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中国应用生理学杂志 ›› 2018, Vol. 34 ›› Issue (4): 289-293.doi: 10.12047/j.cjap.5642.2018.067

• 研究论文 •    下一篇

外源性H2S恢复缺氧后适应对衰老H9C2细胞的保护作用及机制

孙伟铭, 张源洲, 温馨, 席雨鑫, 袁迪, 王跃虹, 魏璨, 徐长庆, 李鸿珠   

  1. 哈尔滨医科大学基础医学院病理生理学教研室, 黑龙江 哈尔滨 150086
  • 收稿日期:2017-10-17 修回日期:2018-01-29 出版日期:2018-07-28 发布日期:2019-02-21
  • 通讯作者: 李鸿珠 E-mail:hongzhuli61@163.com
  • 基金资助:
    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)

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)

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30 μmol/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,pH=6.8)培养3 h,然后正常培养6 h;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mRNA水平。结果:30 μmol/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 mRNA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。

关键词: 硫化氢, 缺氧/复氧, 缺氧后适应, H9C2细胞, 细胞凋亡

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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