第一附属医院风湿科,合肥230031

出　　处：《华中科技大学学报（医学版）》2015年第4期429-433,439,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81173211);国家中医药重点学科中医痹病学建设项目(No.国中医药发[2009]30号);安徽省科技厅科研计划项目(No.09-020304046)

摘　　要：目的观察骨关节炎(OA)大鼠软骨自噬基因(Atg5、Atg7、Atg12)以及自噬相关蛋白mTOR、Beclin-1、PI3K、Akt、LC3-Ⅱ的表达,并分析其与IL-4、IL-10、IL-1β、TNF-α的相关性。方法 20只大鼠随机分为正常组和模型组,每组各10只。模型组大鼠采用关节腔注射木瓜蛋白酶(Papain)和L-半胱氨酸(L-cystine)方法制成骨关节炎大鼠模型。以光镜对大鼠软骨进行Mankin标准评分,以电镜观察OA大鼠软骨中自噬小体的形态及数目;采用荧光定量PCR检测软骨Atg5、Atg7、Atg12的表达,采用免疫印迹法(Western blot)检测软骨mTOR、Beclin-1、PI3K、Akt、LC3-Ⅱ的表达,采用ELISA法检测血清细胞因子IL-4、IL-10、IL-1β、TNF-α水平。结果与正常组大鼠比较,OA大鼠关节软骨Mankin评分明显升高(P<0.01);Atg5在软骨中降低(P<0.01),并与IL-4、IL-10呈正相关(均P<0.01),与IL-1β、TNF-α呈负相关(P<0.05或P<0.01);Atg7在软骨中降低(P<0.01);mTOR在软骨中升高(P<0.05),PI3K、Akt在软骨中均升高(均P<0.01),并与IL-4、IL-10呈负相关(P<0.05或P<0.01),与IL-1β、TNF-α呈正相关(P<0.05或P<0.01);LC3-Ⅱ、Beclin-1在软骨中降低,与IL-4、IL-10呈正相关(P<0.05或P<0.01),与IL-1β、TNF-α呈负相关(P<0.05或P<0.01)。结论 OA大鼠软骨在Mankin评分升高的同时,出现Atg5及Atg7表达的降低,PI3K/Akt-mTOR通路上调,Beclin-1下调,自噬受抑制,此过程可能参与OA软骨病变。Objective To observe the expression of autophagy genes（Atg5,Atg7,Atg12）and autophagy related proteins,Beclin-1mTOR,PI3 K,and Akt,LC3-Ⅱ in cartilage of OA rats,and analyze its correlation with IL-4,IL-10,TNF-αand IL-1β.Methods Twenty rats were randomly divided into normal control group（NC）and model control group（MC）（n=10each group）.In MC group,osteoarthritis rat model was established by articular cavity injection of Papain and L-cysteine.Cartilage of the rats was evaluated by Mankin standard score under light microscopy.Autophagosome form and number in OA rats cartilage were observed with electron microscope.Expression of Atg5,Atg7 and Atg12in cartilage was detected with Real-time PCR;Beclin-1,mTOR,PI3 K,LC3-Ⅱ and Akt in cartilage were detected with Western blot.Serum cytokines IL-4,IL-10,TNF-αand IL-1βwere determined by ELISA.Results As compared with normal group,Mankin score of cartilage of OA rats was increased significantly（P〈0.01）;Atg5in cartilage was significantly reduced（P〈0.01）,and was positively correlated with IL-4and IL-10（P〈0.01）,but was negatively correlated with TNF-αand IL-1β（P〈0.05 or P〈0.01）;Atg7in cartilage was significantly decreased（P〈0.01）;mTOR in cartilage was significantly increased（P〈0.05）;PI3Kand Akt in cartilage were significantly increased（both P〈0.01）,and were negatively correlated with IL-4and IL-10（P〈0.05 or P〈0.01）,but were positively correlated with TNF-αand IL-1β（P〈0.05 or P〈0.01）;LC3-Ⅱ and Beclin-1were reduced in cartilage,and positively correlated with IL-4and IL-10（P〈0.05 or P〈0.01）.Conclusion At the same time with the increase of Mankin score in OA rats,the expression of Atg5 and Atg7is reduced,expression of Atg12 increased,PI3K/Akt-mTOR pathway is up-regulated while Beclin-1is down-regulated,and autophagy is suppressed,which may participate in the pathological changes of OA rats cartilage.

关 键 词：骨关节炎 自噬通路 细胞因子 

分 类 号：R684.3[医药卫生—骨科学]