白藜芦醇缓解低氧/复氧诱导的TCMK1细胞线粒体自噬

Resveratrol relieves hypoxia-reoxygenation-induced mitophagy in TCMK1 cells

目的研究白藜芦醇(Res)对低氧/复氧(H/R)导致小鼠肾小管上皮细胞系TCMK1内线粒体功能及线粒体自噬的影响。方法体外建立H/R细胞模型,经Res或(和)线粒体自噬抑制剂1(Mdivi-1)预处理TMCK1细胞2 h,采用CCK8法检测TCMK1细胞存活率;试剂盒法检测钙离子超负荷情况;流式细胞测量术测定线粒体膜电位(MMP)变化和活性氧(ROS)含量;Western blot检测线粒体融合蛋白(OPA)、分裂蛋白(DRP)以及凋亡相关蛋白cleaved caspase-3,Bax,Bcl2表达;免疫荧光检测线粒体外膜转位酶(TOMM20)和自噬溶酶体相关膜蛋白2(LAMP2)共定位情况。结果与对照组相比,H/R组TCMK1细胞存活率显著减低(P<0.05),细胞内钙离子、ROS及MMP含量显著减低(P<0.05),OPA表达显著减低(P<0.05),同时DRP表达显著增高(P<0.05),线粒体与LAMP2共定位的细胞数目显著减少;与H/R组相比,10μmol/L Res可以显著增加TMCK1细胞的存活率(P<0.05),抑制细胞内ROS的释放、钙离子超负荷及MMP的下降(P<0.05),同时促进OPA表达而抑制DRP表达(P<0.05),增加线粒体与LAMP2共定位的细胞数目。与(H/R+Res)组相比,在其基础上给予Mdivi-1后,凋亡执行蛋白Bax、cleaved caspase-3蛋白表达升高,抗凋亡蛋白Bcl2表达降低(P<0.05)。结论Res缓解H/R诱导的TCMK1细胞损伤,至少部分通过激活线粒体自噬,维护线粒体动力学稳态而实现。

Objective To study the effect of resveratrol(Res)on mitochondrial function and mitophagy in mouse renal tubular epithelial cell line TCMK1 induced by hypoxia-reoxygenation(H/R).Methods The H/R model cell was established in vitro,and TMCK1 cells were pre-treated with Res or(and)mitophagy inhibitor 1(Mdivi-1)for 2 h.CCK-8 method was used to measure cell viability.Flow cytometry was applied to determine cell mitochondrial membrane potential(MMP)and reactive oxygen species(ROS).Western blot was adapted for determining the protein expression levels of optic atrophy(OPA),dynamin-related protein(DRP)and cleaved caspase-3.Cellular immunofluorescence was used to observe the co-localization of mitochondrial outer membrane translocase(TOMM20)and autophagolysosome-associated membrane protein 2(LAMP2).Results There was significantly reduction in the survival rate of TCMK1 cells in H/R group versus control(P<0.05).The intracellular calcium ion,ROS andMMP contents were significantly reduced(P<0.05),and the expression of OPA was significantly reduced(P<0.05).The expression of DRP was significantly improved(P<0.05),and the number of mitochondria and LAMP2 co-localized significantly decreased.As compared to the H/R group,10μmol/L Res could significantly increase the survival rate of TMCK1 cells(P<0.05),and inhibit the release of ROS,calcium overload and the decrease of MMP in TCMK1 cells after H/R injury(P<0.05).While Res could promote the expression of OPA and inhibite the expression of DRP(P<0.05),increase the number of cells with co-localized mitochondria and LAMP2.Compared with(HR+Res)group,the expression of cleaved caspase-3 and Bax increased,while the expression of Bcl2 decreased after Mdivi-1 was administered on the basis(P<0.05).Conclusions Res attenuation of H/R-induced TCMK1 cell damage is medicated at leaet in part by activation of mitophagy and maintaining mitochondrial dynamic homeostasis as one of potential mechanisms.