九味通窍汤对人脑胶质瘤的体外药效学研究及组方分析

In Vitro Pharmacodynamics and Prescription Analysis on Jiuwei Tongqiao Decoction in Human Glioma U251 Cells

目的:对九味通窍汤进行组方拆分,并对各组进行体外抗脑胶质瘤药效学分析,对其体外药效学可能的作用机制进行探索,为探究全方起效的物质基础提供实验依据。方法:根据君臣佐使组合原理将九味通窍汤拆分成7个组方,选用人源脑神经胶质瘤U251细胞进行体外药效筛选,采用噻唑蓝(MTT)法测定各个组方对细胞活力的影响;采用流式细胞术比较所得体外药效最佳组方Ⅳ与全方(10,4,1 g·L^(-1))作用U251细胞24 h对细胞的凋亡影响;采用蛋白免疫印迹法(Western blot)检测全方及组方Ⅳ对凋亡蛋白p53和抗凋亡蛋白B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)表达影响;组方Ⅳ的醇提物用石油醚、三氯甲烷、乙酸乙酯及水饱和正丁醇梯度萃取,采用MTT法测定各萃取部位对U251细胞增殖的抑制作用。结果:组方Ⅳ对细胞的抑制作用最为明显,且具有浓度依赖性;细胞经10,4,1 g·L^(-1)组方Ⅳ,全方处理24 h后凋亡率显著高于空白组(P<0.01);细胞经4 g·L^(-1)组方Ⅳ,全方处理后Bcl-2蛋白表达水平较空白组显著降低(P<0.01),p53蛋白表达较空白组显著提高(P<0.01);组方Ⅳ梯度萃取后部位Ⅲ对细胞活力的抑制作用最为明显,且具有浓度依赖性。结论:组方Ⅳ细胞活力抑制作用明显,诱导细胞凋亡明显,并可以降低Bcl-2,并提高p53蛋白的表达;部位Ⅲ抑制脑胶质瘤细胞活力明显。

Objective： To analyze the compositions of Chinese herbal prescription Jiuwei Tongqiao decoction and its pharmacodynamic in vitro, explore its poaaile mechanism and provide experimental basis for its effectiveness. Method： Jiuwei Tongqiao decoction was separated into seven prescriptions based on the principles of Jun, Chen, Zuo, Shi. After alcohol extract enrichment, U251 cells of anthropogenic brain glioma were used inmethylthiazolyldiphenyl tetrazolium bromide （MTT） assay to determine the effect of different prescriptions on cell survival. Flow cytometry instrument was used to compare the effect of optimal prescription Ⅳ （10, 4, 1 g·L^-1） and whole prescription on cells apoptosis. Western blot was used to detect apoptosis-related protein B-cell lymphoma-2 （Bcl-2） and p53 expression differences between the optimal prescription Ⅳ and the whole prescription. Alcohol extraction of the optimal prescription Ⅳ was concentrated with petroleum ether, chloroform and ethyl acetate, and then MTT was applied to determine the effect of various extraction parts on cell survival. Result： Prescription Ⅳ had the most obvious inhibition effect on U251 cells in a concentration-dependent manner.The apoptosis ratesinprescription Ⅳ treatment group （10, 4, 1 g·L^-1） and whole prescription were all significantly higher than those in blank group after 24 h （P 〈 0.01 ）. The Bcl-2 protein expression levels of prescription Ⅳ group and the whole prescription group were significantly lower than those in blank group （P 〈 0.01） , and p53 protein expression levels were significantly higher （P 〈 0.01 ）. The directive ingredients in part Ⅲ of prescription Ⅳ were more comprehensive after gradient extraction. Conclusion： Prescription Ⅳ is the optimal prescription, and part m is the most effective part with the anti-proliferative effect on U251 glioma cells.