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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 investigate the effect of Wen Dan Tang containing serum on the function of gap junctional intercellular communication (GJIC) and the expression of connexin 43 (Cx43) in 10μmol / ml glutamic acid . Methods: 40 clean SD rats were randomly divided into 5 groups: normal group, clozapine 20 mg / kg group, Weng Dan decoction 40 g / kg group, Wendan decoction 30 g / kg group, Wen Dan Tang 20 g / kg group, 8 in each group. Normal group with the same amount of normal saline ig, clozapine 20 mg / kg group ig clozapine 20 mg / kg, Wendan Decoction 40,30,20 g / kg group were ig Wendan Tang crude drug 40,30,20 g / kg, 1 time / d, 8 days after sacrifice to take blood, preparation containing serum. Astrocytes were treated with drug-containing serum at 10μmol / ml glutamic acid, that is, 10μmol / ml glutamate control group, normal rat serum group, clozapine 20mg / kg serum-containing group and Wendan Decoction 40 , 30 g, 20 g / kg serum group. After treated for 24, 36, 48 h, scrape-loading and dye transfer (SLDT) were used to assess the degree of diffusion of fluorescent dye between cells Gap junction communication indicators; Western blotting assay Cx43 phosphorylation and non-phosphorylation of astrocyte expression. Results: Under the condition of 10μmol / ml glutamate, the number of apoptotic cells was significantly increased after 24 hours of astrocytes, and more than 80% of cells were apoptotic and necrotic after 36h. Scratch-dye labeling was used to directly detect the apoptosis of GJIC In the 40 and 30 g / kg serum groups, GJIC and GJIC were increased at different degrees in three different time points. GJIC was increased only in the 36 h period in the 20 g / kg serum of the Wendan Decoction group. Under the condition of 10 μmol / Dang decoction 40 g / kg containing serum Cx43 phosphorylation and non-phosphorylation increased. CONCLUSION: Wen Dan Tang contains serum can reduce astrocyte apoptosis, promote astrocyte cell gap junctional intercellular communication (GJIC), downregulate astrocyte Cx43 phosphorylation, increase Cx43 non-phosphorylation , Which can promote cell gap junctional communication, which may be related to the abnormal activation of Cx43 in cells.