| ObjectiveEpithelial mesenchymal transition(EMT)of hepatic oval cells is closely related to precancerous lesions.In this study,transforming growth factor-β1(TGF-β1)was used to induce EMT changes in rat hepatic oval cells WB-F344,and to explore the regulatory role of Wnt/β-catenin signaling pathway in this process.On this basis,the serum containing Biejiajian Pills was added to further explore whether Biejiajian Pills could reverse EMT by regulating Wnt/β-catenin signaling pathway to achieve the preliminary anti hepatoma effect.Methods1.EMT was induced by TGF-β1 in WB-F344 cells,and the expression of EMT marker protein was detected by Western blot.2.WB-F344 cells were treated with Wnt/β-catenin signaling pathway inhibitor XAV-939.The migration ability,Wnt/β-catenin signaling pathway and EMT marker protein expression were detected by scratch test,Western blot and immunofluorescence.3.WB-F344 cells of EMT were treated with Biejiajian Pills containing serum.The migration ability,Wnt/β-catenin signaling pathway protein,EMT marker mRNA and protein expression levels were detected by scratch test,qPCR,Western blot and cellular immunofluorescence.Results1.The cell morphology changed significantly,the cell morphology gradually extended from cobblestone to fibroblast like cells after TGF-β1 stimulated WB-F344 cells for 4 days,and the expression of E-cadherin protein decreased(P<0.01),and the expression of N-cadherin protein increased(P<0.01);which indicated that TGF-β1 can successfully induce EMT changes in WB-F344 cells.2.TGF-β1 stimulated WB-F344 cells for 4 days was EMT model group.Compared with the blank group,the expression levels of β-catenin and p-GSK3βprotein in TGF-β1 model group were increased(P<0.01);the fluorescence expression of β-catenin was increased;compared with TGF-β1 group,the fluorescence expression of β-catenin was decreased in XAV-939 group;the expression levels ofβ-catenin and p-GSK3 β protein were decreased(P<0.01),and the expression levels of GSK3β protein in each group had no significant change.Compared with the blank group,the cell migration ability of TGF-β1 model group was enhanced;the expression level of E-cadherin protein was decreased,and the protein levels of N-cadherin and vimentin were increased(P<0.01);compared with the TGF-β1 model group,the cell migration ability of XAV-939 group was decreased;the expression level of E-cadherin protein was increased,and the protein levels of N-cadherin and Vimentin were decreased(P<0.01).It indicated that inhibition of Wnt/β-catenin signaling pathway could reverse EMT and migration ability of WB F344 cells.3.Compared with the TGF-β1 model group,the fluorescence expression ofβ-catenin of low,medium and high dose Biejiajian pills group and compound Biejiaruangan tablets group was decreased,and the expression levels of β-catenin and p-GSK3β protein were decreased(P<0.01),but there was no significant difference in the expression levels of GSK3β protein among the groups.Compared with TGF-β1 model group,the migration ability of WB-F344 cells was decreased(P<0.05),the expression levels of E-cadherin mRNA and protein were increased,and the expression levels of N-cadherin mRNA and protein were decreased of low,medium and high dose Biejiajian pills group and compound Biejiaruangan tablets group(P<0.05),and vimentin protein were decreased of medium and high dose Biejiajian pills group(P<0.05),which indicated that Biejiajian Pills could partially reverse the EMT changes of WB-F344 cells induced by TGF-β1 in a concentration dependent manner.Conclusions1.TGF-β1 can induce EMT in WB-F344 cells,and it can promote the expression of p-GSK3β during EMT in WB-F344 cells,so that β-catenin can not be phosphorylated and degraded by the destruction complex composed of GSK3β,APC,Axin and CK1,which accumulates in the cytoplasm,enters the nucleus and activates Wnt/β-catenin signaling pathway.2.Inhibition of Wnt/β-catenin signaling pathway can inhibit epithelial mesenchymal transition and migration of WB-F344 cells.3.Biejiajian Pills can inhibit the phosphorylation of GSK3β,promote the degradation of β-catenin,inhibit the activation of Wnt/β-catenin signaling pathway,and then partially inhibit the epithelial mesenchymal transition and migration of WB-F344 cells.In conclusion,Wnt/β-catenin signaling pathway is involved in regulating the epithelial mesenchymal transition of WB-F344 cells.Biejiajian Pills can partially reverse the epithelial mesenchymal transition and migration of WB-F344 cells by inhibiting the activation of Wnt/β-catenin signaling pathway,so as to achieve the preliminary anti hepatoma effect,and provide sufficient theoretical basis for its clinical application. |
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