PARP-1通过调节HMGB1乙酰化和核移位促进缺氧/复氧诱导的心肌细胞损伤

PARP-1 promotes hypoxia/reoxygenation-induced cardiomyocyte damage by regulating acetylation and nuclear translocation of HMGB1

目的:探究聚腺苷二磷酸核糖聚合酶-1(PARP-1)对缺氧/复氧(H/R)诱导的心肌细胞损伤的作用及其机制。方法:体外培养大鼠心肌细胞H9c2,将其分为对照组、H/R组、PARP-1特异性抑制剂氨基苯甲酰胺(3-AB)处理组(3-AB组)、3-AB+过表达空载(3-AB+oe-NC)组和3-AB+过表达HMGB1(3-AB+oe-HMGB1)组。除对照组外,其他各组均予H/R处理。采用CCK-8法检测细胞活力,EdU实验检测细胞增殖,流式细胞术检测细胞凋亡,免疫沉淀法检测HMGB1乙酰化,免疫荧光染色检测细胞中HMGB1的表达,western blotting法检测细胞PARP-1、HMGB1、Beclin1、LC3和p62蛋白表达。结果:与对照组比较,H/R组H9c2细胞活力、EdU阳性细胞数、p62蛋白表达明显降低,细胞凋亡率和PARP-1、HMGB1、Beclin1、LC3Ⅱ/LC3Ⅰ蛋白表达明显升高,HMGB1乙酰化水平升高(均P<0.05),胞核HMGB1蛋白转移至胞质。与H/R组比较,3-AB组H9c2细胞活力、EdU阳性细胞数、p62蛋白表达明显升高,细胞凋亡率和PARP-1、HMGB1、Beclin1、LC3Ⅱ/LC3Ⅰ蛋白表达明显降低,HMGB1乙酰化水平降低(均P<0.05),HMGB1蛋白滞留于胞核。3-AB+oe-NC组与3-AB组各指标比较,差异均无统计学意义(均P>0.05)。与3-AB+oe-NC组比较,3-AB+oe-HMGB1组H9c2细胞活力、EdU阳性细胞数、p62蛋白表达明显降低,细胞凋亡率和HMGB1、Beclin1、LC3Ⅱ/LC3Ⅰ蛋白表达及HMGB1乙酰化水平明显升高(均P<0.05),胞核HMGB1蛋白转移至胞质,而两组PARP-1蛋白表达比较无明显差异(P>0.05)。结论:抑制PARP-1通过抑制HMGB1乙酰化和核移位减轻H/R诱导的心肌细胞凋亡和自噬,促进细胞增殖。

Objective:To investigate the effect of polyadenosine diphosphate ribose polymerase-1(PARP-1) on cardiomyocyte damage induced by hypoxia/reoxygenation(H/R) and its possible mechanism.Methods:Rat cardiomyocytes H9 c2 were cultured in vitro,and randomly divided into control group,H/R group,PARP-1 specific inhibitor 3-AB treatment group(3-AB group),3-AB + overexpression no-load(3-AB + oe-NC) group and 3-AB+ overexpression of HMGB1(3-AB + oe-HMGB1) group.Except forthe control group,the other four groups were treated with H/R.Cell Counting Kit-8(CCK-8) was used to detect cell viability.EdU experiment was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Immunoprecipitation was used to detect acetylation of HMGB1.Immunofluorescence staining was used to detect the expression of HMGB1 in cells.Western blotting was used to detect the expressions of PARP-1,HMGB1,Beclin1,LC3 and p62 protein.Results:Compared with the control group,the viabilityof H9 c2 cells,the number of EdU positive cells,and the expression of p62 protein in the H/R group were significantly reduced,whereas the apoptosis rate and the expressions of PARP-1,HMGB1,Beclin1 and LC3Ⅱ/LC3Ⅰ protein as well asthe acetylation level of HMGB1 were increased (P<0.05),and the nuclear HMGB1 protein was transferred to the cytoplasm.Compared with the H/R group,the viability of H9 c2 cells,the number of EdU-positive cells,and the expression of p62 protein were significantly increased in the 3-AB group,while the apoptosis rate and the expressions of PARP-1,HMGB1,Beclin1 and LC3Ⅱ/LC3Ⅰ protein as well as the acetylation level of HMGB1 were reduced(P<0.05),and the HMGB1 protein was retained in the nucleus.Indicatorsof H9 c2 cells had no statistical significance between 3-AB+oe-NC group and 3-AB group(P>0.05).Compared with the 3-AB+oe-NC group,the viability of H9 c2 cells,the number of EdU-positive cells,and the expression of p62 protein in the 3-AB + oe-HMGB1 group were significantly reduced,while the apoptosis rate,the expressions of HMGB1,Beclin1 and LC3Ⅱ/LC3Ⅰ protein,and the acetylation level of HMGB1 were significantly increased(P<0.05),but the difference in PARP-1 protein expression was not statistically significant(P>0.05),and the nuclear HMGB1 protein was transferred to the cytoplasm.Conclusion:Inhibition of PARP-1 reduces H/R-induced cardiomyocyte apoptosis and autophagy and promotes cell proliferation by inhibiting acetylation and nuclear translocation of HMGB1.