Objectives: 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.
Methods: 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.
Results: After I/R, the PGC1α expression was increased in infarcted myocardium. H/R induced H9C2 cell apoptosis (P<0.001). The release of mitoSOX (P<0.001) and protein expression of PGC1α, and apoptosis-related p21, BAX, and caspase-3 were increased. However, knockdown of PGC1α reduced apoptosis (P<0.01) and mitoSOX release (P<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 P<0.001) and mitoSOX release (P<0.01). While overexpression of PGC1α reversed the effects caused by HIF-1α knockdown (all P<0.001). Metformin effectively reduced H/R-induced apoptosis (P=0.013), mitoSOX release (P<0.001), HIF-1α, PGC1α and apoptosis-related protein expression, recovered the cell viability (P<0.001), and reduced myocardial infarction (P=0.003).
Conclusions: 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.
目的: 过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator-activated receptor gamma coactivator 1 alpha，PGC1α)控制线粒体的生物发生，然而其在心血管疾病中的作用并不清楚。本研究旨在探究PGC1α对心肌缺血再灌注(ischemia reperfusion，I/R)损伤的作用及其机制。方法: 通过结扎SD大鼠冠状动脉30 min后复灌2 h制作心肌I/R模型。采用氯化三苯四唑(triphenyl tetrazolium chloride，TTC)染色法测算心肌梗死面积，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(hypoxia/reoxygenation，H/R)，敲低PGC1α或缺氧诱导因子1α(hypoxia-inducible factor 1α，HIF-1α)基因表达，上调PGC1α基因表达，加入二甲双胍等处理，采用蛋白质印迹法检测PGC1α，HIF-1α，p21，BAX和caspase-3的蛋白质表达水平；CCK-8检测细胞的活力；流式细胞仪检测细胞凋亡情况及线粒体超氧化物释放量；定量RT-PCR(RT-qPCR)检测PGC1α和HIF-1α mRNA表达水平；染色质免疫共沉淀(chromatin immunoprecipitation，ChIP)-qPCR和荧光素酶报告基因实验检测HIF-1α对PGC1α的转录调控作用。结果: 大鼠梗死心肌中PGC1α的蛋白质表达水平上调。对H9C2细胞进行H/R处理后，细胞凋亡率上升(P<0.001)，线粒体超氧化物释放量增加(P<0.001)，PGC1α和凋亡相关基因(p21，BAX，caspase-3)的蛋白质表达水平均上调；而敲低PGC1α表达后，细胞凋亡率下降(P<0.01)、线粒体超氧化物释放量减少(P<0.001)，凋亡相关基因的蛋白质表达水平均下调。HIF-1α结合在PGC1α的启动子区域。敲低HIF-1α抑制PGC1α的转录和蛋白质表达，细胞凋亡率下降(P<0.001)，线粒体超氧化物释放减少(P<0.01)；而上调PGC1α的表达逆转敲低HIF-1α导致的上述影响(均P<0.001)。二甲双胍有效减少H/R导致的细胞凋亡(P=0.013)，线粒体超氧化物的释放(P<0.001)，HIF-1α，PGC1α以及凋亡相关基因的表达，恢复细胞活力(P<0.001)，减少大鼠I/R导致的心肌梗死面积(P=0.003)。结论: 心肌I/R后，HIF-1α上调PGC1α的表达，导致ROS的产生增加，损伤心肌；二甲双胍可抑制HIF-1α在缺氧时的积累，有效保护心肌免受I/R损伤。.
Keywords: hypoxia-inducible factor 1α; ischemia reperfusion; metformin; peroxisome proliferator-activated receptor gamma coactivator 1α; reactive oxygen species.