多种细胞自噬调节剂对自噬标记物LC3II及p62表达的影响

利用Western杂交检测自噬标记物LC3II和p62的表达是评价细胞自噬活性的常用方法。为了比较作用于不同靶点的细胞自噬调节剂对LC3II和p62表达的影响，分别利用以mTOR依赖或非依赖方式活化细胞自噬的雷帕霉素或海藻糖、抑制自噬起始的3-甲基腺嘌呤、抑制自噬小体与溶酶体融合的巴弗洛霉素A1以及抑制溶酶体酶活性的E64d和pepstatin A处理HEK293细胞，通过Western杂交检测LC3II和p62的表达。结果显示，雷帕霉素增强LC3I向LC3II转化，促进p62降解，而海藻糖仅引起LC3II表达的上调；阻断细胞自噬过程各个阶段均导致LC3II和p62堆积，并呈现出剂量和时间依赖性。这些结果提示尽管LC3II和p62均为自噬标记物，但不同的调节剂对其表达的调控存在差异。

Effects of various autophagy modulators on the expression of autophagic markers LC3II and p62

Western blotting of autophagic markers LC3II and p62 are widely used for estimating autophagic activity. To compare the regulation of various autophagy modulators on LC3II and p62, HEK293 cells were treated separately with mTOR-dependent autophagy activator rapamycin or -independent autophagy activators trehalose, and autophagy inhibitors including 3-methyladenine(3-MA), bafilomycin A1 or E64d and pepstatin A that inhi-bited the initiation of autophagy, the fusion of autophagosome and lysosome, and the activities of lysosomal enzymes accordingly, and then LC3II and p62 levels were assessed. Western blot results demonstrated that rapamycin enhanced the conversion of LC3I to LC3II, promoted the degradation of p62 simultaneously, while trehalose merely increased the expression of LC3II with no influence on the p62 level. Moreover, inhibition of autophagy commonly led to accumulation of LC3II as well as blockage of p62 degradation in a concentration- and time- dependent manner. These results indicate that obvious differences exist in the regulation of LC3II and p62 by various modulators although both are autophagic markers.