H_2S抑制高浓度ATP诱导的SH-SY5Y细胞凋亡与P2X_7受体的相关性研究

目的:研究H_2S对高浓度ATP诱导的SH-SY5Y细胞凋亡的保护作用和可能的机制。方法:人神经母细胞瘤SH-SY5Y细胞、HEK 293和HEK 293-hP2X_7R细胞,分为对照组,Na HS组,KN-62组,ATP组,ATP+Na HS组和ATP+KN-62组。倒置显微镜观察细胞形态变化,CCK-8法检测细胞活力,Hoechst 33258核染色分析细胞凋亡,流式细胞术检测细胞凋亡率,Western Blot和RT-PCR法分别检测Caspase-3、Bcl-2在蛋白和mRNA水平的表达。结果:与对照组比较,6 mmol/L ATP处理3 h后SH-SY5Y细胞损伤明显,活力降低至62.7%±3.8%(P0.01),而凋亡率升高至30.75%±5.1%(P0.01)。与ATP组比较,用200μmol/L Na HS和500 nmol/L KN-62预处理30 min,SH-SY5Y细胞活力分别升高至90.1%±3.8%和84.6%±3.1%(P0.05),凋亡率则分别降低至14.73%±3.4%和18.32%±3.1%(P0.01)。ATP组SH-SY5Y细胞内Caspase-3表达上调,Bcl-2表达下调,但Na HS和KN-62可抑制Caspase-3表达,促进Bcl-2表达。与对照组和HEK293细胞比较,用2 mmol/L ATP分别处理HEK 293、HEK 293-h P2X7R细胞3 h,可见HEK 293-h P2X7R细胞内Caspase-3表达上调(P0.01)。与ATP组比较,200μmol/L Na HS预处理30 min,HEK 293-h P2X7R细胞内Caspase-3表达明显下调(P0.01)。HEK 293细胞Caspase-3表达在各组无差异(P0.05)。结论:H2S对高浓度ATP诱导的SH-SY5Y细胞凋亡具有抑制作用,且呈浓度依赖性,其作用机制可能与P2X7R相关。

Correlation Study of H2S inhibits the apoptosis of SH-SY5Y cells induced by high concentration of ATP and P2X7 receptors

Objective:To study the protective effect and possible mechanism of H2S on the apoptosis of SH-SY5Y cells induced by high concentration of ATP.Methods:SH-SY5Y human neuroblastoma cells,HEK 293 and HEK 293-hP2X7 R cells,according to the design,divided into control group,NaHS group,KN-62 group,ATP group,ATP + NaHS group and ATP + KN-62 group.The changes of cell morphology were observed by inverted microscope,and the cell viability was detected by CCK-8.Apoptosis was analyzed by Hoechst 33258 staining,and apoptosis rate was detected by flow cytometry.Western Blot and RT-PCR were used to detect the expression of Caspase-3 and Bcl-2 in protein and mRNA levels,respectively.Results:compared with control group,SH-SY5Y cells were injuryed obviously when they were treated with ATP at 6 mmol/L for 3 hours,cell viability decreased to 62.7％ ± 3.8％ (P ＜ 0.01),and the apoptosis rate increased to 30.75％ ±5.1％ (P ＜ 0.01).Compared with ATP group,SH-SY5Y cells were treated with 200 μmol/L NaHS and 500 nmol/L KN-62 pretreatment for 30 min,cell viability were increased to 90.1％ ± 3.8％ and 84.6％ ± 3.1％ (P ＜ 0.01) and apoptosis rate were reduced to 14.73 ± 3.4％ and 18.32％ ± 3.1％ (P ＜ 0.05).Up regulation of Caspase-3 expression and down regulation of Bcl-2 expression in SH-SY5Y cells can be achieved with ATP,but NaHS and KN-62 can inhibit the expression of Caspase-3 and promote the expression of Bcl-2.Compared with control group and HEK 293 cells,HEK 293 and HEK 293-hP2X7 R cells were treated with ATP at 2 mmol/L for 3 hours respectively,which could increase the expression of Caspase-3 in HEK 293-hP2X7 R cells (P ＜ 0.01).Compared with ATP group,that 200 μmol/L NaHS pretreated for 30 min can down regulate the expression of Caspase-3 in HEK cells (P ＜ 0.01).In HEK 293 cells,the expression of Caspase-3 in each group showed no difference (P ＞ 0.05).Conclusion:The results showed that H2S could inhibit the apoptosis of SH-SY5Y cells induced by high concentration of ATP,and the concentration was dependent.Its mechanism may be related to P2X7 R.