白藜芦醇介导AMPK/mTOR信号通路调控线粒体自噬缓解小鼠精原细胞氧化应激损伤和凋亡  

Investigation on Resveratrol Regulating Mitophagy to Alleviate Oxidative Stress Injury and Apoptosis of Gc-1 spg Cells via AMPK/mTOR Signaling Pathway

作　　者：石拴霞 王纪田 宋诚 魏璐晓 甄雪蓉 黄冰雪 阎一鑫 王玲 SHI Shuanxia;WANG Jitian;SONG Cheng;WEI Luxiao;ZHEN Xuerong;HUANG Bingxue;YAN Yixin;WANG Ling(Reproductive Medicine Centre,The 940th Hospital of Joint Logistic Support Force of Chinese People's Liberation Army,Lanzhou 730050,China;School of Clinical Medicine,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Gansu Provincial Key Laboratory of Stem Cells and Gene Drugs,The 940th Hospital of Joint Logistic Support Force ofChinese People's Liberation Army,Lanzhou 730050,China)

机构地区：[1]中国人民解放军联勤保障部队第九四〇医院生殖医学中心,兰州730050 [2]甘肃中医药大学第一临床医学院,兰州730000 [3]中国人民解放军联勤保障部队第九四〇医院,甘肃省干细胞与基因药物重点实验室,兰州730050

出　　处：《中国药学杂志》2024年第5期416-424,共9页Chinese Pharmaceutical Journal

基　　金：甘肃省青年科技基金计划资助(21JR1RA188);甘肃省青年科技基金计划资助(21JR11RA013)。

摘　　要：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-activatedproteinkinase/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(H_(2)O_(2))、H_(2)O_(2)+RES组、H_(2)O_(2)+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H_(2)O_(2)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。OBJECTIVE To observe whether resveratrol can alleviate oxidative stress injury of mouse spermatogonium by regulating mitochondrial autophagy through AMPK/mTOR signaling pathway.METHODS Control group,H2 O2 group,H_(2)O_(2)+RES group,and H_(2)O_(2)+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,MitoTrackerGreen 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 H_(2)O_(2) promoted oxidative stress damage in Gc-1 spg cells,activated the AMPK/mTOR signaling pathway and mitochondrial autophagy,the expressions of p-AMPK/AMPK and LC3,and decreased the expressions of p-mTOR/mTOR and P62.RES could alleviate 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.

关 键 词：白藜芦醇 过氧化氢 小鼠精原细胞 腺苷酸活化蛋白激酶 哺乳动物雷帕霉素靶蛋白 线粒体自噬 凋亡 

分 类 号：R966[医药卫生—药理学]