Protective Effects And Mechanisms Of Sennoside A On CCl₄-induced Hepatic Fibrosis In Mice

Hepatic fibrosis（HF）is a continuous pathological damage-repair process caused by continuous action of multiple injury factors on the liver,which can lead to excessive deposition of extracellular matrix（ECM）in the liver tissue,resulting in changes in the structure and function of the liver,and eventually lead to cirrhosis and even liver cancer.At present,liver fibrosis has no effective treatment.The study demonstrated that the activation of hepatic stellate cell（HSC）is the main source of the abnormal deposition of ECM,and the activation and proliferation of HSC play an important role in the occurrence and development of liver fibrosis,which is regulated by various cytokines and signaling pathways.Therefore,inhibiting the activation and proliferation of HSC regarded as useful therapeutic strategy for liver fibrosis.A large number of studies have shown that DNA methylation can promote the activation of hepatic stellate cells and promote the progress of liver fibrosis.DNA methyltransferase 1（DNMT1）catalyze DNA methylation and is closely related to many fibrotic diseases,including liver fibrosis.Our preliminary experimental results showed that sennoside A（SA）can directly bind to DNMT1 and inhibit its activity.SA has been reported could improve obesity,diabetes,and liver steatosis.However,the effect of SA on liver fibrosis and its mechanism have not been reported in the literature.To further explore the role of SA in liver fibrosis.In this study,C57BL/6J mouse and HSC-T6 cell lines were used as research objects to observe the role of SA in liver fibrosis and its mechanism through in vitro and in vivo experiments.This study mainly includes the following five parts:1.Hepatic protective effects of SA on mice with CCl₄-induced liver fibrosis。Seventy-two C57BL/6J mice（male,20g-22g）were randomly divided into normal group,model group,low,medium and high dose group,and normal and high dose group（12mice in each group）.Mice in model group were intraperitoneally injected with 10%CCl₄olive oil solution to establish liver fibrosis model twice a week for 4 weeks.Low,medium and high doses of SA were administered daily to the drug administration group on the first day of modeling.The normal group was intraperitoneally injected with equal dose of olive oil.After molding,the food was cut off for 24 hours.The mice were killed and liver tissue and serum were collected.In order to detect each group of liver tissue damage,and the degree of fibrosis using HE staining,serum ALT and AST biochemical index detection of liver tissue injury situation,choose Masson staining to detect liver tissue collagen deposition,and selects the immunohistochemical detection of liver fibrosis markers ofαSMA expression level,Western blot detection collagen I（col1α1）in the level of protein expression in the liver tissue.The experimental results showed that the mouse liver fibrosis model was successfully constructed,and SA had a certain therapeutic effect on CCl₄-induced liver fibrosis in mice.2.SA inhibited the activation and proliferation of HSCs.Firstly,MTT assay was used to detect the effect of SA at different concentrations on the cellular activity of HSC-T6 cells,and to determine the cytotoxicity of SA on HSC-T6cells.Then,TGF-β1（10ng/ml）and different concentrations of SA were used to treat the cells,and MTT assay was used to screen out the optimal concentration of SA on HSC-T6 cells.The effect of SA on proliferation and apoptosis of HSC-T6 cells was detected by CCK8 method and flow cytometry.In addition,the expression levels ofα-SMA,col1α1,Cyclin D1,C-myc and CDK5 in HSC-T6 cells were detected by Western blot.The results showed that SA could inhibit the activation and proliferation of HSC-T6 cells and block it in G1 phase,but had no effect on cell apoptosis.3.The effect of SA on PTEN/AKT pathway.The expression of PTEN in liver tissues of normal group,model group and medium dose group were detected by immunofluorescence double staining.HSC-T6 cells were treated with TGF-β1（10ng/ml）and SA（10μM）for 48h.Immunofluorescence and Western blot were used to detect the expression changes of PTEN,and the expression changes of p-AKT and p-ERK were detected by Western blot.HSC-T6 cells were silenced with PTEN small interfering RNA（si RNA-PTEN）and treated with TGF-β1（10ng/ml）and SA（10μM）for 48h.The effect of SA on proliferation of HSC-T6 cells was detected by CCK8 method and flow cytometry.In addition,Western blot was used to detect the expression ofα-SMA,col1α1,Cyclin D1,C-myc,PTEN,p-AKT,and p-ERK in HSC-T6 cells.The experimental results showed that SA could up-regulate the expression of PTEN and inhibit the expression of p-AKT and p-ERK,while when treated with SA after PTEN was silenced,the effects of SA to inhibit the proliferation of HSC-T6 cells was reduced.4.SA directly binds to DNMT1.According to reports in the literature,DNMT1-mediated PTEN hypermethylation leads to the loss of PTEN expression and the activation of HSCs.In order to confirm the interaction between SA and DNMT1,we used SPR to detect the binding affinity of SA and DNMT1.In order to detect the effect of SA on the activity of DNMT1,the activity change of DNMT1 was measured using a DNMT1 assay kit.Experimental results showed that SA could bind to DNMT1 and reduce the activity of DNMT1 in liver fibrosis.5.The effect of SA on the activation and proliferation of HSC-T6 cells after overexpression of DNMT1.DNMT1 was overexpressed by DNMT1 plasmid on HSC-T6 cells,and TGF-β1（10ng/ml）and SA（10μM）were treated for 48h.Western blot was used to detect the overexpression efficiency.CCK8 method was used to detect the influence of overexpressed DNMT1 on the activity of HSC-T6 cells.Flow cytometry was used to detect the influence of overexpression of DNMT1 on the cell cycle of HSC-T6.Western blot assay was used to detect the effect of DNMT1 onα-SMA,col1α1 and related cyclins influences.The experimental results showed that the overexpression of DNMT1in HSC-T6 cells and the ability of SA to inhibit the proliferation of HSC-T6 cells were weakened.