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 (1 μmol/L和10 μmol/L, U1组和U10组)、SP600125 (1 μmol/L和10 μmol/L, SP1组和SP10组)和SB203580 (1 μmol/L和10 μmol/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p38 MAPK(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p38 MAPK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 P2 cells were divided into control group, OGD group and inhibitors of U0126, SB203580 and SP600125 groups. The latter groups underwent OGD for five hours and reoxygenated, the inhibitors of U0126, SB203580 and SP600125 (1 μmol/L and 10 μmol/L, respectively) groups, named U1, U10, SB1, SB10, SP1 and SP10 groups, 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 AQP4 was detected half, one, one and half, eight and twelve hours after reoxygenation with Western blotting. The expression of AQP4, and phosphorylation of extracellular regulated protein kinases (p-ERK), c-Jun N-terminal kinase (p-JNK) and p38 MAPK (p-p38 MAPK) was detected 24 hours 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 AQP4 also increased in OGD group after reoxygenation (P<0.001), especially 1.5 hours of reoxygenation. The expression of AQP4, p-ERK, p-JNK and p-p38 MAPK 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-p38 MAPK decreased in the inhibitors groups compared with those in OGD group (P<0.001), and the expression of AQP4 decreased in SB10 group (P<0.001). The activity of LDH was less in all the inhibitors groups except SP1 and SB1 groups than in OGD group (P<0.01), and was the least in SB10 group (P<0.001). Conclusion MAPKs signal pathway, especially p38 MAPK, may promote AQP4 expression in astrocytes after OGD in rat, and play a role in cells death.
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