香青藤提取物通过Wnt/β-catenin信号通路抑制胶质瘤U87细胞的间质转化

Extract of Illigera aromatica inhibited interstitial transformation of glioblastoma U87 cells through Wnt/β-catenin signaling pathway

目的:探讨香青藤提取物对胶质瘤U87细胞侵袭力及间质转化的影响,并揭示其对Wnt/β-catenin信号通路的调控作用。方法:体外培养U87细胞,将其分为对照组、香青藤提取物低剂量组(3 mg/ml)、香青藤提取物高剂量组(6 mg/ml)。采用CCK-8法检测香青藤提取物对U87细胞的半数抑制量(IC50)以及各组细胞增殖情况,用流式细胞术检测U87细胞凋亡情况,Transwell小室法检测细胞侵袭能力,Western blot检测Wnt1、β-catenin、N-cadherin、Vimentin、E-cadherin蛋白表达情况,采用细胞免疫技术检测Vimentin、N-cadherin表达情况。结果:以不同浓度香青藤提取物作用于U87细胞24 h的IC50值为8.194 mg/ml;与对照组相比,香青藤提取物(3、6 mg/ml)能显著抑制U87细胞的增殖(P<0.01)和侵袭能力(P<0.01),诱导U87细胞发生凋亡(P<0.01)。Western blot结果显示,与对照组相比,香青藤提取物(6 mg/ml)可显著增加E-cadherin表达水平,下调N-cadherin、Vimentin、Wnt1和β-catenin水平(P<0.05,P<0.01)。免疫荧光实验也证实香青藤提取物(6 mg/ml)可以下调U87细胞中间质标志物N-cadherin、Vimentin表达水平。结论:香青藤提取物可能通过抑制Wnt/β-catenin信号,下调间质表型相关标志物N-cad-herin和Vimentin表达,从而抑制U87细胞间质转化和侵袭能力。

Objective:To investigate the effects of the extract of Illigera aromatica on the invasion and interstitial transforma-tion of glioblastoma U87 cells,and to reveal the regulation of Wnt/β-catenin signaling pathway.Methods:U87 cells were cultured and divided into Control group,3 mg/ml of Illigera aromatica extracts group,6 mg/ml of Illigera aromatica extracts group.Cell 50%inhibitory concentration(IC50)and proliferation were detected by CCK-8 assay.Cell apoptosis was evaluated by flow cytometry.Cell invasion was detected by Transwell assay.The expressions of Wnt1,β-catenin,N-cadherin,Vimentin and E-cadherin were detected by Western blot,and the expressions of Vimentin and N-cadherin were detected by cellular immunofluorescence.Results:The IC50 value was 8.194 mg/ml in U87 cells treated with different concentrations of Illigera aromatica extract for 24 h.Compared with the control group,the extract(3,6 mg/ml)could significantly inhibit the proliferation of U87 cells(P<0.01)and invasion ability(P<0.01),and induce apoptosis of U87 cells(P<0.01).Western blot showed that compared with the control group,the extract(6 mg/ml)could significantly increase the expression level of E-cadherin and down-regulate the levels of N-cadherin,Vimentin,Wnt1 andβ-catenin(P<0.05,P<0.01).The immunofluorescence assay also confirmed that the extract of Illigera aromatica(6 mg/ml)could down-regulate the expres-sion levels of intermediate markers N-cadherin and Vimentin in U87 cells.Conclusion:By inhibiting Wnt/β-catenin signaling,ex-tracts of Illigera aromatica may down-regulate the expression of interstitial phenotypic markers N-cadherin and Vimentin,thereby in-hibiting the interstitial transformation and invasion ability of U87 cells.