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目的:探讨孕酮对抗腺苷三磷酸(ATP)诱导的人神经母细胞瘤SH-SY5Y细胞损伤的神经保护作用和机制。方法:取对数生长期的SH-SY5Y细胞按照孕酮或ATP浓度的不同进行分组,CCK-8法检测细胞存活率,YO-PRO-1染色检测细胞膜通透性,Fluo-3染色检测细胞内Ca~(2+)浓度的变化,Western blot法检测嘌呤能P2X_7受体表达的变化。结果:与对照组相比,不同浓度(1、3、5和7 mmol/L)ATP作用2 h,SH-SY5Y细胞存活率显著降低(P<0.05),细胞摄入YO-PRO-1的荧光强度明显增加(P<0.05),且呈剂量依赖性。浓度为3、10和30 nmol/L的孕酮预孵育30 min可减轻ATP损伤作用,细胞存活率较单纯ATP组明显升高(P<0.05或P<0.01)。孕酮(30nmol/L)或P2X_7受体拮抗剂KN-62(500 nmol/L)预孵育30 min均可显著抑制ATP诱导的胞内YO-PRO-1的荧光增强(P<0.01),而孕酮和KN-62两者之间没有明显差异。正常组细胞内钙离子含量少,ATP组细胞内钙离子荧光强度较对照组明显增高(P<0.05),孕酮(30 nmol/L)或KN-62(500 nmol/L)预孵育30 min可明显降低(P<0.05)ATP诱导的胞内钙荧光增强,而孕酮和KN-62两者之间的作用无明显差异。ATP组SH-SY5Y细胞P2X_7受体表达较对照组明显增加(P<0.05),而孕酮(30 nmol/L)预孵育30 min则可显著降低ATP诱导的P2X_7受体表达(P<0.05)。结论:孕酮可抑制ATP诱导的P2X_7受体表达、膜孔形成和胞内Ca~(2+)升高,降低细胞死亡率,明显减轻高浓度ATP对SH-SY5Y细胞的损伤作用。
Objective: To investigate the neuroprotective effects of progesterone on the injury of human neuroblastoma SH-SY5Y cells induced by adenosine triphosphate (ATP) and its mechanism. Methods: SH-SY5Y cells in logarithmic growth phase were divided into groups according to progesterone or ATP concentration. Cell viability was detected by CCK-8 assay, cell membrane permeability was detected by YO-PRO-1 staining and cells were detected by Fluo-3 staining (Ca2 +) concentration in serum was detected by Western blot. Results: Compared with the control group, the survival rate of SH-SY5Y cells was significantly decreased (P <0.05) after treated with different concentrations of 1, 3, 5 and 7 mmol / L ATP for 2 hours. Fluorescence intensity was significantly increased (P <0.05), and in a dose-dependent manner. Preincubation of progesterone at concentrations of 3, 10 and 30 nmol / L for 30 min reduced ATP-induced injury, and cell viability was significantly higher than that of ATP alone (P <0.05 or P <0.01). Preincubation of progesterone (30 nmol / L) or P2X_7 receptor antagonist KN-62 (500 nmol / L) for 30 min significantly inhibited the increase of ATP-induced intracellular YO-PRO-1 fluorescence (P <0.01) There was no significant difference between progesterone and KN-62. Compared with the control group, the intracellular Ca2 + fluorescence intensity in ATP group was significantly increased (P <0.05), while the progesterone (30 nmol / L) or KN-62 (500 nmol / L) (P <0.05) ATP-induced intracellular calcium fluorescence enhancement, while there was no significant difference between progesterone and KN-62. Compared with the control group, the expression of P2X_7 receptor in SH-SY5Y cells in ATP group increased significantly (P <0.05), while the progesterone (30 nmol / L) preincubated for 30 min decreased the expression of P2X_7 receptor (P <0.05) . CONCLUSION: Progesterone can inhibit ATP-induced P2X_7 receptor expression, membrane pore formation and intracellular Ca 2+ increase, reduce cell death rate and significantly reduce the injury of SH-SY5Y cells induced by high concentration of ATP.