Astragalus Polysaccharide Inhibits Autophagy and Apoptosis from Peroxide-Induced Injury in C2C12 Myoblasts
Astragalus Polysaccharide Inhibits Autophagy and Apoptosis from Peroxide-Induced Injury in C2C12 Myoblasts
复制标题
黄芪多糖抑制 C2C12 成肌细胞过氧化物诱导损伤的自噬和凋亡
DOI：
10.1007/s12013-015-0659-8
复制
发表时间：
2015-11-01
影响因子：
2.6
通讯作者：
Wei, Lian-Bo
中科院分区：
文献类型：
--
作者：
Yin, Yi;Lu, Lu;Wei, Lian-Bo
目的：研究黄芪多糖（APS）对体外培养的C2 C12成肌细胞过氧化损伤的影响及其机制。通过甲基噻唑基四唑比色法检测APS存在或不存在下的细胞活力。电镜观察APS对H2 O2诱导的C2 C12细胞自噬的影响，流式细胞仪检测细胞凋亡率。为了进一步证实H2 O2对C2 C12细胞的影响，通过Western blot分析分别作为自噬和凋亡标志物的LC 3和RARP的蛋白表达，以及p-p70 S6 K、p70 S6 K、Bcl-2、Bax、cyto-C和Caspase-3的表达水平，以揭示潜在的机制。我们观察到APS对C2 C12功能的多种影响。1 mg/mL的APS处理C2 C12细胞将细胞活力降低至低于70%，电子显微镜分析显示APS还减少了H2 O2诱导的自噬体形成的数量。同样，APS减弱了H2 O2介导的细胞凋亡增加，这是伴随着抑制LC 3 II和RARP，通常上调H2 O2。而对照组、H2 O2组和H2 O2 + APS组C2 C12细胞中p-p70 S6 K和p70 S6 K的表达无明显变化。此外，APS促进过氧化氢处理的C2 C12细胞中蛋白Bcl-2的表达，但不改变Bax，从而降低Bax/Bcl-2的比例，从而阻止细胞色素c的释放和caspase-3的激活。APS通过两条独立的信号通路抑制过氧化损伤诱导的C2 C12成肌细胞自噬和凋亡：mTOR非依赖性抑制自噬通路和caspase-3依赖性抑制凋亡通路。
The aim is to study the effects and underlying mechanisms of astragalus polysaccharide (APS) on the peroxide-induced injury in C2C12 myoblasts in vitro. Cell viability in the presence or absence of APS was detected by the methyl thiazolyl tetrazolium colorimetric assay. The autophagosomes were observed by electron microscopy to examine the influence of APS on autophagy caused by H2O2 in C2C12 cells, and the percentage of apoptosis cells was measured by flow cytometry. To further confirm the effect of H2O2 on C2C12 cells, the protein expression of LC3 and RARP, which are the markers of autophagy and apoptosis, respectively, was analyzed by Western blot, as well as the expression levels of p-p70S6K, p70S6K, Bcl-2, Bax, cyto-C, and Caspase-3, to reveal the underlying mechanisms. We observed multiple effects of APS on C2C12 functionality. APS treatment of C2C12 cells at 1 mg/mL reduced cell viability to less than 70 %, and analysis by electron microscopy revealed that APS also reduced the number of H2O2-induced autophagosome formation. Similarly, APS abated the H2O2-mediated increase in cell apoptosis, which was accompanied by the inhibition of LC3 II and RARP that are normally upregulated by H2O2. The expression of p-p70S6K and p70S6K, however, remained unchanged in C2C12 cells in the Control, H2O2 and H2O2 + APS groups. In addition, APS promoted the expression of protein Bcl-2 in H2O2-treated C2C12 cells, but did not change Bax, thus reducing the Bax/Bcl-2 ratio that in turn prevented the release of cytochrome c and the activation of caspase-3. APS inhibits the autophagy and apoptosis induced by peroxide injury in C2C12 myoblasts through two independent signaling pathways: the mTOR-independent pathway for the inhibition of autophagy, and the caspase-3-dependent pathway for the suppression of apoptosis.