温胆汤含药血清对谷氨酸条件下星形胶质细胞Cx43和GJIC功能的影响

Effect of Wendan decoction medicated serum on Cx43 and GJIC in astrocytes under glutamate condition

目的:探讨温胆汤含药血清在10μmol/ml谷氨酸条件下对星形胶质细胞细胞缝隙连接通讯(gap junctional intercellular communication,GJIC)功能和连接蛋白43(connexin 43,Cx43)表达的影响。方法:将40只清洁级SD大鼠随机分为5组:正常组、氯氮平20 mg/kg组、温胆汤40 g/kg组、温胆汤30 g/kg组、温胆汤20 g/kg组,每组8只。正常组用等量生理盐水ig,氯氮平20 mg/kg组ig氯氮平20 mg/kg,温胆汤40、30、20 g/kg组分别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。星形胶质细胞在10μmol/ml谷氨酸条件下经含药血清处理,即10μmol/ml谷氨酸对照组、正常鼠血清组、氯氮平20mg/kg含药血清组、温胆汤40、30g、20 g/kg血清组,处理24、36、48 h后,采用细胞划痕染料标记示踪技术(scrape-loading and dye transfer,SLDT),以荧光染料在细胞间的扩散程度作为评价缝隙连接通讯的指标;Western blotting法检测星形胶质细胞中Cx43磷酸化和非磷酸化的表达。结果:10μmol/ml谷氨酸条件下,星形胶质细胞24h之后细胞凋亡数量明显,36h之后80%以上细胞处于凋亡坏死状态;划痕染料标记示踪技术直接检测GJIC中,温胆汤40、30 g/kg血清组在3个时间段不同程度的明显增加细胞GJIC,温胆汤20 g/kg血清组只在36h时间段增加细胞GJIC;10μmol/ml谷氨酸条件下,温胆汤40 g/kg含药血清使Cx43磷酸化下调和非磷酸化升高。结论:温胆汤含药血清可减少星形胶质细胞的凋亡;促进星形胶质细胞细胞缝隙连接通讯(GJIC);下调星形胶质细胞Cx43磷酸化,升高Cx43非磷酸化程度,从而对细胞缝隙连接通讯功能具有促进作用,这可能与其引起细胞内Cx43的异常活化有关。

Objective： To research the effect of Wendan decoction medicated serum on Cx43 and GJIC of astrocytes under 10μmol / ml glutamate condition. Methods： 40 SD rats were randomly divided into five groups： normal group,Clozapine 20 mg / kg group,Wendan decoction 40,30,20 g / kg group. normal groups were given normal saline every day,Clozapine 20 mg / kg group given drug clozapine 20 mg / kg,the Wendan decoction 40,30,20 g / kg groups were respectively given Wendan decoction /40,30,20 g / kg,once a day. After 8 days,the rats were sacrificed to preparation medicated serum. Astrocytes treated by drugs in 10μmol / ml glutamate,namely GLU control group,normal rat serum containing group,clozapine 20 mg / kg serum containing group,Wendan decoction 40,30,20 g / kg serum containing group after 24,36,48 h,using SLDT to detect the GJIC. Indicators fluorescent dye diffusion between cells in the degree as the evaluation of gap junctional intercellular communication. The expressing of Cx43 non-phosphorylated Cx43 and phosphorylation were determined by Western Blotting. Result： Under the condition of 10 μmol / ml glutamate,the number of astrocytes apoptotic cells was increased significantly after 24 hours,more than 80%cells were in the state of apoptosis and necrosis after 36 hours. The technology of scratchingdye-labeled tracer measures directly GJIC in 40,30 g o kg-1 Wendan decoction serum class in three different time periods have significantly increased the functions of GJIC,20 g / kg Wendan decoction medicated serum increases the function of GJIC in 36 h period only. Under the condition of 10 μmol / ml glutamate,40 g / kg Wendan decoction medicated serum make Cx43 phosphorylated down and unphosphorylated up. Conclusion： Wendan decoction medicated serum can reduce the apoptosis of astrocytes,promote the function of astrocyte gap junctional intercellular communication（ GJIC）,reduce astrocyte Cx43 phosphorylation,elevate Cx43 degree of non-phosphorylated and thus the cell gap junction intercellular communication has been promoted,this may be related to abnormal activation of intracellular Cx43.