温胆汤含药血清对10 mmol/L谷氨酸环境下星型胶质细胞PI3K、Akt、mTOR表达的影响

Effect of Serum Containing Wendan Decoction(温胆汤)on Expressions of PI3K,Akt and mTOR in Astrocytes Under Environment of 10 mmol/L Glutamic Acid

目的 探讨温胆汤含药血清对10 mmol/L谷氨酸诱导的大鼠星型胶质细胞凋亡、周期及磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)、蛋白激酶B(protein kinase B,Akt)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)表达的影响。方法 将60只SPF级SD雄性大鼠随机分为5组,其中正常组20只,氯氮平组和温胆汤高、中、低剂量组各10只。正常组予20 g/kg 0.9%氯化钠溶液灌胃,氯氮平组予氯氮平原药20 mg/kg灌胃,温胆汤高、中、低剂量组分别予40、20、10 g/kg温胆汤灌胃,1次/d,共8 d。处死大鼠后取血,离心取血清,灭活除菌,EP管分装备用。将大鼠星型胶质细胞分为正常血清组、模型血清组,氯氮平含药血清组和温胆汤高、中、低剂量含药血清组,除正常血清组外,其余各组予10μmol/mL谷氨酸处理48 h后,流式细胞仪检测细胞凋亡和细胞周期;Western bloting、Real-time PCR分别测定细胞PI3K、Akt、mTOR蛋白和mRNA表达。结果 温胆汤含药血清可明显降低谷氨酸环境下大鼠星型胶质细胞凋亡率(P<0.01)和G0/G1期细胞占比(P<0.05),升高S期及G2/M期细胞占比;降低细胞P-P13K/P13K、P-ATK/AKT、P-mTOR/mTOR蛋白表达比值(温胆汤低剂量血清组除外,P<0.05)和细胞PI3K、Akt、mTOR mRNA表达(温胆汤低剂量血清组除外,P<0.01)。结论 温胆汤含药血清可有效调节大鼠星形胶质细胞PI3K、Akt、mTOR表达,达到保护神经细胞的目的,这可能是温胆汤治疗精神分裂症的作用机制之一。

Objective To investigate the effect of serum containing Wendan Decoction(温胆汤)on apoptosis,cell cycle and expressions of phosphoinositide 3-kinase(PI3K),protein kinase B(Akt)and mammalian target of rapamycin(mTOR)of rat astrocytes induced by glutamate.Methods Sixty male SPF SD rats were randomly divided into 5 groups,including 20 in the normal group,10 in the clozapine group and 10 in the high,middle and low dose groups of Wendan Decoction.The normal group was given 20 g/kg normal saline by gavage,the clozapine group was given 20 mg/kg clozapine technical drug by gavage,and the high,middle and low dose groups of Wendan Decoction were given 40,20 and 10 g/kg Wendan Decoction by gavage,once a day,for a total of 8 days.After the rats were killed,the blood was taken out,the serum was centrifuged,inactivated and sterilized,and EP tubes were used for equipment.The astrocytes of rats were divided into normal serum group,model serum group,clozapine drug containing serum group and Wendan Decoction high,medium and low dose drug containing serum groups.In addition to the normal serum group,the other groups were given 10 mol/mL glutamic acid.After 48 hours of treatment,cell apoptosis and cell cycle were detected by flow cytometry.Western blotting and Real time PCR were used to detect the expressions of PI3K,Akt,mTOR protein and mRNA.Results The serum containing Wendan Decoction could significantly reduce the apoptosis rate of rat astrocytes induced by glutamate(P<0.01)and the percentage of cells in G0/G1 phase(P<0.05),and increase the percentage of cells in S phase and G2/M phase.It decreased the protein expression ratios of P-P13K/P13K,P-ATK/AKT and P-mTOR/mTOR in cells(except the low dose group of Wendan Decoction,P<0.05)and the mRNA expressions of PI3K,Akt and mTOR in cells(except the low dose group of Wendan Decoction,P<0.01).Conclusion The serum containing Wendan Decoction can effectively regulate the expressions of PI3K,Akt and mTOR in rat astrocytes,which may be one of the mechanisms of Wendan Decoction treating schizophrenia.