钙络合剂BAPTA-AM对胞外酸化诱导大鼠关节软骨细胞自噬作用的影响及其可能机制  

Effects of BAPTA-AM on acid-induced autophagy of rat articular chondrocytes and its possible mechanisms

作　　者：高文凡[1] 陈飞虎[1] 葛金芳[1] 邓子云[1] 雷静[1] 周仁鹏[1] 王志森[1] 

机构地区：[1]安徽医科大学药学院,安徽合肥230032

出　　处：《中国药理学通报》2015年第5期655-659,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81271949)

摘　　要：目的观察BAPTA-AM对胞外酸化诱导大鼠关节软骨细胞自噬作用的影响,探讨其可能的作用机制。方法体外使用胰酶-Ⅱ型胶原酶消化法分离提取大鼠关节软骨细胞,分为正常组(p H 7.4)、酸化组(p H 6.0)、以及分别经BAPTA-AM处理的正常组和酸化组,激光共聚焦技术检测软骨细胞胞内钙离子变化,实时荧光定量PCR法检测细胞自噬基因Beclin-1、ULK1 mRNA的表达,Western blot法检测自噬蛋白LC3的表达,吖啶橙染色分析细胞自噬溶酶体形成情况。结果与p H 7.4正常组比较,p H 6.0酸化刺激明显增加大鼠关节软骨细胞内Ca2+的浓度,且自噬标志物Beclin-1、ULK1 mRNA及LC3Ⅱ蛋白表达均明显升高,酸性自噬溶酶体形成增多,同时酸化刺激能引起Ca MKKβ及pAMPK蛋白表达水平增高,磷酸化蛋白p-m TOR水平明显降低。BAPTA-AM酸化组自噬水平和Ca MKKβ及p-AMPK表达明显降低,p-m TOR表达明显升高。结论 BAPTA-AM能明显减弱胞外酸化诱导软骨细胞自噬作用,其机制可能与抑制胞内Ca2+有关。Aim To observe the effect of BAPTA-AM on extracellular acid-induced autophagy in rat articular chondrocytes and its possible mechanisms.Methods Rat articular chondrocytes were isolated from Sprague-Dawley rats and incubated with different pH medium. The states of autophagy were examined by acridine or-ange （AO ） staining .Moreover,the expressions of LC3 ,Beclin-1 ,ULK1 ,CaMKKβ,AMPK and mTOR were detected using Western blot or quantitative real-time PCR （qRT-PCR ）. Intracellular calcium （[Ca2＋]i ）was analyzed by a Ca2＋-imaging method. Results Compared with pH 6.0 group,BAPTA-AM could significantly decrease the activation of autophagyinduced by acid exposure,and the expressions of autophagy markers including LC3 Ⅱ,Beclin1 and ULK1were also decreased,accompanied with reduced acidinduced [Ca2 ＋]i influx,decreased proteins expressionof CaMKKβand phosphorylatedAMPK,and increasedphosphorylation of mTOR.Conclusion BAPTAAMcan significantly restrain acidinduced autophagy in ratarticular chondrocytes,the mechanism of which may beassociated with decreased Ca2 ＋ influx.

关 键 词：BAPTA-AM 类风湿关节炎 关节软骨细胞 细胞自噬 CA2+ 胞外酸化 

分 类 号：R-332[医药卫生] R322.72