Mechanism Of Sal B Regulating Hippo/YAP Signaling Pathway Against Liver Fibrosis

BackgroundsThe evolution of liver fibrosis resulting from abnormal manifestations of the chronic liver injury-repair process in clinical practice is a common phenomenon,manifested by the accumulation of extracellular matrix（ECM）proteins,which in turn replace the damaged normal tissue and form fibrous scarring.Liver fibrosis is a dynamic disease that can be reversed if the underlying liver injury is cleared or effectively treated,according to preclinical models and clinical studies.Therefore,the development of drugs to reverse or treat liver fibrosis has far-reaching implications for the treatment of liver fibrosis.In recent years,with the development of network pharmacology and molecular biology,some herbal monomers or complexes have been shown to have significant effects in improving liver function impairment and inflammation,and do not contain adverse effects and toxic side effects,which have obvious advantages for drug use.Salvia miltiorrhiza is a well-known Chinese herb containing highly bioactive salvianolic acid B（Sal B）,which has important pharmacological effects on the liver and kidney,and previous studies have shown that salvianolic acid B has the mighty ability to boost liver fibrosis in patients with chronic hepatitis B virus（HBV）liver fibrosis and rats with liver fibrosis model constructed with tetrachloromethane（CCl₄）,inhibit cell proliferation of primary hematopoietic stem cells in rats and collagen production and induced apoptosis in mouse hepatic fibrosis cells,which indicated that Sal B has strong anti-fibrotic,oxidative,and inflammatory properties,in addition to inhibiting the activation of hepatic stellate cells,suggesting that Sal B is a potential option for the treatment of hepatic fibrosis.Previous researchs have reveled that the Hippo/YAP signaling pathway is an important pathway controling the process of liver fibrosis,and it has been reported that YAP（Yes associated protein）,TAZ（PDZ-binding motif）plays a vital effector protein downstream of the Hippo signaling pathway and plays a crucial character in the process of liver fibrosis.YAP and TAZ combine and interact with transcription factors,such as TEADs（1-4）,and accelerate the expression of target genes after aggregation in the nucleus.thereby activating hepatic stellate cells.Therefore,targeting the Hippo pathway is a potential antifibrotic target,but whether Sal B,despite its antifibrotic efficacy,regulates liver fibrosis through the Hippo/YAP signaling pathway is unclear.Based on the above background,this project adopts DEN/CCl₄/C2H5 OH to construct an in vivo model of liver fibrosis,and TGF-β₁ and XP-1 to stimulate the activation of HSC-T6 cells in vitro to verify the effect of Sal B on liver fibrosis via Hippo/YAP signaling pathway in vivo and in vitro and to investigate the mechanism.Objectives1.To clarify the effect of Sal B on DEN/CCl₄/C2H5OH-induced liver fibrosis in C57BL/6J mice,and to investigate whether the mechanism of action of Sal B is related to the regulation of Hippo/YAP signaling pathway.2.To clarify the effects of Sal B on proliferation and migration of TGF-β₁ and XP-1 stimulated HSC-T6 cells,and to investigate whether the mechanism of action of Sal B is related to the regulation of Hippo/YAP signaling pathway.Methods1.In vivo investigation of the effect and mechanism of action of Sal B on DCCinduced liver fibrosis model（1）Establishment of mouse liver fibrosis model using DCC（DEN/CCl₄/C2H5OH）induction: DEN（100 mg/kg,i.p.）was given once in the 1st week of modeling and the 2nd time in the 2nd week;20% CCl₄ dissolved in olive oil（0.05 ml/10 g,2 t/w,i.g.）was given in the 3rd week-7th week of modeling;in the 8th week-12 th week of modeling,20% CCl₄ dissolved in olive oil was given.20% CCl₄ dissolved in olive oil（0.06 ml/10 g,2 t/w,i.g.）was given;from the 3rd week,a solution containing 10% ethanol was used as drinking water for mice to drink on their own.（2）Animal administration: 36 healthy male C57BL/6J mice（6-8 weeks old,weight 18-23 g）were stochastically segments into 6 experimental groups: control group,model group,Sal B low,Sal B medium and Sal B high doses（7.5,15,30 mg/kg/d,i.g.）,positive control group:colchicine（0.1mg/ kg/d,i.g.）,each group include 6 animals,were taken after 12 weeks of continuous administration.2.In vitro investigation of the effect of Sal B on the proliferation and migration of TGF-β₁ and XP-1 stimulated HSC-T6 cells and the mechanismThe cells were cultured to logarithmic growth phase and grouped as follows: control group,TGF-β₁ group,TGF-β₁ group + Sal B group,XP-1 group（5 μM）,XP-1 group（5 μM）+ Sal B group,XP-1 group（10 μM）,XP-1 group（10 μM）+ Sal B group.The corresponding concentrations of the drugs were given sequentially according to the subgroups,and the control group was given the same volume of lysis medium.After drug action,CCK8 and scratch tests were used to explore the effects on their ability to add value and migrate in the presence of Sal B.The effect of Sal B on the expression level of YAP m RNA in TGF-β₁ and XP-1 stimulated HSC-T6 cells was probed using real-time quantitative PCR.Western blot and fluorescence assays were used to explore the effects of their protein expression levels and distribution regulated by Sal B.Results1.Sal B ameliorated DCC-induced liver injury and liver fibrosis process in miceLiver tissue biopsies of mice showed that the liver of mice given Sal B drug was significantly normal compared to the model group and close to the control mice.HE staining showed that the liver lobules of mice in the model group were disordered and the tissue was infiltrated with a large amount of inflammatory factors.In contrast,Sal B effectively improved the level of liver inflammation induced by DCC in mice.The results of ALT and AST assays also showed that the expression levels of both were abnormally increased in mice after DCC induction,and there was significant liver function impairment.After the administration of Sal B treatment,the expression levels of both decreased in mice.The results of Masson experiment showed that compared with the control group,the model group deposited a large number of collagen fibers and the collagen fibers wrapped around the liver lobules,resulting in pseudobullets.In addition there was a significant improvement in liver lesions in both Sal B administration groups compared to the model group.Immunohistochemical results showed（α-SMA）that,similar to Masson,α-SMA expression was elevated in the liver of the model group of mice induced by DCC,whereas it was decreased in the Sal B-treated group.2.Sal B regulates Hippo/YAP signaling pathway to inhibit liver fibrosisWestern blot and immunofluorescence experiments showed that after 12 weeks of DCC induction,the expression of YAP,TAZ increased in the model mice compared with the control group.p YAP,TAZ,MST1 expression increased in the Sal B-treated group.The above results indicated that Sal B could regulate the expression of Hippo/YAP signaling pathway proteins.Subsequent immunofluorescence experiments also verified the results of this experiment.3.Sal B inhibited the proliferation and migration of TGF-β₁ and XP-1-stimulated HSC-T6 cellsThe results of functional-type experiments（CCK8,wound closure）showed that: compared with the control group,the proliferation and migration of the experimental group given TGF-β₁ and XP-1 stimulation were significantly increased,while the proliferation and migration of the group coadministered Sal B treatment were significantly inhibited.This suggests that Sal B can inhibit the proliferation and migration of TGF-β₁ and XP-1-stimulated HSC-T6 cells in vitro.4.Sal B regulates Hippo/YAP pathway in TGF-β₁ and XP-1-stimulated HSC-T6 cellsThe results of q RT-PCR experiments showed that YAP m RNA expression was significantly elevated in the experimental group stimulated by TGF-β₁ and XP-1 relative to the control group,while its expression was suppressed after administration of Sal B co-treatment,suggesting that Sal B can reduce YAP m RNA transcript levels.Western blot and immunofluorescence assays showed that the expression of YAP,TAZ,p Smad3 L and other proteins increased under TGF-β₁ and XP-1 stimulation compared with the control group,while the expression of p YAP,p TAZ,MST1,p Smad3 C and other proteins decreased after Sal B co-treatment,and increased after Sal B cotreatment.The subsequent fluorescence experiments were consistent with this,and the results of in vivo and in vitro experiments were also consistent.Conclusion1.Sal B inhibited the development of DCC-induced hepatic fibrosis in C57BL/6J mice,and may exert antifibrotic function in vivo by regulating Hippo/YAP signaling pathway.2.Sal B inhibited the proliferation and migration of TGF-β₁ and XP-1 stimulated HSCT6 cells,and may exert antifibrotic function in vitro by regulating Hippo/YAP signaling pathway.