Effects Of Kangxian Ruangan Granule-containing Serum On Epithelial-mesenchymal Transformation Of Hepatic Oval Cells And The Intervention Mechanism Of Wnt-1/β-catenin Signal Pathway

Part Ⅰ Effect of serum containing Kangxian ruangan granule on MNNG induced epithelial-mesenchymal transformation of hepatic oval cell line WB-F344Objective:To study the effect of Kangxianruangan granule containing serum(KXRG)on the epithelial-mesenchymal transformation of hepatic oval cell line WB-F344 induced by n-methyl-n-nitro-n-nitrosoguanidine(MNNG).Methods:WB-F344 cell line was treated with MNNG to prepare the precancerous lesion cell type.The experimental group was stimulated with KXRG of different concentration,while the normal group and the model group were supplemented with blank serum.the proliferation ability of cells was detected by CCK-8 method,The expression of E-cadherin,N-cadherin and Vimentin was detected by Western blot,and AFP was detected by enzyme-linked immunosorbent assay(Elisa).in supernatant,N-cadherin and Vimentin protein expression in model group were significantly increased(p<0.05),while E-cadherin protein expression was significantly decreased(p< 0.05).Compared with model group,OD value,AFP concentration in supernatant,N-cadherin and Vimentin expression were significantly decreased(p<0.05),and the higher the concentration was,the more obvious the decrease was.The expression level of E-cadherin increased obviously(p<0.05),and the higher the concentration of drug-containing serum,the higher the expression level of E-cadherin.Conclusion:KXRG can inhibit the epithelial-mesenchymal transformation of WB-F344 cells.Results:Compared with normal group,the OD value,AFP concentrationPart Ⅱ The mechanism of Kangxianruangan granule-containing serum in inhibiting differentiation of hepatic oval cells into hepatoma cells via Wnt-1/β-catenin pathwayBackground: Hepatocellular carcinoma is highly malignant and has a poor clinical prognosis.However,hepatic oval cells tend to differentiate into hepatoma cells in the tumor microenvironment.The purpose of this study is to explore Kangxianruangan granule-containing serum(KXRG)in inhibiting differentiation of hepatic oval cells into hepatoma cells via Wnt/β-catenin pathway.Methods: In this study,Nmethyl-N’-nitro-N-nitrosoguanidine(MNNG)was used to induce rat liver oval cell line WB-F344 tohepatocarcinoma cells,and further use plasmids to overexpress the Wnt-1 molecule in the cells.Subsequently,high concentration,medium concentration and low concentration of KXRG were used to intervene in the cell model.Flow cytometry was used to detect the cell cycle and apoptosis rate.Immunofluorescence double staining method was used to detect the expression of Wnt-1 and β-catenin in cells.Flow cytometry was used to detect the expression of CK-19 protein in cells.Elisa detected the content of AFP in the cell supernatant.RT-PCR and western blot were used to detect the m RNA and protein levels of Wnt-1,β-catenin,Cyclin D1,C-myc,MMP-7,Axin2,and Ep CAM in cells.Results: Compared with normal group,the apoptosis rate,the proportion of G2/M phase cells,the concentration of AFP in the cell supernatant,the level of CK-19 protein,Wnt-1,β-catenin,Cyclin D1,C-myc,MMP-7,Axin2,and Ep CAM m RNA and protein levels in model group were significantly increased.After intervention with KXRG,the above indicators were significantly reduced.After intervened with overexpressed plasmid of Wnt-1,the above indicators were significantly increased.However,KXRG still has inhibitory effects on the above indexes in model cells after overexpressing Wnt-1.Conclusion: Kangxianruangan granule-containing serum could inhibit the differentiation of hepatic oval cells into hepatoma cells via wnt-1/β-catenin pathway,which has important clinical guiding significance for the clinical use of Kangxian Ruangan granules to prevent hepatocellular carcinoma.