【摘要】 目的:探讨外源性硫化氢（H₂S）恢复缺氧后适应对衰老H9C2细胞的保护作用及相关机制。方法:H9C2细胞（心肌细胞系）用30μmol/L过氧化氢（H₂O₂）处理2 h后再培养3 d,诱导生成衰老细胞。衰老H9C2细胞被随机分5组（n=8）:正常组（Control）、缺氧/复氧组（H/R）、H/R+NaHS组、缺氧后适应（PC）组、PC+NaHS组。缺氧/复氧（H/R）模型:衰老H9C2细胞用缺氧液（无血清、无糖培养基,p H=6.8）培养3 h,然后正常培养6h;缺氧后适应（PC）模型:方法同H/R模型,缺氧结束复氧前连续进行3次5 min间隔的复氧/再缺氧处理,随后复氧6 h。ELISA试剂盒分别检测大鼠晚期糖基化终末产物（AGEs）含量和caspase-3活性; CCK-8试剂盒检测细胞活力; DCFH-DA染色检测活性氧（ROS）水平; Hoechst 33342染色检测细胞凋亡率; Real-time PCR检测相关基因m RNA水平。结果:30μmol/L H₂O₂可诱导H9C2细胞衰老但不会导致其凋亡;与Control组比较,H/R和PC均降低细胞活力,增加细胞凋亡率、ROS水平及caspase-3、caspase-9和Bcl-2 m RNA水平（P<0.01）;且PC组与H/R组比较,上述指标变化无明显差异;在H/R和PC组加入NaHS,可显著提高细胞活力,降低细胞凋亡率和氧化应激; PC+NaHS对上述指标的作用明显强于H/R+NaHS。结论:外源性H₂S能够恢复PC对衰老H9C2细胞的保护作用,其机制与抑制氧化应激和细胞凋亡有关。更多还原

【Abstract】 Objective: To investigate the recovery of protective effects of exogenous hydrogen sulfide（ H₂S） on hypoxia post-conditioning in aged H9C2 cells and its mechanism. Methods: H9C2 cells（ cardiomyocytes line） were treated with 30 μmol/L hydrogen peroxide（ H₂O₂） for 2 hours,then cultured for 3 days in order to induce cellular aging. Aged H9C2 cells were randomly divided into5 groups（ n = 8） : Control group（ Control）,hypoxia/reoxygenation group（ H/R）,H/R + NaHS group,hypoxia post-conditioning（ PC） group,PC+NaHS group. H/R model: the cells were exposed to hypoxic culture medium（ serum and sugar free medium,p H=6.8） for 3 hours and then cultured at normal condition for 6 hours. PC model: at the end of hypoxia for 3 hours,the cells were exposed to normoxic culture solution for 5 minutes,then the cells were placed in hypoxic solution for 5 minutes,the cycle above-mentioned was repeated 3 times and followed by reoxygenation for 6 hours. Advanced glycation end products（ AGEs） content and caspase-3 activity were detected by ELISA. The cell viability was observed by cell counting kit-8（ CCK-8）. The reactive oxygen species（ ROS） levels were analyzed using 2,7-dichlorodihydrofluorescein diacetate（ DCFH-DA） staining. The apoptotic rate was determined through Hoechst33342 staining. The m RNA levels of relative gene expression were detected by real-time PCR. Results: Thirty μmol/L H₂O₂ induced H9C2 cell senescence while did not lead to apoptosis. Compared with control group,cell viability was decreased,the apoptotic rate、levels of ROS and the m RNA of caspase-3,caspase-9 and Bcl-2 were increased in H/R and PC groups（ P < 0. 01）. There were no differences in the above indexes between PC group and H/R group. Supplementation of NaHS increased cell viability and decreased apoptotic rate and oxidative stress. The effects of PC + NaHS on the above indexes were better than those of H/R+NaHS group. Conclusion: Exogenous H₂S can restore the protective effect of PC on the aged H9C2 cells,and its mechanism is related to the inhibition of oxidative stress and apoptosis.更多还原