目的 基于"厥阴伏邪"理论探讨加味连理汤抑制肝癌细胞HepG2浸润侵袭和血管生成的作用机制.方法 肝癌细胞HepG2体外培养,采用6孔培养板传代接种细胞,将细胞分为4组,空白组、阴性对照组、缺氧诱导因子(Hypoxia inducible factor,HIF)组与中药干预HIF组,其中阴性对照组造模前转染HIF阴性对照片段,HIF组转染HIF反义RNA-2,中药干预HIF组的干预药物为加味连理汤.细胞转染后24、36 h MTT法检测细胞增殖抑制作用,Transwell小室法检测细胞侵袭能力,流式细胞仪检测细胞凋亡情况,Western Blot法检测DEP结构域含有雷帕霉素靶蛋白相互作用蛋白(DEP domain containing mTOR-interae-ting protein,DEPTOR)、程序性细胞死亡 4(Programmed cell death 4,PDCD4)及e-Jun(AP-1)蛋白表达水平.结果 细胞转染后24、36 h HIF组与中药干预HIF组细胞增殖指数及侵袭指数均显著低于空白组、阴性对照组,细胞凋亡指数均显著高于空白组、阴性对照组,差异均有统计学意义(P＜0.05),且中药干预HIF组细胞增殖指数及侵袭指数均显著低于HIF组,细胞凋亡指数均显著高于HIF组,差异均有统计学意义(P＜0.05).细胞转染后24、36 h,HIF组与中药干预HIF组DEPTOR蛋白表达水平显著低于空白组、阴性对照组,PDCD4及e-Jun(AP-1)蛋白表达水平均显著高于空白组、阴性对照组,差异均有统计学意义(P＜0.05),且中药干预HIF组DEPTOR蛋白表达水平显著低于HIF组,PDCD4及e-Jun(AP-1)蛋白表达水平均显著高于HIF组,差异均有统计学意义(P＜0.05).结论 加味连理汤温清并用,能够抑制肝癌细胞HepG2的增殖和侵袭,促进其凋亡,介导DEPTOR蛋白表达,激活PDCD4/e-Jun(AP-1)转录激活因子形成的信号轴,抑制血管生成.
Mechanism of Jiawei Lianli Decoction(加味连理汤)Inhibiting Invasion and Angiogenesis of Hepatocellular Carcinoma HepG2 Cells Based on"Jueyin Fuxie Latent Evils"Theory
Objective To investigate the mechanism of Jiawei Lianli Decoction(加味连理汤)inhibiting invasion and angio-genesis of hepatocellular carcinoma HepG2 cells based on"Jueyin Fuxie latent evils"theory.Methods Hepatocellular carcinoma HepG2 cells were cultured in vitro and inoculated in 6-well culture plates.The cells were divided into 4 groups:blank group,negative control group,hypoxia inducible factor(HIF)group and traditional Chinese medicine intervention HIF group.The nega-tive control group was transfected with HIF negative pair before modeling.The HIF group was transfected with HIF antisense RNA-2,and the intervention drug of the traditional Chinese medicine intervention HIF group was Jiawei Lianli Decoction.The inhibitory effect of cell proliferation was detected by MTT assay at 24 h and 36 h after transfection.The cell invasion ability was detected by Transwell chamber assay.The apoptosis was detected by flow cytometry.The protein expression levels of DEP do-main containing rapamycin target protein interacting protein(DEPTOR),programmed cell death 4(PDCD4)and e-Jun(AP-1)were detected by Western Blot.Results At 24 h and 36 h after transfection,the cell proliferation index and invasion index of the HIF group and the traditional Chinese medicine intervention HIF group were significantly lower than those of the blank group and negative control group,and the apoptosis index was significantly higher than that of the blank group and negative control group,and the differences were statistically significant(P＜0.05).The cell proliferation index and invasion index of the traditional Chi-nese medicine intervention HIF group were significantly lower than those of the HIF group,and the apoptosis index was signifi-cantly higher than that of the HIF group,and the differences were statistically significant(P＜0.05).At 24 h and 36 h after transfection,the expression level of DEPTOR protein in the HIF group and the traditional Chinese medicine intervention HIF group was significantly lower than that in the blank group and the negative control group,and the expression levels of PDCD4 and AP-1 protein were significantly higher than those in the blank group and the negative control group(P＜0.05).The expression level of DEPTOR protein in the traditional Chinese medicine intervention HIF group was significantly lower than that in the HIF group,and the expression levels of PDCD4 and AP-1 protein were significantly higher than those in the HIF group(P＜0.05).Conclusion Jiawei Lianli Decoction which can warm and clear can inhibit the proliferation and invasion of HepG2 cells,promote the apoptosis,mediate the expression of DEPTOR protein,activate the signal axis formed by PDCD4/e-Jun(AP-1)transcrip-tion activator,and inhibit angiogenesis.
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