硫化氢抑制高糖诱导的内皮细胞损伤的研究

Mechanisms of hydrogen sulfide that attenuate high glucose-induced injury in endothelial cells

目的观察高糖环境下细胞内硫化氢的生成及硫化氢对高糖诱导的内皮细胞损伤的影响并探讨其机制。方法将人脐静脉内皮细胞分为4组:A组为正常糖浓度组(5.5 mmol/L),B组为高糖组(25 mmol/L),C组为高糖(25 mmol/L)+硫氢化钠(50μmol/L)组,D组为正常糖浓度(5.5 mmol/L)+硫氢化钠(50μmol/L)组,处理48 h后,测定细胞内硫化氢的含量,M TT检测细胞存活率,Western Blot检测细胞凋亡相关蛋白Bax、Bcl-2和Cleaved caspase-3的表达。结果①与A组相比,B组硫化氢生成量明显减少(P<0.05),与B组相比,C组硫化氢生成量明显增多(P<0.05);②与A组相比,B组的细胞存活率明显减少(P<0.05),而C组的细胞存活率较B组显著升高(P<0.05);③与A组相比,B组Bax表达显著升高(P<0.05),Bcl-2的表达显著降低(P<0.05),而Caspase-3活性明显增强(P<0.05);与B组相比,C组Bax水平显著降低(P<0.05),Bcl-2水平显著升高(P<0.05),Caspase-3活性显著下降(P<0.05)。与A组相比,D组各指标表达差异无统计学意义(P>0.05)。结论高糖对内皮细胞硫化氢的生成具有抑制作用;外源性硫化氢可保护高糖诱导的内皮细胞损伤,其机制可能与抑制细胞凋亡有关。

Objective To observe the production of hydrogen sulfide in endothelial cells exposed to high glucose as well as the effects of hydrogen sulfide on high glucose-induced injury of endothelial cells,and to explore the possible mechanisms.Methods Human umbilical vein endothelial cells were divided into 4 groups： A 5.5 mmol/l glucose,B 25 mmol/L glucose,C 25 mmol/L glucose ＋ 50 μmol/L NaHS,and D 5.5 mmol/L glucose＋ 50 μmol/L NaHS.After treatment for 48 h,the production of H2S was measured,cell viability was detected by MTT,and the expressions of apoptosis-related proteins,such as Bax,Bcl-2 and cleaved caspase-3,were examined by Western Blot analysis.Results ① Compared with that of group A,the production of H2S distinctly decreased in group B（P〈0.05）,however,the production of H2S distinctly increased in group C when compared with that of group B（P〈0.05）.② Compared with that of group A,the percentage of cell viability obviously decreased in group B（P〈0.05）,but NaHS could reverse the viability of HUVECs which was induced by high glucose in group C（P〈0.05）.③ High glucose significantly increased Bax protein expression（P〈0.05） and decreased the Bcl-2 protein expression（P〈0.05）,and the level of cleaved Caspase-3,the activated form of Caspase-3,was higher in group B compared with that of group A（P〈0.05）.Compared with that of group B,decrease in Bax（P〈0.05） and cleaved Caspase-3 expressions（P〈0.05） and increase in Bcl-2 expression（P〈0.05） were observed in group C.There was no significant difference between group A and group D in cell viability,Bax,Bcl-2,cleaved Caspase-3 expressions and the production of H2S.Conclusions High glucose can inhibit the production of H2S in endothelial cells.Supplementation of extrinsic H2S can protect against high glucose-induced injury in human umbilical vein endothelial cells possibly through the inhibition of cell apoptosis.