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CJAP ›› 2018, Vol. 34 ›› Issue (4): 289-293.doi: 10.12047/j.cjap.5642.2018.067

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Protective effects of exogenous H2S to the recovery of hypoxia post-conditioning on aged H9C2 cells and the underling mechanisms

SUN Wei-ming, ZHANG Yuan-zhou, WEN Xin, XI Yu-xin, YUAN Di, WANG Yue-hong, WEI Can, XU Chang-qing, LI Hong-zhu   

  1. Departmentof Pathophysiology, Harbin Medical University, Harbin 150086, China
  • Received:2017-10-17 Revised:2018-01-29 Online:2018-07-28 Published:2019-02-21
  • Supported by:
    国家自然科学基金(81770486,81270273);哈尔滨医科大学研究生创新基金(YJSCX2016-35HYD)

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 into 5 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, 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. 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 mRNA 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.

Key words: hydrogen sulfide, hypoxia/reoxygenation, hypoxia post-conditioning, H9C2 cells, apoptosis

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