桑根皮素调控Wnt/β-catenin信号通路影响胶质瘤U251细胞增殖的实验研究

Experimentalstudy on the effect of morusin on the proliferation of glioma U251 cells by regulating Wnt/β-catenin signaling pathway

目的探讨桑根皮素调控Wnt/β-catenin信号通路对脑胶质瘤U251细胞增殖的影响。方法体外培养人脑胶质瘤细胞U87,采用不同浓度桑根皮素(1、5、25、50、100μmol/L)作用于U87细胞,分别采用MTT法、细胞集落形成法、流式细胞术检测细胞增殖和凋亡活性。采用Western blotting法和细胞免疫荧光法检测Wnt-5a、β-catenin、糖原合酶激酶3β(GSK3β)、BIRC5(survivin)、Bcl-2相关X蛋白(BAX)表达情况以及β-catenin的核转位情况。结果基于网络药理学分析,Wnt/β-catenin信号通路是桑根皮素治疗脑胶质瘤的关键通路。桑根皮素呈剂量依赖方式和时间依赖方式抑制U87细胞的增殖。1、5、25、50、100μmol/L桑根皮素处理后,U87细胞集落形成数[(421±33)个、(380±41)个、(305±34)个、(207±56)个、(147±49)个]较空白对照组[(450±33)个]减少(P<0.001);细胞凋亡率[(12.76±1.18)%、(18.45±1.31)%、(25.02±1.69)%、(27.41±2.31)%、(38.16±2.19)%]较空白对照组[(2.18±0.19)%]增加(P<0.001)。桑根皮素可降低U87细胞中Wnt-5a、β-catenin、BIRC5、GSK3β蛋白表达量,同时上调BAX蛋白的表达;并且经细胞免疫荧光分析,桑根皮素可抑制β-catenin核转位。结论桑根皮素可能通过调节Wnt/β-catenin信号通路抑制U87细胞增殖,其作用机制可能为抑制β-catenin由胞浆向细胞核内转位,从而下调BIRC5表达并上调BAX表达。

Objective To investigate the effect of morusin on Wnt/β-catenin signaling pathway on proliferation of brain glioma U251 cells.Methods U87 cells were cultured in vitro and treated with different concentrations of morusin(1,5,25,50,100μmol/L).MTT method,cell colony formation and FCM were respectively used for the determination of the growth and apoptosis activities.Beside,the Wnt-5a,β-catenin,Glycogen synthase kinase 3β(GSK3β),BIRC5(survivin),Bcl-2 associated X protein(BAX)proteins expressions were detected by Western blotting.And the immunofluorescence assay were used to detect changes of intracellularβ-catenin protein content in U87 cells.Results Based on the network pharmacology,Wnt/β-catenin was a key pathway of morusin on glioma.Morusin could inhibit the proliferation of U87 cells in a time-and dose-dependent manner.After 7d of treatment with morusin,the colonies formations(421±33,380±41,305±34,207±56,147±49)were less than that in control group(450±33),(P<0.001);while the apoptosis rates[(12.76±1.18)%,(18.45±1.31)%,(25.02±1.69)%,(27.41±2.31)%,(38.16±2.19)%]were also higher than that in control group(2.18±0.19)%,(P<0.001).Compared with the control group,Wnt-5a,β-catenin,BIRC5,GSK3βproteins levels in morusin groups were lower and BAX protein levels were up-regulated(P<0.05).And the distribution ofβ-catenin in nucleus was decresed in morusin groups by immunofluorescence assay.Conclusion Morusin can inhibit the growth of glioma cells by regulation Wnt/β-catenin pathway.Its mechanism is possible inhibiting the distribution ofβ-catenin in nucleus and down-regulating BIRC5 expression,up-regulating BAX expression,to induce the apoptosis of glioma cells.