芹黄素通过PI3K/Akt通路抑制肾癌786-0细胞增殖研究

Study on Apigenin Inhibiting Proliferation of Renal Carcinoma 786-0 Cells Through PI3K/Akt Pathway

目的:探讨芹黄素对肾癌细胞的作用及其潜在机制。方法:利用中药系统药理学技术平台(TCMSP)寻找芹黄素潜在的作用靶点;利用注释可视化数据库(DAVID)分析潜在靶点的通路富集情况。用不同浓度的芩黄素干预人肾癌786-0细胞,利用CCK8法检测芹黄素对人肾癌786-0细胞增殖的作用;流式细胞术检测芹黄素对786-0细胞周期的影响;Western blot法检测细胞中蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)蛋白的表达;利用人肾癌786-0细胞构建的实体瘤裸鼠模型验证芹黄素对肿瘤的作用。结果:共筛选出82个芹黄素的潜在靶点。通路富集分析结果显示符合条件的通路有102条,绝大部分通路与肿瘤密切相关,其中包括肾癌、细胞周期及磷脂酰肌醇激酶(PI3K)/Akt通路等。用不同浓度的芩黄素干预786-0细胞后,与0μmol/L组比较,10μmol/L组、20μmol/L组、40μmol/L组细胞增殖抑制率升高(P<0.05,P<0.01);20μmol/L组和40μmol/L组G1期细胞比例及p-Akt蛋白下降(P<0.05),G2期细胞比例升高(P<0.05);40μmol/L组S期细胞比例下降(P<0.05)。动物实验结果显示,与对照组比较,12天、15天、18天时实验组裸鼠肿瘤体积明显减小(P<0.05)。结论:网络药理学及细胞、动物实验均表明芹黄素可能通过调控PI3K/Akt通路抑制肾透明细胞癌细胞的增殖。

Objective:To discuss the function and potential mechanism of apigenin on renal carcinoma cells.Methods:Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP)was applied to find potential action targets of apigenin.The Database for Annotation,Visualization and Integrated Discovery(DAVID)was applied to analyzed the pathway enrichment of potential targets.Human renal carcinoma 786-0 cells were treated with different concentrations of apigenin.CCK8 assay was used to measure the effect of apigenin on the proliferation of human renal carcinoma 786-0 cells.The effect of apigenin on 786-0 cell cycle was detected by flow cytometry.The protein expression of protein kinase B(Akt)and phosphorylated protein kinase B(p-Akt)of the cells was detected by Western blot,and the effect of apigenin on the tumor was verified by using nude mouse model with solid tumors constructed from human renal carcinoma 786-0 cells.Results:A total of 82 potential targets of apigenin were screened out.Pathway enrichment analysis showed that there were 102 eligible pathways,most of which were closely related to tumors,including renal carcinoma,cell cycle and phosphatidylinositol kinase(PI3K)/Akt pathway.After the intervention of 786-0 cells with different concentrations of apigenin,compared with the 0μmol/L group,the inhibition rate of cell proliferation was increased in the 10μmol/L group,the 20μmol/L group and the 40μmol/L group(P<0.05,P<0.01);the proportion of cells in G1 phase and the expression of p-Akt protein were decreased in the 20μmol/L group and the 40μmol/L group(P<0.05),and the proportion of cells in G2 phase was increased(P<0.05);the proportion of cells in S phase was decreased in the 40μmol/L group(P<0.05).The results of animal experiments showed that compared with the control group,the tumor volume of nude mouse in the experimental group was significantly reduced on the 12th,15th and 18th days(P<0.05).Conclusion:Network pharmacology,cell experiment and animal experiment have indicated that apigenin may inhibit the cell proliferation of renal clear cell carcinoma by regulating the PI3K/Akt pathway.