紫草素减轻低氧-复氧致大鼠心肌细胞系H9c2损伤

Shikonin attenuates hypoxia-reoxygenation-induced injury of cardiomyocyte cell line H9c2

目的探索紫草素对低氧-复氧(H/R)损害的H9c2心肌细胞的保护作用。方法将心肌细胞系H9c2分为对照组、H/R组、紫草素(0.1、1和10μmol/L)干预组,每组3个平行孔;MTT法检测紫草素对H9c2细胞毒性;流式细胞计量术检测细胞凋亡和周期;DCFH-DA检测细胞活性氧水平;生化检测细胞中MDA、8-OHdG和GSH含量;qPCR检测细胞γ-GCS、HO-1和NQO1 mRNA表达;Western blot检测细胞中Bax、Bcl-2、caspase-3、cleaved caspase-3、Keap1和Nrf2蛋白表达;免疫荧光检测细胞Nrf2蛋白进核情况。结果紫草素呈浓度-时间依赖性抑制H9c2细胞增殖(P<0.05);H/R诱导H9c2细胞凋亡、细胞周期阻滞及促进Bax及cleaved caspase-3蛋白表达,抑制Bcl-2蛋白表达(P<0.05),紫草素呈浓度依赖性抑制细胞凋亡、解除细胞周期阻滞及cleaved caspase-3和Bax蛋白表达,增加Bcl-2蛋白表达;紫草素可降低H/R所致活性氧水平、MDA及8-OHdG升高,增高H/R所致GSH含量以及γ-GCS、HO-1和NQO1 mRNA的表达下调,导致Nrf2蛋白的升高及Keap1下调,并促进Nrf2蛋白进核(P<0.05)。结论紫草素减轻低氧-复氧致H9c2心肌细胞的损伤。

Objective To explore the protective effect of shikonin on H9c2 cardiomyocytes damaged by hypoxiareoxygenation(H/R).Methods H9c2 cells were divided into control group,H/R group,shikonin(0.1,1 and10μmol/L)group,including 3 parallel wells in each group;MTT assay was used to detect the toxicity of shikonin on H9c2 cells;Apoptosis and cell cycle were detected by flow cytometry;DCFH-DA was used to detect the level of reactive oxygen species;biochemical detection of MDA and 8-OHdG in cells And GSH content;qPCR was used to detect the expression ofγ-GCS,HO-1 and NQO1 mRNA;Western blot was used to detect the expression of Bax,Bcl-2,caspase-3,cleaved caspase-3,Keap1 and Nrf2 proteins;immunofluorescence was used to detect Nrf2 cells.Protein into the nuclear situation.Results Shikonin inhibited the proliferation of H9c2 cells in a concentrationtime-dependent manner(P<0.05).H/R induced apoptosis of H9c2 cells,cell cycle arrest and promoted the expression of Bax and cleaved caspase-3 proteins,inhibited the expression of Bcl-2 protein(P<0.05).Shikonin inhibited apoptosis,relieved cell cycle arrest,and cleaved caspase-3 and Bax protein expression,and increased Bcl-2 protein expression in a concentration-time-dependent manner;Shikonin decreased the active oxygen,MDA and8-OHdG induced by H/R,increased the GSH content induced by H/R and down-regulated the expression ofγ-GCS,HO-1 and NQO1 mRNA,caused an increase in Nrf2 protein and down-regulation of Keap1,and promoted Nrf2 protein entry into the nucleus(P<0.05).Conclusions Shikonin attenuates myocardial cell injury induced by hypoxia-reoxygenation in H9c2 cells.