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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%(P<0.01),而凋亡率升高至30.75%±5.1%(P<0.01)。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%(P<0.05),凋亡率则分别降低至14.73%±3.4%和18.32%±3.1%(P<0.01)。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(P<0.01)。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(P<0.01)。HEK 293细胞Caspase-3表达在各组无差异(P>0.05)。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。
Objective: To investigate the protective effect and possible mechanism of H_2S on apoptosis of SH-SY5Y cells induced by high concentration of ATP. METHODS: Human neuroblastoma SH-SY5Y cells, HEK 293 and HEK 293-hP2X_7R cells were divided into control group, Na HS group, KN-62 group, ATP group, ATP + Na HS group and ATP + KN-62 group . The morphological changes of cells were observed by inverted microscope. Cell viability was determined by CCK-8 assay. Apoptosis was analyzed by Hoechst 33258 nuclear staining. Flow cytometry was used to detect the apoptosis rate. Caspase-3 and Bcl-2 were detected by Western Blot and RT- Expression at protein and mRNA levels. Results: Compared with the control group, SH-SY5Y cells were significantly damaged after treated with 6 mmol / L ATP for 3 h and their viability decreased to 62.7% ± 3.8% (P <0.01) and apoptosis rate increased to 30.75% ± 5.1% P <0.01). Compared with the ATP group, the viability of SH-SY5Y cells increased to 90.1% ± 3.8% and 84.6% ± 3.1% (P <0.05) after pretreatment with 200 μmol / L Na HS and 500 nmol / L KN-62 for 30 min, The apoptotic rates were reduced to 14.73% ± 3.4% and 18.32% ± 3.1%, respectively (P <0.01). Caspase-3 was up-regulated and Bcl-2 was down-regulated in SH-SY5Y cells in ATP group, but Na HS and KN-62 inhibited Caspase-3 expression and promoted Bcl-2 expression. Compared with the control group and HEK293 cells, the HEK 293-h P2X7R cells were treated with 2 mmol / L ATP for 3 h, respectively. The expression of Caspase-3 in HEK 293-h P2X7R cells was up-regulated (P <0.01). Compared with ATP group, Caspase-3 expression in HEK 293-h P2X7R cells was significantly down-regulated after pretreatment with 200 μmol / L Na HS for 30 min (P <0.01). The expression of Caspase-3 in HEK 293 cells was not different in all groups (P> 0.05). CONCLUSION: H2S can inhibit the apoptosis of SH-SY5Y cells induced by high concentration ATP in a concentration-dependent manner, and its mechanism may be related to P2X7R.