基于毒痰瘀虚理论的抗癌中药对HepG2细胞Wnt/β-catenin信号通路的影响

Study on the effect of anticancer Chinese medicine on Wnt/βcatenin pathway of HepG2 cells based on toxin,thlegm,blood stasis and virtual theory

目的:分析基于毒痰瘀虚理论的抗癌中药对HepG2细胞凋亡及Wnt/β-catenin信号通路相关蛋白表达的体外作用。方法:实验大鼠随机分空白组、阴性血清组、索拉菲尼组、全方组、脾虚组、肾虚组、肾阳虚组、肾阴虚组及毒痰瘀组9组,予相应药物灌胃,制备不同含药血清并与HepG2细胞共培养,以流式细胞术检测HepG2细胞凋亡率,Western Blot检测Wnt/β-catenin信号通路相关蛋白的表达。结果:不同含药血清作用于HepG2细胞24 h后,与空白组或阴性血清组比较,药物组细胞凋亡率显著升高(P<0.05或P<0.01);全方组促进HepG2细胞凋亡效果优于拆方组方组(P<0.05)。以Western Blot检测不同组Wnt/β-catenin信号通路相关蛋白的表达,与空白组和阴性血清组比较,各药物组p-GSK-3β、c-myc、cyclinD1、β-catenin、MMP2表达水平明显下降(P<0.05或P<0.01),索拉菲尼组、全方组、脾虚组、肾虚组、肾阳虚组中p-GSK-3β、c-myc、cyclinD1、β-catenin表达下降更为显著(P<0.01);药物组中,索拉菲尼组与全方组相关蛋白表达下降最为显著(两组中相关蛋白表达量差异无统计学意义,分别与拆方组比较时P<0.01)。结论:基于毒痰瘀虚理论的抗癌中药可明显促进HepG2细胞凋亡、下调Wnt/β-catenin信号通路相关蛋白的表达,全方效果明显优于其拆方组方,全方对Wnt/β-catenin信号通路的抑制作用接近于索拉菲尼。

Objective:To study the in vitro effects of anticancer Chinese medicine based on the theory of Toxin,Thlegm,Blood Stasis and Virtual on the apoptosis of HepG2 cells and the expression of proteins related to Wnt/βcatenin signaling pathway.Methods:The rats were randomly divided into blank group,negative serum group,sorafenib group,toxic thlegm and blood stasis whole prescription group,spleen deficiency group,kidney deficiency group,kidney yang deficiency group,kidney yin deficiency group and toxic thlegm and blood stasis group,and the corresponding drugs were administered by gavage.Serum containing different drugs was prepared and co cultured with HepG2 cells.The apoptosis rate of the cells was detected by Flow cytometry,and the expression of proteins related to Wnt/βcatenin signaling pathway was detected by Western blot.Results:After the treatment of HepG2 cells with different serum containing drugs for 24h,the apoptosis rate of HepG2 cells in the drug group was significantly increased compared with that in the blank group or the negative serum group(P<0.05 or P<0.01).The effect of promoting apoptosis of HepG2 cells in the whole prescription group was better than that in the Separated and Combined Recipes group(P<0.05).WB was used to detect the expression of Wnt/β-catenin signaling pathway related proteins in different groups.Compared with the blank group and the negative serum group,the expression of p-GSK-3β,c-myc,cyclinD1,β-catenin,MMP2 in each drug group was significantly decreased(P<0.05 or P<0.01),and the expression of p-GSK-3β,c-myc,cyclinD1 andβ-catenin in the sorafenib-group,the whole prescription group,the spleen deficiency group,the kidney deficiency group and the kidney yang deficiency group was more significantly decreased(P<0.01).In the drug group,the expression of related proteins decreased most significantly in the sorafenib group and the whole drug group(there was no statistically significant difference in the expression of related proteins between the two groups,and P<0.01 when Separately compared with the Separated and Combined Recipes group).Conclusion:the anticancer Chinese medicine based on the theory of Toxin,Thlegm,Blood Stasis and Virtual can significantly promote the apoptosis of HepG2 cells and down-regulate the expression of proteins related to Wnt/βcatenin signaling pathway.The effect of the whole recipe is obviously superior to that of the Separated and Combined Recipes group,and its inhibition of Wnt/world-catenin pathway is similar to that of sorafenib.