目的 观察白藜芦醇(resveratrol,RES)是否通过腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白(amp-activatedproteinki-nase/mammalian target of rapamycin,AMPK/mTOR)信号通路调控线粒体自噬缓解小鼠精原细胞(Gc-1 spg)氧化应激损伤.方法 设置空白对照组(Control)、过氧化氢组(H2O2)、H2O2+RES组、H2O2+RES+AMPK抑制剂(Compound C,CC)组,各组给予相应处理后.CCK-8检测细胞活力,试剂盒检测过氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、谷胱甘肽过氧化物(glutathione peroxidase,GSH-PX)、乳酸脱氢酶(lactate dehydrogenase,LDH)和三磷酸腺苷(adenosine triphosphate,ATP)SOD、MDA、GSH-PX、LDH 和 ATP 水平,JC-1 检测线粒体膜电位,MitoTracker ® Green FM 检测活细胞线粒体形态,Hoechst 33342染色检测细胞核凋亡率,透射电镜观察细胞超微结构及线粒体自噬变化,Western blot检测凋亡蛋白Bcl-2关联 X 蛋白(Bcl-2-associated X protein,Bax)、B 细胞淋巴瘤-2 蛋白(B-cell lymphoma 2 protein,Bcl-2)、半胱氨酸天冬氨酸蛋白酶 3(cysteinyl aspartate specific proteinase,Caspase-3),自噬蛋白微管相关蛋白轻链 3(light chain 3,LC3)、P62,通路相关蛋白AMPK、p-AMPK、mTOR、p-mTOR表达水平.结果 H2O2促进Gc-1 spg细胞氧化应激损伤,激活AMPK/mTOR信号通路和线粒体自噬,提高p-AMPK/AMPK和LC3表达并降低p-mTOR/mTOR和P62表达;RES能缓解Gc-1 spg细胞氧化应激损伤,进一步提高p-AMPK/AMPK和LC3表达,降低p-mTOR/mTOR和P62表达,促进Gc-1 spg细胞线粒体自噬;AMPK抑制剂能逆转RES对Gc-1 spg细胞氧化应激损伤的保护作用,抑制SOD和GSH-PX活性并增加MDA和LDH水平,降低细胞线粒体膜电位,增加线粒体损伤,破坏细胞核,增加Bax、Caspase-3和P62表达水平并降低Bcl-2和LC3表达水平,增加细胞凋亡率,降低线粒体自噬.结论 RES可能通过AMPK/mTOR信号通路促进Gc-1 spg细胞线粒体自噬缓解线粒体氧化应激损伤和细胞凋亡.
Investigation on Resveratrol Regulating Mitophagy to Alleviate Oxidative Stress Injury and Apoptosis of Gc-1 spg Cells via AMPK/mTOR Signaling Pathway
OBJECTIVE To observe whether resveratrol can alleviate oxidative stress injury of mouse spermatogonium by regula-ting mitochondrial autophagy through AMPK/mTOR signaling pathway.METHODS Control group,H2 O2 group,H2 O2+RES group,and H2O2+RES+Compound C(AMPK inhibitor)group were set up,and each group was given the appropriate treatment.Cell viability was detected by CCK-8,SOD,MDA,GSH-PX,LDH and ATP levels were determined by kits,mitochondrial membrane potential was measured by JC-1,MitoTracker® Green FM was used to detect the morphology of live cell mitochondria,Hoechst 33342 staining was used to detect the rate of nuclear apoptosis,cell ultrastructure and mitochondrial autophagy changes were observed by transmission electron microscopy,and Western blotting was used to detect the expression levels of apoptotic proteins Bax,Bcl-2,Caspase-3,autophagy proteins LC3,P62,and pathway-related proteins AMPK,p-AMPK,mTOR,and p-mTOR.RESULTS H2 O2 promoted oxidative stress damage in Gc-1 spg cells,activated the AMPK/mTOR signaling pathway and mitochondrial autophagy,increased the expressions of p-AMPK/AMPK and LC3,and decreased the expressions of p-mTOR/mTOR and P62.RES could allevi-ate oxidative stress damage in Gc-1 spg cells,further increase the expressions of p-AMPK/AMPK and LC3,reduce the expressions of p-mTOR/mTOR and P62,and promote mitochondrial autophagy in Gc-1 spg cells.AMPK inhibitor can reverse the protective effect of RES on oxidative stress injury of Gc-1 spg cells,inhibit the activities of SOD and GSH-PX and increase the levels of MDA and LDH,reduce the mitochondrial membrane potential,increase mitochondrial damage,destroy the nucleus,increase the expression levels of Bax,Caspase-3 and P62,and reduce the expression levels of Bcl-2 and LC3,increase cell apoptosis rate and reduce mitophagy.CONCLUSION RES may promote mitochondrial autophagy and alleviate mitochondrial oxidative stress damage and apoptosis in Gc-1 spg cells through AMPK/mTOR signaling pathway.
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