Effects Of Heshuxiaoji Formula On Proliferation And Apoptosis Of Human Hepatoma Cell Line By Regulating AKT/GSK-3β/mTOR Signaling Pathway With AsTP3 Positive Feedback

Objective:To investigate the effect of Heshuxiaoji formula on the proliferation and apoptosis of human hepatocellular carcinoma cell line by regulating the Arsenic trioxide-activated Protein 3(AsTP3)Methods:SMMC-7721 hepatocellular carcinoma cells were cultured in vitro,and the optimal concentration of CCK8 was screened and disaggregated,cell proliferation,cell scar,cell migration and invasion ability were measured by CCK8 and plate cloning assay,cell apoptosis was measured by flow cytometry,and AsTP3 m RNA level was detected by q RT-PCR.The protein expression levels of AsTP3,P-Akt,P-GSK-3β,P-MTOR,BCL2 and BAX were detected by Western blot.Results:1.Effect of Heshuxiaoji formula on proliferation and apoptosis of hepatocellular carcinoma cells:(1)CCK8 experiment:Compared with 0ug/ml of the control group,when the drug concentration was 18ug/ml,P*<0.05,the difference was statistically significant,so 18ug/ml was selected as the optimal drug concentration of this experiment,and 1/2 times and 1/4 of the optimal drug concentration were selected to set the medium dose and low dose groups.Be and pivot negative side respectively intervention SMMC-7721 cells for 24 h,48h,72 h,compared with the control group,and pivot product can significantly reduce the cell vitality;(2)Plate clone formation experiment: Cell clone formation experiment showed that with the increase of drug concentration,the colony of cell formation decreased significantly;(3)Flow cytometry:compared with the control group,the apoptosis rate increased significantly with the increase of drug concentration;Western-blot analysis showed that Heshuxiaoji formula could down-regulate anti-apoptotic protein BCL2 and up-regulate the expression of pro-apoptotic protein BAX.2.Effetcs of Heshuxiaoji formula on invasion and migration of hepatoma cells:(1)Transwell experiment:Compared with the control group,the invasion and migration of cells were inhibited in a dose-dependent manner after 24 h of treatment with And Heshuxiaoji formula;(2)Cell scratch experiment: Compared with the control group,24 hours after Heshuxiaoji formula intervened in cells,the healing ability of scratch gradually decreased with the increase of drug concentration,among which,the high-dose group showed significant difference compared with the control group.3.Effects of AKT/GSK-3β/m TOR signaling pathway and effects on AsTP3:(1)q RT-PCR: Compared with the control group,drug concentration was negatively correlated with AsTP3 m RNA level,and only the high-dose group was statistically significant;(2)Western-blot assay:The protein levels of AsTP3,P-Akt,P-GSK-3β,P-MTOR protein was upregulated by Heshuxiaoji formula.Conclusion: 1.Compared with the control group,Heshuxiaoji formula can restrain the proliferation,migration,invasion and cloning capacity of liver cancer cells and promote the apoptosis of HCC cells;2.The m RNA expression and protein level of AsTP3,P-Akt,P-GSK-3β,P-MTOR and BCL2 were down-regulated and the expression of BAX protein was up-regulated;3.Heshuxiaoji formula may down-regulate the expression of AsTP3 and inhibit the AKT/GSK-3β/m TOR signaling pathway,thereby to promot human hepatoma cell lines and inhibit the invsion、migration of cells.