外源性H_(2)S对氧糖剥夺再灌注后SH-SY5Y细胞损伤的保护作用

Protective effect of exogenous H_(2)S on oxygen-glucose deprivation and reoxygenation-induced cell injury of SH-SY5Y cells

目的探讨外源性硫化氢(H_(2)S)对氧糖剥夺再灌注(OGD/R)后SH-SY5Y细胞损伤的保护作用。方法建立SH-SY5Y细胞OGD/R模型,SH-SY5Y细胞分为对照组、OGD/R模型组、OGD/R+不同浓度NaHS(H_(2)S供体)处理组;OGD/R制备:用不含血清的低糖DMEM培养基在37℃、930 mL/L N_(2)、20 mL/L O_(2)的低氧条件下培养各组细胞12 h,然后用完全培养液复氧培养24 h。采用CCK-8检测存活率,流式细胞技术检测细胞凋亡水平,蛋白印迹方法(Western blotting)检测细胞中内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)及NF-κBP65、COX-2蛋白表达水平。结果OGD/R模型组SH-SY5Y细胞活性较对照组降低(P<0.05)。0~0.5 mmol/L浓度的NaHS处理组细胞活性随着给予NaHS浓度的增加较OGD/R模型组逐渐升高(P<0.05),超过这个浓度范围,细胞活性较OGD/R模型组降低(P<0.05)。模型组细胞的早期凋亡水平均显著高于对照组(P<0.05)。0.2、0.4 mmol/L NaHS处理组细胞的早期凋亡水平较OGD/R模型组显著降低、4 mmol/L NaHS处理组细胞的早期凋亡水平较OGD/R模型组显著升高(P<0.05);Western blotting检测结果显示,OGD/R模型组细胞的GRP78、CHOP、NF-κBp65及COX-2蛋白表达水平均显著高于对照组(P<0.05);与OGD/R模型组比较,0.2、0.4、4 mmol/L浓度NaHS处理组细胞的GRP78、CHOP、NF-κBp65及COX-2表达水平显著降低(P<0.05),但各剂量组之间比较,差异无统计学意义(P>0.05)。结论NaHS可以提高经OGD/R损伤后细胞的活性,可能是通过抑制内质网应激诱导的NF-κBp65、COX-2蛋白的激活从而减少细胞凋亡水平来实现。

Objective To explore the protective effects of exogenous hydrogen sulfide(H_(2)S)on oxygen glucose deprivation and re-oxygenation(OGD/R)-induced SH-SY5Y cell injury.Methods OGD/R model was established in SH-SY5Y cells.Based on interferences,SH-SY5Y cells were divided into control group(no interference),OGD/R model group,and treatment groups(OGD/R model treated with different concentrations of NaHS(H_(2)S donor).The cells were cultured in low glucose medium without fetal bovine serum under 37℃,93%N_(2) and hypoxia(2%O_(2))for 12 h,then in complete medium under normoxia(5%O_(2))for 24 h.CCK-8 was used to detect cell viability,and flow cytometry was used for assaying the level of cellular apoptosis.Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins including glucose regulatory protein 78(GRP78),CCAAT/enhancer-binding homologous protein(CHOP),NF-κB P65 and COX-2.Results The cell viability was lower in OGD/R model group than in the control group(P<0.05).NaHS treatment at a concentration of 0-0.5 mmol/L increased cell viability in OGD/R model group in a dose-dependent manner(P<0.05).In contrast,NaHS(C>0.5 mmol/L)decreased cell viability of OGD/R(P<0.05).The apoptosis level of early stage in OGD/R model group was significantly higher than that in control group(P<0.05).NaHS treatment at concentrations of 0.2 and 0.4 mmol/L lowered the apoptosis level of early stage in OGD/R model group(P<0.05).Western blot showed that the protein expression levels of GRP78,CHOP,NF-κB p65,and COX-2 were significantly higher in OGD/R model group than those in control group(P<0.05).When compared with OGD/R model group,the expression levels of GRP78,CHOP,NF-κBp65 and COX-2 were decreased by NaHS at concentrations of 0.2,0.4 and 4 mmol/L(P<0.05),but there was no statistically significant difference among the treatment groups(P>0.05).Conclusion NaHS can rescue cell viability of OGD/R-induced cell injury.It may be achieved by inhibiting the activation of NF-κBp65 and COX-2 proteins induced by endoplasmic reticulum stress to reduce the level of cell apoptosis.