P38信号通路在星形胶质细胞氧糖剥夺/复氧后水通道蛋白4表达及细胞水肿形成中的作用

Role of P38 signaling pathway in neonatal rat astrocyte swelling and aquaporin-4 expression after oxygen-glucose deprivation and recovery

目的探讨P38信号通路在星形胶质细胞氧糖剥夺,复氧后水通道蛋白4表达及细胞水肿形成中发挥的作用。方法原代培养的星形胶质细胞分为正常组,模型组和P38抑制剂组。模型组:细胞接受5 h氧糖剥夺/复氧处理;P38抑制剂组:细胞在5 h氧糖剥夺后的复氧过程中加入P38抑制剂(SB203580,10μmol/L)处理。在5 h氧糖剥夺/复氧后的不同时间点,用RT-PCR及Westem blotting法测定P38、磷酸化P38及水通道蛋白4(AQP4)的表达变化,光镜观察细胞形态变化,乳酸脱氢酶测定反应细胞损伤程度。结果与正常组相比,星形胶质细胞在5 h氧糖剥夺/复氧后,其磷酸化P38的水平明显上升,并在复氧lh时达到峰值(P<0.01),AQP4 mRNA和蛋白的表达也明显升高(P<0.01),同时细胞水肿在氧糖剥夺/复氧后2 h时达到高峰,LDH漏出率也明显升高(P<0.01)。与模型组相比,加入P38抑制剂可明显降低5 h氧糖剥夺/复氧后P38磷酸化的水平(P<0.01),以及复氧后各时间点AQP4增高的水平(P<0.01),并可明显缓解氧糖剥夺/复氧后细胞水肿及LDH上升的程度(P<0.01)。结论 P38信号通路的激活参与了星形胶质细胞5 h氧糖剥夺/复氧后AQP4上调及细胞水肿形成,抑制p38的激活可降低AQP4表达的上调,减轻细胞水肿。

Objective To explore the role of P38 signaling pathway in neonatal rat astrocyte swelling and the expression of aquaporin-4（AQP4） after oxygen-glucose deprivation（OGD） and recovery（OGD/R）.Methods Primarily cultured neonatal rat astrocytes were subject to OGD for 5 h followed by oxygen-glucose recovery in the presence or absence of the P38 inhibitor SB203580（10μmol/L）.The astrocytes were investigated at 0.5,2,8 and 24 h after oxygen-glucose recovery for morphological changes and cell injuries using lactate dehydrogenase（LDH） assay.The expressions of P38,P-P38,and AQP4 mRNAs and proteins in the astrocytes were detected using RT-PCR and Western blotting.Results OGD/R caused significantly enhanced expression of P-P38 protein,and this effect was blocked by SB203580.AQP4 mRNA and protein expression declined transiently at 0.5 h after OGD and increased gradually to reach the peak level at 8 h（P〈0.05）.Application of the SB203580 significantly lowered OGD-induced AQP4 mRNA and protein up-regulation（P〈0.05）.Astrocyte swelling occurred after OGD/ R but was obviously lessened by SB203580.LDH release increased markedly after OGD/R,and was attenuated by treatment with SB203580（P0.01）.Conclusion P38 signaling pathway participates in astrocyte swelling after OGD/R,and blocking this pathway can attenuate AQP4 up-regulation and ameliorate the cell swelling.