Institute of Integrated Traditional Chinese and Western Medicine, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China.
2 School of Nursing, Gansu University of Chinese Medicine, Lanzhou 730000, China.
J Tradit Chin Med. 2022 Apr;42(2):176-186. doi: 10.19852/j.cnki.jtcm.20220311.002.
To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/ reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2 (PINK1/PARKIN) signaling pathway.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells; the hypoxia/ reoxygenation (H/R) model was established in tri-gas incubator; 2', 7'-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species (ROS); the changes of mitochondrial membrane potential was determined by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining; the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits; flow cytometry was used to detect the ratio of apoptotic cells; transmission electron microscope was used to observe the ultrastructure of H9C2 cells; Western blot was used to detect the protein changes of mitochondrial 20 kDa outer membrane protein (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), presenilins associated rhomboid-like protein (PARL), PINK1, PARKIN and mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa (P62), microtubule-associated protein 1 light chain 3 beta (LC3B); the mRNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction; immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.
Resveratrol could inhibit the proliferation of H9C2 cells in a time- and concentration- dependent manner; however, pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels, alleviate the loss of mitochondrial membrane potential induced by H/R, inhibit H/R-induced apoptosis of H9C2 cells, and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage. Resveratrol could further increase the levels of p62, PINK1, PARKIN protein, the expression of PINK1, PARKIN mRNA and the ratio of LC3BⅡ/LC3BⅠin H/R-induced H9C2 cells, inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells, and further reduce the expression of TOM20,TIM23, PARL, Mfn1 and Mfn2 protein in H/R-induced H9C2 cells. The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells.
Resveratrol can protect H9C2 cells from H/R injury, which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.
基于 PTEN 诱导的假定激酶蛋白 1/帕金森病蛋白 2（PINK1/PARKIN）信号通路，探讨白藜芦醇对缺氧/复氧干预后心肌细胞的保护作用。
采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐检测白藜芦醇对 H9C2 细胞活力的影响；在三气孵箱中建立缺氧/复氧（H/R）模型；2',7'-二氯荧光素二乙酸酯染色法测定活性氧（ROS）含量；5,5',6,6'-四氯-1,1',3,3'-四乙基碘化羰花青碘染色法测定线粒体膜电位变化；酶活性试剂盒评估线粒体呼吸链复合体活性变化；流式细胞术检测细胞凋亡率；透射电子显微镜观察 H9C2 细胞超微结构；Western blot 检测线粒体 20kDa 外膜蛋白（TOM20）、内膜转位酶 23（TIM23）、早老素相关环指蛋白（PARL）、PINK1、PARKIN 和融合蛋白 1（Mfn1）、融合蛋白 2（Mfn2）、62kDa 微管相关蛋白 1 轻链 3β（LC3B）磷酸化酪氨酸非依赖性 Lck SH2 结构域配体（P62）的蛋白变化；采用实时定量聚合酶链反应检测 PINK1 和 PARKIN 的 mRNA 水平；免疫沉淀法检测 PARKIN 与泛素的相互作用。
白藜芦醇可呈时间和浓度依赖性抑制 H9C2 细胞增殖；然而，低细胞毒性预处理白藜芦醇可降低 H/R 诱导的细胞 ROS 水平升高，减轻 H/R 诱导的线粒体膜电位丧失，抑制 H/R 诱导的 H9C2 细胞凋亡，保护 H9C2 细胞线粒体结构和呼吸链免受 H/R 损伤。白藜芦醇还可进一步增加 H/R 诱导的 H9C2 细胞中 p62、PINK1、PARKIN 蛋白水平、PINK1、PARKIN mRNA 表达水平和 LC3BⅡ/LC3BⅠ比值，抑制 H/R 诱导的 H9C2 细胞中 PARKIN 与泛素的相互作用，并进一步降低 H/R 诱导的 H9C2 细胞中 TOM20、TIM23、PARL、Mfn1 和 Mfn2 蛋白的表达。白藜芦醇的作用与自噬激活剂对 H/R 诱导的 H9C2 细胞的作用一致。
白藜芦醇可保护 H9C2 细胞免受 H/R 损伤，这可能与白藜芦醇通过激活 PINK1/PARKIN 信号通路促进线粒体自噬有关。
