尿石素B对人神经胶质瘤U118 MG细胞生物学行为的影响及其机制

Effect of urolithin B on biological behaviors of human glioblastome U118 MG cells and its mechanism

目的:观察尿石素B(UB)对人神经胶质瘤(GBM)的U118 MG细胞增殖、迁移、侵袭和凋亡的影响,并探讨其作用机制。方法:体外培养U118 MG细胞,将细胞分为对照组(0μmol·L^(-1)UB)和不同浓度(20、40、80、120、160和200μmol·L^(-1))UB组,培养24、48和72 h后,采用CCK-8法检测各组细胞增殖率。将U118 MG细胞分为对照组(0μmol·L^(-1)UB)和不同浓度(40、80和120μmol·L^(-1))UB组,采用细胞克隆形成实验检测各组U118 MG细胞克隆形成率,划痕愈合实验检测各组U118 MG细胞划痕愈合率,Transwell小室实验检测各组U118 MG细胞侵袭百分率,流式细胞术检测各组U118 MG细胞凋亡率和不同细胞周期U228 MG细胞百分率,Western blotting法检测各组U118 MG细胞中波形蛋白(Vimentin)、上皮型钙黏附蛋白(E-cadherin)、B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)表达水平。结果:CCK-8法检测,作用24、48和72 h后,与对照组比较,不同浓度UB组U118 MG细胞增殖率明显降低(P<0.01),且呈浓度和时间依赖性。细胞克隆形成实验,与对照组比较,40、80和120μmol·L^(-1)UB组U118 MG细胞克隆形成率降低(P<0.01),且呈浓度依赖性;划痕愈合实验,作用24和48 h后,与对照组比较,40、80和120μmol·L^(-1)UB组U118MG细胞划痕愈合率明显降低(P<0.05或P<0.01),且呈浓度依赖性;Transwell小室实验,与对照组比较,40、80和120μmol·L^(-1)UB组侵袭U118 MG细胞百分率明显降低(P<0.05或P<0.01),且呈浓度依赖性;流式细胞术,与对照组比较,80和120μmol·L^(-1)UB组U118 MG细胞凋亡率升高(P<0.05或P<0.01),且呈浓度依赖性,G2/M期细胞百分率升高(P<0.05或P<0.01);Western blotting法检测,与对照组比较,不同浓度UB组U118 MG细胞中Vimentin和Bcl-2蛋白表达水平降低(P<0.05或P<0.01),E-cadherin和Bax蛋白表达水平升高(P<0.01)。结论:UB能够抑制人GBM的U118 MG细胞增殖、迁移和侵袭,抑制U118 MG细胞中Bcl-2蛋白表达并上调Bax蛋白表达,诱导细胞凋亡,并引起细胞周期G2/M期阻滞。

Objective:To observe the effect of urolithin B(UB)on the proliferation,migration,invasion and apoptosis of human glioblastoma(GBM)U118 MG cells,and to discuss its mechanism.Methods:The U118 MG cells were cultured in vitro and divided into control group(0μmol·L^(-1)UB)and different concentrations(20,40,80,120,160 and 200μmol·L^(-1))of UB groups.After cultured for 24,48 and72 h,the proliferation rates of U118 MG cells in various groups were detected by CCK-8 method.The U118 MG cells were divided into control group(0μmol·L^(-1)UB)and different concentrations(40,80 and120μmol·L^(-1))of UB groups,and clone formation experiment was used to detect the rates of clone formation of U118 MG cells in various groups;cell scratch healing test was used to detect the scratch healing rates of U118 MG cells in various groups;Transwell chamber assay was used to detect the percentages of invasion U118 MG cells in various groups;flow cytometry was used to detect the apoptotic rates of U118 MG cells and the percentages of U118 MG cells at different cell cycles in various groups;Western blotting method was used to detect the expression levels of Vimentin,epithelial adhesive protein(E-cadherin),B cell lymphoma-2(Bcl-2),and Bcl-2 related X protein(Bax)in the U118 MG cells in various groups.Results:The CCK-8 results showed that compared with control group,the proliferation rates of U118 MG cells in different concentrations of UB groups at 24,48 and 72 h after treatment were decreased(P<0.01)in a dose and time-dependent manner.The clone formation experiment results showed that compared with control group,the rates of clone formation of U118 MG cells in 40,80 and120μmol·L^(-1)UB groups were decreased(P<0.01)in a dose-dependent manner.The cell scratch test results showed that compared with control group,the scratch healing rates of U118 MG cells in 40,80 and120μmol·L^(-1)UB groups at 24 and 48 h after treatment were significantly decreased(P<0.05 or P<0.01)in a dose-dependent manner.The Transwell chamber assay results showed that compared with control group,the percentages of invasion U118 MG cells in 40,80 and 120μmol·L^(-1)UB groups were decreased significantly(P<0.05 or P<0.01)in a dose-dependent manner.The flow cytometry results showed that compared with control group,the apoptotic rates of U118 MG cells in 80 and 120μmol·L^(-1)UB groups were increased(P<0.05 or P<0.01)in a dose-dependent manner,and the percentages of U118 MG cells at G2/M phase were increased(P<0.01).The Western blotting results showed that compared with control group,the expression levels of Vimentin and Bcl-2 proteins in U118 MG cells in different concentrations of UB groups were decreased(P<0.05 or P<0.01),and the expression levels of E-cadherin and Bax proteins were increased(P<0.01).Conclusion:UB can inhibit the proliferation,migration and invasion of human GBM U118 MG cells,inhibit the expression of Bcl-2 protein,increase the expression of Bax protein,induce the apoptosis,and result in G2/M phase arrest.