从PI3K/Akt/p53通路探讨藤茶总黄酮抗肝癌的作用机制

Mechanism of Total Flavonoids of Ampelopsis Grossedentata in Inhibiting Liver Cancer by PI3K/Akt/p53 Pathway

目的:研究藤茶总黄酮(TF)对肝癌裸鼠移植瘤的抑制作用,预测其作用机制可能与调控细胞凋亡肌醇磷脂-3-激酶/蛋白激酶B/p53(PI3K/Akt/p53)通路的相关因子有关。方法:建立BEL-7404肝癌裸鼠移植瘤模型,实验分为模型组,5-氟尿嘧啶(5-FU,1.0 g·L^(-1))组及TF高、中、低质量浓度(30,15,7.5 g·L^(-1))组,给药干预2周后处死裸鼠,剥离出瘤组织,根据瘤重和瘤体积计算抑瘤率(IR)及相对肿瘤增殖率(T/C);采用逆转录聚合酶链式反应(RT-PCR)检测PI3K/Akt/p53中相关基因胞内PI3K,Akt1,p53,半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)mRNA表达;采用免疫组化检测相关蛋白PI3K,Akt1,p53,Caspase-3,Bcl-2,Bax蛋白的表达。结果:TF高、中、低质量浓度组IR分别为53.26%,35.94%,26.74%,T/C分别为59.74%,69.66%,84.82%;逆转录聚合本科链式反应(RT-PCR)表明,与模型组比较,TF 30 g·L^(-1)能明显下调PI3K,Akt1,Bcl-2 mRNA表达,显著上调抑癌基因p53,Capsase-3,Bax mRNA表达;免疫组化表明,与模型组比较,TF 30,15 g·L^(-1)均明显下调PI3K,Akt1,Bcl-2蛋白的表达,显著上调p53,Capsase-3,Bax蛋白的表达。结论:TF有明显的体内抗肝癌活性,其机制可能与上调p53,Caspase-3的表达,激活细胞凋亡PI3K/Akt/p53通路,从而抑制Bcl-2,提高Bax的表达,促进肝癌细胞凋亡有关。

Objective:To study inhibitory effect of total flavonoids from Ampelopsis grossedentata(TF)on transplanted tumors of human hepatocellular carcinoma in nude mice,and predict that its mechanism may be related to relevant factors regulating phosphatidylinositol 3 kinase(PI3K)/protein kinases B(Akt)/p53pathway in apoptosis.Method:The nude mice transplanted BEL-7404 hepatoma model was established and divided into model group,5-fluorouracil(5-FU)group(1.0 g·L-1)and TF(30,15,7.5 g·L-1)groups.Nude mice were put to death after two weeks of administration.The tumor tissues were excised,and tumor inhibition rate(IR)and relative tumor proliferation rate(T/C)were calculated.Reverse transcription PCR(RT-PCR)was used to detect PI3K,Akt1,p53 gene(p53),Caspase-3,B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2 associated X protein(Bax)mRNA expressions,immunohistochemical method was used to detect expressions of relevant proteins PI3K,Akt1,p53,Caspase-3,Bcl-2,Bax.Result:The establishment of xenograft tumor in mice showed that TF was administered orally once per day for two consecutive weeks.IRs were 53.26%,35.94%,and 26.74%,respectively.T/Cs were 59.74%,69.66%,and 84.82%,respectively.RT-PCR experiments showed that compared with model group,when TF concentration was 30 g·L-1,mRNA expressions of PI3K,Akt1,and Bcl-2 were significantly down-regulated,and mRNA expressions of tumor suppressor genes p53,Capsase-3,and Bax were significantly up-regulated.Immunohistochemical method results showed that compared with model group,at TF concentrations of 30,15 g·L-1,all PI3K,Akt1,Bcl-2 protein expressions were significantly down-regulated,while p53,Capsase-3,Bax protein expressions were significantly increased.Conclusion:TF has an obvious antiliver cancer activity in vivo.Its mechanism may be correlated with up-regulation of expressions of p53,Caspase-3,and activation of apoptosis PI3K/Akt/p53 pathway,thereby inhibiting Bcl-2,increasing expression of Bax,and promoting hepatocellular apoptosis.