温胆汤含药血清对谷氨酸条件下原代星形胶质细胞P38MAPK及PKC的影响

Effect of Wendan decoction medicated serum on P38MAPK and PKC in primary astrocytes under glutamate condition

目的:探讨温胆汤含药血清在谷氨酸(Glu)环境条件下对星形胶质细胞P38MAPK和PKC表达的影响。方法:将40只SD大鼠随机分为5组:正常组、氯氮平20mg/kg组、温胆汤40g/kg组、温胆汤30g/kg组、温胆汤20g/kg组,每组8只。正常组用等量生理盐水ig,氯氮平组ig 20 mg/kg,ig温胆汤生药40、30、20 g/kg,1次/d,8天后处死取血,制备含药血清。胶质原纤维酸性蛋白免疫染色法鉴定培养的星形胶质细胞纯度。星形胶质细胞在谷氨酸(0、5、10μmol/ml)条件下经含药血清处理,即谷氨酸对照组、正常血清组、氯氮平20mg/kg含药血清组、温胆汤40g/kg、30g/kg、20g/kg血清组,处理24h、36h和48h后,采用四甲基偶氮唑盐(MTT)法检测细胞存活率;ELISA法检测P38MAPK磷酸化和非磷酸化及PKC蛋白表达的影响。结果:培养的星形胶质细胞纯度在95%以上。谷氨酸(0μmol/ml)条件下,温胆汤40g/kg、30g/kg含药血清组在3个时间段不同程度增加星形胶质细胞存活率;谷氨酸(5、10μmol/ml)条件下,温胆汤含药血清处理24h、36h后,温胆汤40g/kg、30g/kg、20g/kg含药血清组不同程度增加的细胞存活率;48h后,温胆汤40g/kg含药血清组增加的细胞存活率。谷氨酸(0、5、10μmol/ml)条件下,氯氮平20mg/kg含药血清组、温胆汤30g/kg含药血清组能显著上调星形胶质细胞P38MAPK蛋白磷酸化和非磷酸化的表达。谷氨酸(0、5μmol/ml)条件下,温胆汤40g/kg、30g/kg、20g/kg含药血清组显著降低星形胶质细胞PKC表达;谷氨酸(10μmol/ml)条件下,温胆汤40g/kg含药血清组显著降低星形胶质细胞PKC表达。结论:一定谷氨酸环境下温胆汤能不同程度的增加星形胶质细胞的存活率,能上调P38MAPK磷酸化和非磷酸化的表达及降低PKC蛋白的表达,从而间接反映出温胆汤能调节星形胶质细胞GJIC(细胞缝隙连接)的功能。

Objective： To research the effect of serum containing Wendan decoction on P38 MAPK and PKC of astrocytes in glutamate（ Glu） environmental conditions. Methods： 40 SD rats were randomly divided into five groups： normal group,clozapine 20 mg / kg group,Wendan decoction 40 g / kg group,Wendan decoction 30 g / kg group and Wendan decoction 10 g / kg group,egivenht rats in each group. normal group groups were given normal saline every day,clozapine 20 mg / kg group given orgiveninal drug clozapine 20 mg / kg,the Wendan decoction 40 g / kg,Wendan decoction 30 g / kg and Wendan decoction 10 g / kg groups were respectively given Wendan decoction（ 40,20,10 g / kg）,1 times / d,After 8 days,the rats were sacrificed to preparation containing serum. Glial fibrillary acidic protein immunostaining identified cultured astrocytes purity.Astrocytes in glutamate（ 0,5,10μmol / ml） conditions treated by the medicated serum drugs,namely glutamate in the control group,normal serum containing group,clozapine 20 mg / kg serum containing group,Wendan decoction 40 g / kg,30 g / kg,20 g / kg serum containing group,after 24 h,36h and 48 h treatment,Using tetrazolium salt（ MTT） assay to detect cell viability; The contents of serum P38 MAPK phosphorylated and non-phosphorylated,PKC were detected by double antibody clip ELASA method. Results： cultured astrocytes purity of more than 95%. Under glutamate（ 0μmol / ml） conditions,Wendan decoction 40 g / kg group containing serum significantly increased the survival rate of astrocytes in three time periods（ P 0. 05）; Under glutamate（ 5,10μmol / ml） conditions,Wendan decoction containing serum treatment 24 h,and 36 h,Wendan decoction 30 g / kg,20 g / kg medicated serum cell viability was significantly increased; 48 h later,Wendan decoction 40 g / kg medicated serum cells was significantly increased. After the first treatment with serum containing 24 h,Under glutamate（ 0,5,10μmol / ml） conditions,clozapine 20 mg / kg medicated serum group,Wendan decoction 30 g / kg medicated serum significantly upregulated astrocytes P38 MAPK protein phosphorylation and expression of non-phosphorylated; Under glutamate（ 0,5μmol / ml） acid conditions,Wendan decoction 40 g / kg,30 g / kg,20 g / kg medicated serum significantly reduced astrocyte PKC expression,under glutamate（ 10μmol/ml） conditions,Wendan decoction medicated serum 40g/kg group was significantly reduced astrocyte PKC expression. Conclusion： Under glutamic acid conditions,Wendan decoction can increase the survival rate of astrocytes,can upregulate the expression of P38 MAPK phosphorylation and non-phosphorylated and PKC,reflecting Wendan decoction astrocytes can increase GJIC（ gap junction intercellular communication） function.