The Molecular Mechanisms Of Zuojinwan On Human Hepatocellular Carcinoma Based On The PI3K/AKT And Wnt/β-catenin Signaling Pathways

Objective:Zuojin Wan,as a classic prescription for clearing and purging liver fire,has achieved certain research results in anti-tumor effect.This subject has Zuojin Wan as the research object.On the basis of verifying its pharmacodynamic effect on hepatocellular carcinoma,explore whether Zuojin Wan regulates PI3K/AKT and Wnt/β-Catenin dual pathway plays an anti-tumor role,and its specific molecular mechanism is clarified,so as to provide a theoretical basis for the anti-liver cancer effect of Zuojin Wan.Methods:The study was divided into three parts:anti-cancer effect in vitro,molecular mechanisms in vitro and anti-cancer effect and molecular mechanism in vivo.1.The effect of alcohol extract of Zuojin Wan on the proliferation of human hepatoma cells Hep G2,BEL-7402 and HCC-LM3 was detected by CCK-8 method;BEL-7402 cell line was selected for follow-up experiments.Pharmacodynamic evaluation included microscopic observation of cell morphology,observation of cytoskeleton by rhodamine labeled phalloidin/DAPI staining,evaluation of cell proliferation by trypan blue exclusion staining and calcein AM/PI staining;Flow cytometry method and Hoechst 33258 staining were used to evaluate and observe apoptosis and apoptotic morphology.2.The mitochondrial membrane potential of BEL-7402 cells was detected by JC-1;Western blot was used to observe the effect of Zuojin Wan on the expression of Bcl-2 family related proteins,Caspase family related proteins,NF-κB/i NOS-Cox-2 signaling pathway related proteins,PI3K/AKT and Wnt/β-catenin signaling pathway related proteins of BEL-7402 cells were detected by Western blot.3.The H₂₂ hepatoma Xenograft hepatoma model derived from BALB/c mice was established,and the HCC therapeutic drug 5-fluorouracil was used as the positive control drug.The general situation and appearance,the tumour weights,animal body weights and inhibition rates were measured after 14 days;histopathological staining was used to evaluate histopathological features,apoptosis was detected by TUNEL method,immunohistochemistry method was used to detect the expression level of PCNA,Ki67 and CD31 in H₂₂ hepatoma Xenograft hepatoma model;The effects of Zuojin Wan on proliferation related proteins,serum markers,related signal pathways and apoptosis related proteins in H₂₂Tumor bearing mice were detected by immunohistochemistry,ELISA kit and Western blot.Results:1.In vitro anti-cancer results showed that Zuojin Wan had inhibitory activity on three HCC cell lines in comparison of control group,and BEL-7402 was the most sensitive to Zuojin Wan;After BEL-7402 cells were treated with different concentrations of Zuojin Wan for 24 hours,Zuojin Wan affected the cell morphology of BEL-7402.The double staining experiment with rhodamine-labeled phalloidin/DAPI demonstrated that Zuojin Wan changed the cytoskeleton of BEL-7402 cell line.Trypan blue exclusion assay illustrated that Zuojin Wan could remarkably inhibit the cell proliferation ability;The results of calcein AM/PI staining showed that the proportion of living and dead cells decreased significantly with the increase of Zuojin Wan concentration.It was further found that Zuojin Wan could induce apoptosis of BEL-7402 cells by flow cytometry and Hochest33258 staining.2.The results of mechanism in vitro showed that Zuojin Wan could induce apoptosis by reducing mitochondrial membrane potential of BEL-7402 cell line;Compared with the control group,the results of western blot showed that the expression of protein p53 in Zuojin Wan treated groups were significantly up-regulated,the expression of protein Bax and Bad were significantly up-regulated,the expression of protein Bcl-2 and Bcl XL protein were significantly down-regulated,the expression of protein cytochrome C,caspase-9 and caspase-3 protein were significantly up-regulated,and then shearing of PARP and induce apoptosis;Compared with the control group,the expression of p-PI3K and p-AKT in PI3K/AKT pathway and the express of p-GSK-3β、Wnt3a、β-catenin and Cyclin D1 in Wnt/β-catenin pathway in Zuojin Wan treated group were significantly down-regulated,the expression of protein NF-κB、i NOS and Cox-2 in NF-κB/i NOS-COX-2 pathway in Zuojin Wan treated group were significantly down-regulated,the expression of protein TNF-αwas up-regulated.3.The results of anti-cancer and mechanism of action in vivo showed that Zuojin Wan could significantly inhibit the tumor growth of H₂₂ tumor bearing mice dependent on concentration effect,and the tumor inhibition rate in Zuojin Wan treated group were gradually down-regulated;The histopathological study showed that Zuojin Wan had a certain effect on the histomorphology of transplanted tumor.Compare with the model group,the expression levels of PCNA,Ki67 and CD31 in transplanted tumor tissue in Zuojin Wan treated group were significantly up-regulated,and showed a dose-response relationship;the expression levels of TNF-αand IL-2 in serum were effectively up-regulated,and AFP in serum was downregulated;the proteins expression of PI3K/AKT（p-AKT,p-PI3K,p-GSK-3β）and Wnt/β-catenin（Wnt3a、β-catenin and Cyclin D1）in transplanted tumor tissue treated with Zuojin Wan group were significantly down-regulated,the expression of Caspase-3 and Bax protein were up-regulated,and the expression of Bcl-2 and NF-κB were down-regulated.Conclusion:1.Zuojin Wan can significantly inhibit the growth of different hepatoma cells in vitro,especially BEL-7402 cells,with a dose-response relationship.The staining results show that Zuojin Wan can inhibit the proliferation and induce apoptosis of BEL-7402 cells;2.Zuojin Wan can inhibit PI3K/AKT and Wnt of BEL-7402 cells/β-Catenin pathway has double inhibitory effect,which activates mitochondrial apoptosis pathway and induces apoptosis;At the same time,Zuojin Wan has no effect on NF-κB/inos-cox-2 pathway protein can inhibit the abnormal proliferation of liver cancer cells and play an anti liver cancer role.3.Zuojin Wan can significantly inhibit the proliferation of tissue cells and induce apoptosis of HCC cells,and to inhibit the growth of transplanted tumor.Its mechanism may be related to the inhibition of PI3K/AKT and Wnt/β-Catenin signaling pathway.