白藜芦醇通过Zn^(2+)促进自噬发挥心肌细胞保护作用  

Resveratrol promotes autophagy through zinc ion and induces cardiomyocyte protection

作　　者：王培 马虎 杨颖 刘天宇 郭佳宝 胡友成 贺永贵 习瑾昆 WANG Pei;MA Hu;YANG Ying;LIU Tian-yu;GUO Jia-bao;HU You-cheng;HE Yong-gui;XI Jin-kun(School of Public Health,North China University of Science and Technology,Tangshan,Hebei 063210,China;Dept of Physical Education,North China University of Science and Technology,Tangshan,Hebei 063210,China;School of Basic Medical Sciences,North China University of Science and Technology,Tangshan,Hebei 063210,China;North China University of Science and Technology Affiliated Hospital,Tangshan,Hebei 063210,China)

机构地区：[1]华北理工大学公共卫生学院,河北唐山063210 [2]华北理工大学体育部,河北唐山063210 [3]华北理工大学基础医学院,河北唐山063210 [4]华北理工大学附属医院,河北唐山063210

出　　处：《中国药理学通报》2023年第6期1048-1054,共7页Chinese Pharmacological Bulletin

基　　金：河北省自然科学基金资助项目(No H2020209172,H2021209061);河北省社会科学基金资助项目(No HB20TY012);河北省教育厅项目(No ZD2020110);河北省留学回国项目(No C20200508);唐山市高层次人才项目(No A202006016);大学生创新性实验研究计划(No X20222141)。

摘　　要：目的探讨白藜芦醇(resveratrol,Res)是否通过锌离子(Zinc,Zn^(2+))促进自噬,抑制内质网应激(endoplasmic reticulum stress,ERS),发挥心肌细胞保护作用及可能的信号机制。方法常规培养H9c2细胞,2-脱氧葡萄糖(2-deoxyglucose,2-DG)建立ERS模型。随机分为Control组、2-DG组、Res+2-DG组、锌离子螯合剂TPEN+Res+2-DG组、自噬抑制剂3-MA+Res+2-DG组。MTT和CCK-8法检测细胞活性;Western blot法检测ERS分子伴侣蛋白葡萄糖调节蛋白78(GRP78)、葡萄糖调节蛋白94(GRP94)和自噬蛋白LC3Ⅱ/Ⅰ、p62、p-AMPK的表达水平;免疫荧光检测LC3蛋白的表达;激光扫描共聚焦显微镜检测线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)和细胞内Zn^(2+)水平。结果与Control组相比,2-DG使细胞活性降低,Res抑制2-DG引起的变化,此作用被TPEN所逆转;2-DG使GRP78、GRP94表达明显增多,Res抑制2-DG引起的变化,此作用被TPEN逆转;2-DG使LC3Ⅱ/Ⅰ、p-AMPK表达增多,p62表达减少,Res促进2-DG引起的变化,此作用被TPEN逆转;2-DG使LC3的荧光强度升高,Res增强2-DG的作用,此作用被TPEN和3-MA所逆转;2-DG使线粒体TMRE红色荧光及细胞内Zn^(2+)的绿色荧光强度明显降低,Res抑制2-DG引起的荧光强度变化,这些作用同样被TPEN和3-MA所逆转。以上结果差异均具有统计学意义(P<0.05)。结论Res通过增加细胞内Zn^(2+)促进自噬,抑制ERS进而阻止mPTP的开放,发挥心肌细胞保护作用。Aim To investigate whether resveratrol(Resveratrol,Res)induces cardiomyocyte protection by increasing intracellular zinc ion and its possible signal mechanism.Methods H9c2 cells were routinely cultured and 2-deoxyglucose(2-DG)was used to establish an endoplasmic reticulum stress(ERS)model.The experiment was randomly divided into control group,2-DG group,Res+2-DG group,TPEN+Res+2-DG group and 3-MA+Res+2-DG group.Cell vi-ability was detected by MTT and CCK-8;the expression levels of ERS molecular chaperone proteins glucose-regulated protein 78(GRP78),glucose-regulated protein 94(GRP94)and autophagy proteins LC3Ⅱ/Ⅰ,p62 and p-AMPK were detected by Western blot;the expression of LC3 protein was measured by cellular immunofluorescence;the mitochondrial membrane potential(Δψm)and the intracellular zinc ion level were measured by laser scanning confocal microscope.Results Compared with the control group,2-DG reduced cell activity and resveratrol inhibited the changes caused by 2-DG,which was reversed by zinc chelator TPEN.2-DG increased GRP78 and GRP94 expression and resveratrol inhibited the protein changes caused by 2-DG,which was reversed by TPEN.2-DG increased the expression of LC3Ⅱ/Ⅰ,p-AMPK and decreased the expression of p62,and resveratrol promoted the effect of 2-DG.2-DG increased the fluorescence intensity of LC3,and resveratrol enhanced the effect of 2-DG,which was reversed by TPEN and 3-MA.2-DG reduced the red fluorescence intensity of mitochondrial TMRE and green fluorescence intensity of intracellular zinc ions,and resveratrol inhibited these changes caused by 2-DG,which was also reversed by TPEN and 3-MA.The above differences were all statistically significant(P<0.05).Conclusion Resveratrol increases intracellular zinc to promote ERS-induced autophagy and prevent the mPTP opening in H9c2 cardiac cells.

关 键 词：白藜芦醇 2-脱氧葡萄糖 锌离子 自噬 内质网应激 心肌保护 

分 类 号：R284.1[医药卫生—中药学] R322.11[医药卫生—中医学] R329.24R348.5