Xiangya Hospital of Centre-south University, Changsha, Hunan 410000, China.
Department of Pharmacology, Guilin Medical University, Guilin, Guangxi 541004, China.
Phytomedicine. 2019 Aug;61:152845. doi: 10.1016/j.phymed.2019.152845. Epub 2019 Jan 30.
This study was designed to investigate whether EGCG prevents cardiac I/R mitochondrial impairment and cell apoptosis by regulating miR-30a/p53 axis.
The H9c2 cardiomyocytes hypoxia/reoxygenation (H/R) model in vitro and myocardial ischemia /reperfusion (I/R) model in vivo were made, with or without EGCG treatment. The levels of I/R-induced creatine kinase-MB (CK-MB) and the release of lactate dehydrogenase (LDH), as well as the adenosine triphosphate (ATP) and cardiac functional impairment were examined. Stablely transfecting miR-30a mimic or inhibitor in H9c2 cardiomyocytes was built. The expression of miR-30a, p53 and related proteins in cells was measured by western blotting and qRT-PCR. Cell viability and apoptosis were examined using CCK-8 assay and flow cytometry. The content of reactive oxygen species (ROS), mitochondrial permeability transition pores (MPTP) opening and mitochondrial transmembrane potential (ΔΨm) in cells was measured by fluorescent probes. The levels of miR-30a and p53, some related proteins expression and apoptosis in the cardiac muscle tissues were determined by quantitative real-time PCR (qRT-PCR), H&E staining, western blotting and TUNEL assays.
We found that EGCG preconditioning significantly decreased the levels of CK-MB and LDH, increased the activity of ATP, reduced the apoptotic rate and partially preserved heart function. Furthermore, EGCG decreased ROS levels, MPTP opening and depolarization of ΔΨm, and improved the activity of post-I/R cardiomyocyte. The beneficial effect of EGCG was associated with restored levels of miR-30a expression in the I/R injury that correspond to p53 mRNA downregulation. The regulatory effect of EGCG was greatly enhanced by miR-30a mimic and suppressed by miR-30a inhibitor. More importantly, EGCG pretreatment inhibited the expression of mitochondrial apoptotic related proteins downstream of the miR-30a/p53 pathway.
This study demonstrated that EGCG pretreatment may attenuate mitochondrial impairment and myocardial apoptosis by regulation of miR-30a/p53 axis.
本研究旨在探讨 EGCG 是否通过调节 miR-30a/p53 轴来防止心脏缺血再灌注（I/R）引起的线粒体损伤和细胞凋亡。
采用体外 H9c2 心肌细胞缺氧/复氧（H/R）模型和体内心肌缺血/再灌注（I/R）模型，观察 EGCG 处理前后细胞培养液中肌酸激酶同工酶-MB（CK-MB）水平、乳酸脱氢酶（LDH）释放、三磷酸腺苷（ATP）水平和心功能变化；构建 H9c2 心肌细胞 miR-30a 模拟物或抑制剂的稳定转染细胞系，采用 Western blot 和 qRT-PCR 检测细胞中 miR-30a、p53 及其相关蛋白的表达；采用 CCK-8 法和流式细胞术检测细胞活力和凋亡；采用荧光探针检测细胞内活性氧（ROS）、线粒体通透性转换孔（MPTP）开放和线粒体跨膜电位（ΔΨm）；采用 qRT-PCR、H&E 染色、Western blot 和 TUNEL 检测心肌组织中 miR-30a 和 p53 水平、部分相关蛋白表达和细胞凋亡。
结果发现，EGCG 预处理可显著降低 CK-MB 和 LDH 水平，提高 ATP 活性，降低细胞凋亡率，部分改善心功能。此外，EGCG 还可降低 ROS 水平，减少 MPTP 开放和 ΔΨm 去极化，改善 I/R 后心肌细胞活力。EGCG 的这种有益作用与 I/R 损伤后 miR-30a 表达水平的恢复有关，这与 p53 mRNA 下调相一致。miR-30a 模拟物可增强 EGCG 的调节作用，而 miR-30a 抑制剂则抑制其作用。更重要的是，EGCG 预处理可抑制 miR-30a/p53 通路下游的线粒体凋亡相关蛋白的表达。
本研究表明，EGCG 预处理可能通过调节 miR-30a/p53 轴来减轻线粒体损伤和心肌细胞凋亡。
