孕酮减轻ATP诱导的SH-SY5Y细胞损伤  

Effect of progesterone on SH-SY5Y cells injured by ATP

作　　者：李秀娟[1] 沈慧[2] 魏林郁[1] 王国红[1] 张利彬[1] 李超堃[1] 李新娟[1] 王璐[1] 赵红岗[1] 宋景贵[3] 李东亮[1] 

机构地区：[1]新乡医学院生理学与神经生物学教研室,河南新乡453003 [2]新乡医学院第三附属医院,河南新乡453003 [3]新乡医学院第二附属医院,河南新乡453003

出　　处：《中国病理生理杂志》2017年第9期1587-1592,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81371346;No.81271376);河南省高等学校重点科研项目计划(No.16A310011;No.16A310013)

摘　　要：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。AIM： To investigate the neuroprotective effect of progesterone against adenosine triphosphate（ATP）-injured human neuroblastoma SH-SY5Y cells. METHODS： The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca^（2＋）concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X_7 receptor was assessed by Western blot. RESULTS： The viability of the SH-SY5Y cells was significantly decreased（ P 0. 05） and YO-PRO-1 uptake was obviously increased（ P 0. 05） in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1,3,5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3,10 and 30 nmol/L for 30 min（ P 0. 05） as compared with ATP group.YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced（ P 0. 05） by progesterone（30 nmol/L） or P2X_7 receptor antagonist KN-62（500 nmol/L） pretreatment for 30 min,and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca^（2＋） fluorescence intensity in normal group was a little,but that in ATP group was increased（ P 0. 05）. Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca^（2＋） induced by ATP（ P 0. 05）. However,no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X_7 receptor in ATP group was significantly higher than that in control group（ P 0. 05）,and progesterone inhibited ATP-induced P2X_7 receptor expression（ P 0. 05）. CONCLUSION：Progesterone inhibits P2X_7 receptor expression,membrane pore formation,intracellular Ca^（2＋） increase and cell death induced by ATP,so prog

关 键 词：孕酮 腺苷三磷酸 嘌呤能P2X7受体 SH-SY5Y细胞 

分 类 号：R[医药卫生]