Effect Of SiNiSan On Regulating Wnt/β-catenin Pathway To Induce Proliferation And Differentiation Of Hepatic Stem Cells In The Treatment Of Liver Injury

Aim:Liver diseases are one of the main health risks for human beings,and a large number of clinical trials have proved that Si Ni San has good therapeutic effects on liver diseases.In this study,we constructed a mice models of acute and chronic liver injury to study whether the mechanism of the effect of Si Ni San anti-liver injury is related to hepatic stem cells,and then investigated the role of Wnt signaling in the proliferation and differentiation of hepatic stem cells induced by Si Ni San-medicated serum.Meanwhile,in vitro experiments were established to induce hepatic stem cell production in a rat partial hepatectomy model,and to investigate whether the promotion of liver regeneration by Si Ni San is related to hepatic stem cell differentiation.To provide an experimental basis and scientific evidence for the further demonstration of the therapeutic mechanism of Si Ni San on liver injury.Methods:In order to investigate the mechanism of the effect of Si Ni San on anti-liver injury,CCl₄ was selected to induce acute liver injury and the alcohol and sucrose to were selected to induce chronic liver injury,which were treated with Si Ni San by gavage.After that,the expression of AST and ALT in the serum of the mice were measured by relevant kits,and the expression of LDL-c and HDL-c in the serum of the mice with chronic liver injury were also measured.H&E staining was used to detect the pathological changes of liver tissue in acute and chronic liver injury models;HPLC-MS technique to detect the components of the aqueous decoction of Si Ni San and drug-medicated serum;MTT was used to detect the cellular activity of Si Ni San-medicated serum on CCl₄-induced L-02 cells injury;Immunofluorescence staining was carried out to detect changes in the levels of AFP and ALB in WB-F344 cells treated with Si Ni San-medicated serum;RT-PCR technique was tested to detect changes in the levels of m RNA of AFP and ALB in the cells.To investigate the role of Wnt/β-catenin signaling in the induced cell differentiation by drug-medicated serum,Western blot technique was firstly designed to detect the expression levels of Wnt signaling-related proteins in cells;Immunofluorescence staining technique was chosen to detect the protein localization of GSK3βandβ-catenin in cells;RT-PCR technique was designed to detect the changes of Wnt signaling-related m RNA levels.Next,we investigated the effects of Si Ni San on liver regeneration in rats by constructing a rat partial hepatectomy model;HPLC-MS technique was established to identify metabolite components in rat liver;H&E staining was applied to detect structural changes in rat liver tissue;Western blot technique was carried out to detect ALB,AFP and CK19 protein levels in liver tissue;Western blot technique was conducted to detect the expression of Wnt/β-catenin signaling in liver tissue.Results:The effect of Si Ni San on both CCl₄-induced acute liver injury and the alcohol and sucrose-induced chronic liver damage were alleviated,as evidenced by the down-regulation of AST and ALT levels in mice serum and up-regulation of HDL-c levels in mice serum of chronic liver injury model after Si Ni San treatment.H&E staining showed that the liver tissue of mice were improved.Meanwhile,the HPLC-MS analysis revealed that the Si Ni San-medicated serum contained a variety of Si Ni San water decoction components.However,the in vitro experiments demonstrated that Si Ni San-medicated serum had no alleviating effect on CCl₄-induced L-02 cell activity and was able to alleviate oxidative stress injury in WB-F344 cells,while promoting the proliferation and differentiation of WB-F344 cells.Immunofluorescence staining results showed that Si Ni San-medicated serum increased the expression of ALB and decreased the expression of AFP in the cells.Next,we further investigated the role of Wnt/β-catenin signaling in the induced differentiation of WB-F344 cells by Si Ni San-medicated serum.Western blot data showed no significant changes in total GSK3βandβ-catenin proteins in the cells,but phos-GSK3β,DVL2 and phos-β-catenin at Ser33/Ser37/Thr41 were upregulated in the cells.After continuing the culture until 21days,GSK3β,β-catenin and DVL2 proteins were significantly decreased.Immunofluorescence staining results indicated thatβ-catenin protein translocated to the nucleus after 21 days of treatment with Si Ni San-medicated serum in WB-F344 cells,which inducedβ-catenin to bind to the LEF1 receptor in the nucleus,thereby decreasing c-Myc and Cycling D1 protein expression to induce cell differentiation.In vivo results showed that Si Ni San increased liver volume.The metabolic components of liver tissues in the Si Ni San group were significantly different compared with the control group.H&E results showed that Si Ni San had no effect on liver tissues during the promotion of liver regeneration.As well,Si Ni San reduced the expression of AFP and increased the expression of CK19 in liver tissues.Immunohistochemical results showed that Si Ni San increased the expression levels of PCNA and TGF-βin liver tissues and promoted the degree of cell proliferation and differentiation.In addition,Si Ni San reduced the protein expression levels of phos-GSK3βand phos-β-catenin in liver tissues.Similarly,the protein levels of c-Myc and Cyclin D1 were significantly decreased with the increase of Si Ni San treatment time.Conclusion:SiNiSan was shown to alleviate acute and chronic liver injury in mice.This effect of anti-liver injury was possibly related to hepatic stem cells,as demonstrated by the failure of Si Ni San-medicated serum to alleviate normal hepatocyte injury,whereas it was able to inhibit oxidative stress injury in hepatic stem cells.In vitro studies suggested that Si Ni San-medicated serum activated Wnt/β-catenin signaling and induced hepatic stem cell differentiation.In vivo results indicated that Si Ni San increased the volume of rat liver and improved the activity of liver tissue cell proliferation and differentiation.At the same time,it was revealed that the potential mechanism by which Si Ni San promotes liver regeneration in rats is related to the activation of Wnt/β-catenin signaling.Together,the above results demonstrate that the hepatoprotective effect of Si Ni San is closely related to the proliferation and differentiation of hepatic stem cells.