Nie Jungang, Ta Na, Liu Lijuan, Shi Guoxiang, Kang Ting, Zheng Zeqi
Department of Cardiovascular Medicine, First Affiliated Hospital of Nanchang University, Nanchang 330006.
Graduate School of Nanchang University, Nanchang 330006, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2020 Oct 28;45(10):1155-1163. doi: 10.11817/j.issn.1672-7347.2020.190215.
Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) controls mitochondrial biogenesis, but its role in cardiovascular diseases is unclear. The purpose of this study is to explore the effect of PGC1α on myocardial ischemia-reperfusion injury and the underlying mechanisms.
The transverse coronary artery of SD rat was ligated for 30 minutes followed by 2 hours of reperfusion. Triphenyltetrazolium chloride (TTC) staining was performed to measure the area of myocardial infarction. Immunohistochemistry and Western blotting were used to detect the PGC1α expression in myocardium. The rat cardiomyocyte H9C2 was subjected to hypoxia/reoxygenation (H/R) with the knockdown of PGC1α or hypoxia- inducible factor 1α (HIF-1α), or with treatment of metformin. Western blotting was used to detect the expression of PGC1α, HIF-1α, p21, BAX, and caspase-3. CCK-8 was performed to detect cell viability, and flow cytometry was used to detect apoptosis and mitochondrial superoxide (mitoSOX) release. RT-qPCR was used to detect the mRNA expression of PGC1α and HIF-1α. Besides, chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter gene assay were applied to detect the transcriptional regulation effect of HIF-1α on PGC1α.
After I/R, the PGC1α expression was increased in infarcted myocardium. H/R induced H9C2 cell apoptosis (<0.001). The release of mitoSOX (<0.001) and protein expression of PGC1α, and apoptosis-related p21, BAX, and caspase-3 were increased. However, knockdown of PGC1α reduced apoptosis (<0.01) and mitoSOX release (<0.001), and decreased protein expression of apoptosis-related gene. HIF-1α is bound to the promoter region of PGC1α. Knockdown of HIF-1α inhibited the transcription and protein expression of PGC1α and further to reduce the apoptosis (all <0.001) and mitoSOX release (<0.01). While overexpression of PGC1α reversed the effects caused by HIF-1α knockdown (all <0.001). Metformin effectively reduced H/R-induced apoptosis (=0.013), mitoSOX release (<0.001), HIF-1α, PGC1α and apoptosis-related protein expression, recovered the cell viability (<0.001), and reduced myocardial infarction (=0.003).
After I/R, HIF-1α up-regulates the expression of PGC1α, leading to an increase in ROS production and aggravation of injury. Metformin can inhibit the accumulation of HIF-1α during hypoxia and effectively protect myocardium from ischemia/reperfusion injury.
过氧化物酶体增殖物激活受体γ共激活因子1α（PGC1α）控制线粒体生物合成，但其在心血管疾病中的作用尚不清楚。本研究旨在探讨PGC1α对心肌缺血再灌注损伤的影响及其潜在机制。
结扎SD大鼠冠状动脉30分钟，随后再灌注2小时。采用氯化三苯基四氮唑（TTC）染色法测量心肌梗死面积。免疫组织化学和蛋白质印迹法检测心肌中PGC1α的表达。对大鼠心肌细胞H9C2进行缺氧/复氧（H/R）处理，同时敲低PGC1α或缺氧诱导因子1α（HIF-1α），或用二甲双胍处理。蛋白质印迹法检测PGC1α、HIF-1α、p21、BAX和半胱天冬酶-3的表达。采用CCK-8法检测细胞活力，流式细胞术检测细胞凋亡和线粒体超氧化物（mitoSOX）释放。RT-qPCR检测PGC1α和HIF-1α的mRNA表达。此外，应用染色质免疫沉淀（ChIP）-qPCR和荧光素酶报告基因检测法检测HIF-1α对PGC1α的转录调控作用。
缺血/再灌注（I/R）后，梗死心肌中PGC1α表达增加。H/R诱导H9C2细胞凋亡（<0.001）。mitoSOX释放增加（<0.001），PGC1α蛋白表达以及凋亡相关的p21、BAX和半胱天冬酶-3表达均增加。然而，敲低PGC1α可减少细胞凋亡（<0.01）和mitoSOX释放（<0.001），并降低凋亡相关基因的蛋白表达。HIF-1α与PGC1α的启动子区域结合。敲低HIF-1α可抑制PGC1α的转录和蛋白表达，并进一步减少细胞凋亡（均<0.001）和mitoSOX释放（<0.01）。而PGC1α的过表达可逆转HIF-1α敲低所引起的效应（均<0.001）。二甲双胍可有效减少H/R诱导的细胞凋亡（=0.013）、mitoSOX释放（<0.001）、HIF-1α、PGC1α和凋亡相关蛋白表达，恢复细胞活力（<0.001），并减少心肌梗死面积（=0.003）。
I/R后，HIF-1α上调PGC1α的表达，导致活性氧生成增加，加重损伤。二甲双胍可抑制缺氧时HIF-1α的蓄积，有效保护心肌免受缺血/再灌注损伤。
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