羟基红花黄色素A对缺氧缺糖/复氧复糖损伤的大鼠H9C2心肌细胞氧化应激和细胞凋亡的影响

Effects of hydroxysafflor yellow A on oxidative stress and apoptosis of H9C2 cardiomyocytes injured by oxygen-glucose deprivation/reperfusion

目的探讨羟基红花黄色素A(hydroxysafflor yellow A,HSYA)对大鼠H9C2心肌细胞缺氧缺糖/复氧复糖损伤(oxygen-glucose deprivation/reperfusion injury,OGD/R)后氧化应激及细胞凋亡的影响,并探索其可能的作用通路。方法大鼠H9C2心肌细胞稳定传代培养至第三代后,随机分为空白组,缺氧/复氧模型组,给药组分为HSYA低、高剂量组(HSYA40、HSYA80,40、80μmol/L),每组设10个复孔。各组细胞在经药物干预6 h后,采用CCK-8法检测细胞活性;生化仪检测培养基中天门冬氨酸转移酶(AST)、肌酸激酶(CK)、乳酸脱氢酶(LDH)活性;测定细胞中过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量;采用流式细胞仪检测细胞凋亡状况并计算凋亡率;通过蛋白质免疫印迹(Western blot)方法检测细胞中凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)并计算Bcl-2/Bax表达,同时检测半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达并进行半定量分析。结果与正常组相比,缺氧缺糖/复氧复糖组细胞活性明显低于正常组,培养基中AST、CK、LDH活性明显升高,SOD,CAT活性明显降低,MDA含量升高,细胞凋亡率显著升高,细胞中Bcl2和Bax mRNA表达明显升高,Bcl2/Bax表达显著降低,Caspase-3蛋白表达水平显著升高。与OGD/R组相比,羟基红花色素A给药组细胞活性明显升高;培养基中AST、CK和LDH活性明显降低,SOD、CAT活性明显升高,MDA含量降低;细胞凋亡率显著降低;细胞中抑凋亡相关基因Bcl-2表达上调,而凋亡相关基因Bax表达明显下调,Bcl2/Bax表达显著升高,Caspase-3蛋白表达水平显著降低。结论羟基红花色素A能够通过抑制H9C2心肌细胞的氧化应激损伤和凋亡从而对缺氧缺糖/复氧复糖损伤的H9C2心肌细胞起到一定保护作用;其作用机制可能与其改善抗氧化酶活性,上调抑制凋亡基因Bcl-2表达、下调促凋亡基因Bax表达,提高Bcl-2/Bax表达,以及抑制促凋亡蛋白Caspase-3表达有关。

ObjectiveTo investigate the effect of hydroxysafflor yellow A(HSYA)on oxidative stress and apoptosis of H9C2 cardiomyocytes after oxygen-glucose deprivation/reperfusion injury and to explore its possible mechanism.MethodsAfter rat H9C2 cardiomyocytes were stably subcultured to the third passage,they were randomly divided into control,hypoxia/reoxygenation model(OGD/R),low-dose HSYA(40μmol/L;HSYA40),and high-dose HSYA(80μmol/L;HSYA80)groups with 10 duplicate wells in each group.After 6 hours of treatment,the cells were analyzed by CCK-8 assays and aspartate transferase(AST),creatine kinase(CK),and lactate dehydrogenase(LDH)activities in the medium were measured by a biochemical analyzer.Corresponding kits were used to measure the contents of superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)in cells.Flow cytometry was used to assess the apoptosis rate.Western blot was used to detect apoptosis-related proteins B-cell lymphoma/leukemia-2(Bcl2)and Bcl2-related X Protein(Bax)in cells.Caspase-3 expression was analyzed semiquantitatively.ResultsCompared with the normal group,cell proliferation in the OGD/R group was significantly lower,AST,CK,and LDH activities in the medium were significantly increased,SOD and CAT activities were significantly decreased,and the MDA content was increased.The apoptosis rate was significantly increased,Bcl2 and Bax mRNA expression was significantly increased,Bcl2/Bax protein expression was significantly decreased,and Caspase-3 protein expression was significantly increased.Compared with the OGD/R group,cell proliferation in the hydroxysafflor A administration group was significantly increased,AST,CK,and LDH activities in the medium were significantly decreased,SOD and CAT activities were significantly increased,and the MDA content was decreased.The apoptosis rate was significantly reduced and expression of anti-apoptotic gene Bcl-2 was upregulated,while the expression of proapoptotic gene Bax was significantly downregulated,Bcl2/Bax protein expression was significantly increased,and caspase-3 protein expression was significantly decreased.ConclusionsHSYA protects H9C2 cardiomyocytes from oxygen-glucose deprivation/reperfusion injury by inhibiting oxidative stress injury and apoptosis.Its mechanism may be related to improving the activity of antioxidant enzymes,upregulating Bcl-2 gene expression,downregulating Bax gene expression,increasing Bcl-2/Bax protein expression,and inhibiting the expression of pro-apoptotic protein Caspase-3.