当归多糖对缺氧-复氧损害H9c2心肌细胞的保护作用

Protective effect of angelica sinensis polysaccharide on H9c2 cardiomyocytes damaged by hypoxia-reoxygenation

目的探讨当归多糖(angelica sinensis polysaccharide,ASP)对缺氧-复氧损害H9c2心肌细胞的保护作用。方法噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide,MTT]检测ASP对H9c2细胞增殖的影响;在体外ASP培养后,再缺氧-复氧培养H9c2心肌细胞为实验组,以正常培养细胞为对照组,以仅缺氧-复氧培养细胞为缺氧-复氧组。观察细胞形态;流式细胞仪检测细胞凋亡情况;2′,7′-二氯荧光黄双乙酸盐(2′,7′-dichlorofluorescin diacetate,DCFH-DA)检测细胞活性氧水平;生化检测细胞丙二醛(malondialdehyde,MDA)、8-羟基脱氧鸟苷(8-hydroxy-2 deoxyguanosine,8-OHdG)浓度;实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测细胞γ-谷氨酰半胱氨酸合成酶(human γ-glutamyl systeine synthetase,γ-GCS)、红素加氧酶-1(heme oxygenase-1,HO-1)、醌氧化还原酶1[NAD(P)H dehydrogenase 1,NQO1]mRNA表达;Western bloting实验检测细胞B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)、Bcl-2相关X(Bcl-2-Associated X,Bax)、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)蛋白浓度。结果在常氧环境中ASP可浓度-时间依赖地抑制H9c2细胞增殖;缺氧-复氧诱导H9c2细胞显著凋亡(P<0.05)、活性氧水平显著升高(P<0.05),同时造成活性氧清除相关酶γ-GCS、HO-1、NQO1mRNA表达及Nrf2蛋白表达显著下调(P<0.05);而ASP可浓度依赖地显著抑制缺氧-复氧诱导的H9c2细胞凋亡(P<0.05),降低缺氧-复氧诱导的H9c2细胞活性氧水平(P<0.05),显著提高缺氧-复氧诱导的H9c2细胞中γ-GCS、HO-1、NQO1mRNA表达及Nrf2蛋白表达(P<0.05)。结论ASP可上调Nrf2表达,提高心肌细胞内抗氧自由基酶的表达,降低由缺氧-复氧所导致心肌细胞的氧化应激水平,抑制心肌细胞凋亡。

Objectives To explore the protective effect of angelica sinensis polysaccharide(ASP)on H9c2 cardiomyocytes damaged by hypoxia-reoxygenation.Methods 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT)was used to detect the proliferation of ASP on H9c2 cells.H9c2 cardiomyocytes after cultured with ASP in vivo and then hypoxic-reoxygenation cultured were used as experimental group,normal cultured cells were used as control group,and only hypoxic-reoxygenated cultured cells were used as hypoxic-reocygerated group.The morphology of the cells was observed;the apoptosis was detected by flow cytometry;the level of reactive oxygen species was detected by 2′,7′-dichlorofluorescin diacetate(DCFH-DA);the concentrations of malondialdehyde(MDA)and 8-hydroxy-2 deoxyguanosine(8-OHdG)were detected by biochemical assay;human γ-glutamyl systeine synthetase (γ-GCS),heme oxygenase-1(HO-1)and NAD(P)H dehydrogenase 1(NQO1)mRNA expression were detected by real time polymerase chain reaction(rtPCR);Western blotting assay was used to detect B cell lymphoma/lewkmia-2 (Bcl-2),concentrations of Bcl-2-Associated X(Bax)and nuclear factor erythroid 2-related factor 2(Nrf2)proteins.Results In the normoxic environment,ASP inhibited the proliferation of H9c2 cells in a concentration-time dependent manner.Hypoxia-reoxygenation induced significant apoptosis of H9c2 cells(P<0.05),and significantly elevation of the level of reactive oxygen species(P<0.05),and significantly down-regulation of oxygen scavenging related enzymes γ-GCS,HO-1,NQO1 mRNA expression and Nrf2 protein expression(P<0.05);while ASP significantly inhibited hypoxiareoxygenation-induced H9c2 cell apoptosis in a concentration-dependent manner(P<0.05),decreased the level of hypoxia-reoxygenation-induced reactive oxygen species in H9c2 cell(P<0.05),significantly increased hypoxia-reoxygenation-induced γ-GCS,HO-1,NQO1 mRNA expression and Nrf2 protein expression in H9c2 cells(P<0.05).Conclusions ASP can up-regulate the expression of Nrf2,increase the expression of anti-oxidative enzymes in cardiomyocytes,decrease the level of oxidative stress induced by hypoxia-reoxygenation,and inhibit the apoptosis of cardiomyocytes.