Sun Wei-Ming, Zhang Yuan-Zhou, Wen Xin, Xi Yu-Xin, Yuan Di, Wang Yue-Hong, Wei Can, Xu Chang-Qing, Li Hong-Zhu
Departmentof Pathophysiology, Harbin Medical University, Harbin 150086, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2018 Apr 8;34(4):289-293. doi: 10.12047/j.cjap.5642.2018.067.
To investigate the recovery of protective effects of exogenous hydrogen sulfide (HS) on hypoxia post-conditioning in aged H9C2 cells and its mechanism.
H9C2 cells (cardiomyocytes line) were treated with 30 μmol/L hydrogen peroxide (HO) for 2 hours, then cultured for 3 days in order to induce cellular aging. Aged H9C2 cells were randomly divided into 5 groups (=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, pH=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 Hoechst 33342 staining. The mRNA levels of relative gene expression were detected by real-time PCR.
Thirty μmol/L HO 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 mRNA of caspase-3, caspase-9 and Bcl-2 were increased in H/R and PC groups (<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.
Exogenous HS 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.
探讨外源性硫化氢（HS）对衰老H9C2细胞缺氧后处理保护作用的恢复情况及其机制。
用30 μmol/L过氧化氢（HO）处理H9C2细胞（心肌细胞系）2小时，然后培养3天以诱导细胞衰老。将衰老的H9C2细胞随机分为5组（=8）：对照组（Control）、缺氧/复氧组（H/R）、H/R +硫氢化钠（NaHS）组、缺氧后处理（PC）组、PC + NaHS组。H/R模型：将细胞置于缺氧培养基（无血清无糖培养基，pH = 6.8）中3小时，然后在正常条件下培养6小时。PC模型：在缺氧3小时结束时，将细胞置于常氧培养液中5分钟，然后将细胞置于缺氧溶液中5分钟，重复上述循环3次，然后复氧6小时。采用酶联免疫吸附测定法（ELISA）检测晚期糖基化终末产物（AGEs）含量和半胱天冬酶-3（caspase-3）活性。使用细胞计数试剂盒-8（CCK-8）观察细胞活力。采用2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）染色分析活性氧（ROS）水平。通过Hoechst 33342染色测定凋亡率。采用实时荧光定量聚合酶链反应（real-time PCR）检测相关基因表达的mRNA水平。
30 μmol/L HO诱导H9C2细胞衰老但未导致凋亡。与对照组相比，H/R组和PC组细胞活力降低，凋亡率、ROS水平以及caspase-3、caspase-9和Bcl-2的mRNA水平升高（<0.01）。PC组与H/R组上述指标无差异。补充NaHS可提高细胞活力，降低凋亡率和氧化应激。PC + NaHS对上述指标的影响优于H/R + NaHS组。
外源性HS可恢复PC对衰老H9C2细胞的保护作用，其机制与抑制氧化应激和凋亡有关。
