有丝分裂原活化蛋白激酶信号通路介导大鼠星形胶质细胞氧糖剥夺后水通道蛋白4的表达

Role of Mitogen-activated Protein Kinase Pathways in Expression of Aquaporin-4 in Astrocytes after Oxygen-glucose Deprivation in Rats

目的探讨有丝分裂原活化蛋白激酶(MAPKs)是否介导缺血后大鼠星形胶质细胞水通道蛋白4(AQP4)的表达。方法分离培养新生Sprague-Dawley大鼠脑星形胶质细胞。第2代细胞分成对照组、氧糖剥夺(OGD)组和阻断组。后两组复制OGD 5 h后复氧模型,12 h后阻断组分别换入含U0126(1μmol/L和10μmol/L,U1组和U10组)、SP600125(1μmol/L和10μmol/L,SP1组和SP10组)和SB203580(1μmol/L和10μmol/L,SB1组和SB10组)的培养液。复氧后0.5 h、1 h、1.5 h、2 h、3 h、4 h、8 h和12 h检测OGD组细胞体积,复氧后0.5 h、1 h、1.5 h、8 h和12 h Western blotting法检测OGD组AQP4表达;复氧后24 h,检测各组乳酸脱氢酶(LDH)活性,Western blotting法检测各组AQP4,磷酸化细胞外调节蛋白激酶(p-ERK)、c-Jun氨基末端激酶(p-JNK)和p38MAPK(p-p38 MAPK)表达。结果复氧后1.5 h、2 h、3 h和4 h时,OGD组细胞体积较对照组显著增大(P<0.001),OGD组AQP4水平较对照组显著升高(P<0.001),并在复氧后1.5 h达到峰值。OGD组p-ERK、p-JNK和p-p38 MAPK、AQP4水平较对照组显著增加(P<0.001),阻断组p-ERK、p-JNK和p-p38 MAPK较OGD组显著下降(P<0.001),SB10组AQP4较OGD组显著下降(P<0.001)。除SP1组、SB1组外,各干预组LDH活性较OGD组显著下降(P<0.01),SB10组最低(P<0.001)。结论 MAPK信号通路,特别是p38MAPK可介导大鼠星形胶质细胞AQP4蛋白表达,加重细胞坏死。

Objective To investigate whether mitogen-activated protein kinases(MAPKs),which were involved in changes in osmolality,might mediate aquaporin-4(AQP4)expression in astrocytes after oxygen-glucose deprivation(OGD)in rats.Methods Astrocytes were obtained from new born Sprague-Dawley rats.The P2cells were divided into control group,OGD group and inhibitors of U0126,SB203580and SP600125groups.The latter groups underwent OGD for five hours and reoxygenated,the inhibitors of U0126,SB203580and SP600125(1μmol/L and10μmol/L,respectively)groups,named U1,U10,SB1,SB10,SP1and SP10groups,respectively,were cultured with the inhibitors for twelve hours.The volume of cells in OGD group was measured half,one,one and half,two,three,four,eight and twelve hours after reoxygenation,and the expression of AQP4was detected half,one,one and half,eight and twelve hours after reoxygenation withWestern blotting.The expression of AQP4,and phosphorylation of extracellular regulated protein kinases(p-ERK),c-Jun N-terminal kinase(p-JNK)and p38MAPK(p-p38MAPK)was detected24hours after reoxygenation in all the groups,while the activity of lactate dehydrogenase(LDH)was measured.Results The volume of cells increased in OGD group one and half,two,three and four hours after reoxygenation compared with those in the control group(P<0.001),and the expression of AQP4also increased in OGD group after reoxygenation(P<0.001),especially1.5hours of reoxygenation.The expression of AQP4,p-ERK,p-JNK and p-p38MAPK increased in OGD group after five hours of OGD compared with those in the control group(P<0.001),and the expression of p-ERK,p-JNK and p-p38MAPK decreased in the inhibitors groups compared with those in OGD group(P<0.001),and the expression of AQP4decreased in SB10group(P<0.001).The activity of LDH was less in all the inhibitors groups except SP1and SB1groups than in OGD group(P<0.01),and was the least in SB10group(P<0.001).Conclusion MAPKs signal pathway,especially p38MAPK,may promote AQP4expression in astrocytes after OGD in rat,and play a role in cells death.