蛋白激酶C对氯化铵上调大鼠脑星形胶质细胞AQP-4表达的影响

Effects of protein kinase C on aquaporin-4 over-expression induced by ammonia in astrocytes of rats

目的:研究经氯化铵处理的大鼠脑星形胶质细胞水通道蛋白-4(AQP-4)的表达情况和蛋白激酶C(PKC)对AQP-4表达的调节作用.方法:取出生1～2d的SD大鼠大脑皮质,原代培养星形胶质细胞.将培养的细胞随机分为对照组,用等量培养基培养24h;NH4CL组,用终浓度为5mmol/L氯化铵培养6,12,24,48h;DMSO组:用10g/L二甲基亚砜(DMSO)预处理30min,余处理同NH4CL组;TPA组:蛋白激酶C激动剂TPA(1μmol/L)预处理30min,余处理同NH4CL组;RT组:蛋白激酶C拮抗剂Ro31-8220(1μmol/L)预处理30min后,余处理同TPA组.用光学显微镜观察细胞的形态学变化,用WesternBlot法检测各组细胞AQP-4的表达情况.结果:AQP-4表达量NH4CL组与DMSO组分别为(0.69±0.03),(0.67±0.03),与对照组相比较分别增加约90%,87%,细胞肿胀明显,差异显著(P<0.05);而NH4Cl组与DMSO组组间差异不显著;TPA组AQP-4表达量(0.40±0.02)与NH4CL组相比较,降低约41%(P<0.05);RT组AQP-4表达量(0.68±0.02)与TPA组相比较,明显增加,而与NH4CL组相比较差异无统计学意义.结论:经5mmol/L氯化铵处理的星形胶质细胞其AQP-4的表达明显增加,而PKC的激活能下调AQP-4的表达.

AIM：To examine the effects of protein kinase C（PKC）on the expression of aquaporin-4（AQP-4）in ammonia-treated astrocytes.METHODS：Cultured astrocytes were randomly divided into 5 groups：Control group：cultured with normal medium for 24 h;NH4Cl group：Incubated in culture medium with 5 mmol/L NH4Cl for 24 h;DMSO group：Pretreated with 10 g/L dimethyl sulfoxide（DMSO）for 30 min,and then treated as NH4Cl group;TPA group：Pretreated with 12-o-tetradecanoylphorbol 13-acetate（TPA）（1 μmol/L）,a PKC activator,for 30 min,subsequently treated as NH4Cl group.RT group：Pretreated with a PKC inhibitor Ro31-8220（1 μmol/L）for 30 min,and then treated as the TPA group;Cell morphology was assessed by light microscopy,and Western Blot analysis was used to detect AQP-4 expression.RESULTS：The AQP-4 expression in NH4Cl group and DMSO group was（0.69±0.03）and（0.67±0.03）,compared with the Control group,they increase significantly respectively（P〈0.05）,but there is no differences between them,and the astrocytes in these 2 group swell notably.The expression of AQP-4 in TPA group was（0.40±0.02）,compared with the NH4Cl group,it decreasesignificantly（P〈0.05）.And there was no difference in AQP-4 expression between RT group and NH4Cl group.CONCLUSION：The expressionof AQP-4 in astrocytes was increased following treatment of ammonia and the activation of protein kinase C could attenuate this AQP-4 over-expression.