淫羊藿苷通过Akt/GSK3β/CDK通路抑制CLC5肝癌细胞增殖  

Icariin Inhibits Proliferation of CLC5 Hepatoma Cells via Akt/GSK3β/CDK Pathway

作　　者：毕瑜婷 花东明 林佳成 游丽萍 郑超[1] 邬海龙 孙学华[1] BI Yu-ting;HUA Dong-ming;LIN Jia-cheng;YOU Li-ping;ZHENG Chao;WU Hai-long;SUN Xue-hua(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai University of Medicine&Health Sciences,Shanghai 201318,China)

机构地区：[1]上海中医药大学附属曙光医院,上海201203 [2]上海健康医学院,上海201318

出　　处：《中国实验方剂学杂志》2022年第12期96-102,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82074336、81904164)。

摘　　要：目的研究淫羊藿苷对肝细胞癌细胞CLC5增殖能力的影响及其可能的机制。方法通过网络药理学筛选淫羊藿苷作用靶点,构建靶点网络及构建蛋白质-蛋白质相互作用(PPI)网络,预测淫羊藿苷可能作用靶点及相关通路;使用梯度浓度(0、6.25、12.5、25、50μmol·L^(-1))淫羊藿苷刺激肝细胞癌CLC5细胞,通过细胞活性及增殖检测(CCK-8)法检测淫羊藿苷对CLC5细胞活力的影响;使用不同浓度(0、25、50μmol·L^(-1))淫羊藿苷处理肝细胞癌CLC5细胞,通过Edu-488及克隆形成实验(Clone Forming)检测淫羊藿苷对CLC5细胞增殖的影响;使用不同浓度淫羊藿苷分别刺激CLC5细胞不同时间,通过蛋白免疫印迹法(Western blot)验证淫羊藿苷对蛋白激酶B(Akt)/糖原合成酶激酶3β(GSK3β)/细胞周期依赖激酶(CDK)通路蛋白表达水平的影响情况。结果网络药理学分析发现淫羊藿苷作用于肝细胞癌的机制与阻滞细胞周期抑制肿瘤细胞增殖有关;CCK-8法结果表明,与空白组比较,淫羊藿苷组CLC5细胞活力显著下降(P<0.01),呈时间-浓度依赖性;与空白组比较,淫羊藿苷组Edu-488阳性率及克隆形成菌落数量明显减少(P<0.05,P<0.01);与空白组比较,淫羊藿苷组p-Akt、p-GSK3β、CDK4、细胞周期蛋白D_(1)(CyclinD_(1))的蛋白表达水平明显下降(P<0.05,P<0.01)。结论淫羊藿苷可抑制阻滞肝细胞癌细胞周期,抑制肝细胞癌增殖,作用机制可能与抑制Akt/GSK3β/CDK通路有关。Objective To study the effect of icariin on the proliferative capacity of hepatocellular carcinoma cell line CLC5 and the underlying mechanism.Method The targets of icariin were screened out by network pharmacology,and the target network and protein-protein interaction(PPI)network were constructed to predict the possible targets and pathways of icariin.CCK-8 assay was employed to explore the effects of different concentrations(0,6.25,12.5,25,50μmol?L^(-1))of icariin on the viability of CLC5 cells.Further,CLC5 cells were treated with 0,25,50μmol·L^(-1) icariin,and the effect of icariin on CLC5 cell proliferation was examined by Edu-488 assay and clone formation assay(CFA).Western blot was employed to measure the expression levels of proteins in the protein kinase B(Akt)/glycogen synthase kinase 3β(GSK3β)/cell cycle-dependent kinase(CDK)pathway in the CLC5 cells exposed to different concentrations of icariin.Result Network pharmacological analysis revealed that icariin may inhibit the hepatocellular carcinoma via cell cycle arrest and inhibition of tumor cell proliferation.Compared with the blank group,icariin decreased the viability of CLC5 cells in a time-and concentration-dependent manner(P<0.01)and reduced the positive rate of Edu-488 and the colonies in CFA(P<0.05,P<0.01).Moreover,icariin down-regulated the protein levels of p-Akt,p-GSK3β,CDK4,and CyclinD_(1)(P<0.05,P<0.01).Conclusion Icariin may block cell cycle to suppress the proliferation of CLC5 cells via inhibiting the Akt/GSK3β/CDK pathway.

关 键 词：淫羊藿苷 肝细胞癌 增殖 蛋白激酶B(Akt)/糖原合成酶激酶3β(GSK3β)/细胞周期依赖激酶(CDK)通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]