Study On The Mechanism Of Tanshinone ⅡA In Ameliorating Liver Fibrosis Based On PI3K/Akt-Nrf2/HO-1 Signaling Pathway

Objective:Chronic liver disease caused by various factors such as hepatitis viruses,autoimmune diseases,and cholestasis,may develop into liver fibrosis.Tanshinone ⅡA（Tan ⅡA）,an extract of Salvia miltiorrhiza,has been reported to protect the liver.However,the potential target and mechanism of Tan ⅡA in the treatment of liver fibrosis are still unclear.The purpose of this study is to observe the improvement of liver fibrosis under the preventive treatment of Tan ⅡA through conducting in vitro and in vivo experiments related to liver fibrosis,combined with the results of network pharmacological analysis,and further explore its pharmacological mechanism,providing relevant scientific basis for the theoretical research of Tan ⅡA in the treatment of liver fibrosis.Methods:1.Through network pharmacological analysis,the target information of Tan ⅡA was screened and matched with the genes related to liver fibrosis.Gene Ontology（GO）enrichment analysis and Kyoto Encyclopedia of Genes and Genome（KEGG）pathway enrichment methods were used to conduct biological function enrichment analysis of intersection genes between Tan ⅡA and liver fibrosis（drugs and diseases）.GO enrichment analysis mainly includes molecular function（MF）,cell component（CC）and biological process（BP）analysis,while KEGG pathway enrichment analysis mainly focuses on signaling pathways.Then the correlation analysis of protein-protein interactions（PPI）was carried out,and the PPI network was constructed to analyze the relationship between Tan ⅡA and liver fibrosis related proteins.2.In vivo experiment,we constructed CCl₄-induced liver fibrosis disease model in mice,and simultaneously used Tan ⅡA for preventive drug intervention.Sixty SPF grade male C57BL/6J mice were randomly divided into 6 groups,with 10 mice in each group.The Control group and the Model group were given the same dose of physiological saline,the Tan ⅡA 10 group,Tan ⅡA 20 group,and Tan ⅡA 40 group were given 10 mg/kg,20 mg/kg,and 40 mg/kg of Tan ⅡA suspension solution,and the Colchicine group was given 0.2mg/kg of colchicine solution.In addition to the Control group,mice in each group were given 20%CCl₄ olive oil solution of 1 m L/kg twice a week for 12 weeks to check their general condition.Through calculating the liver index,and detecting serum AST,ALT,LDH andγ-GT activity to evaluate the degree of liver injury in mice.Mouse liver tissue was fixed with 4%paraformaldehyde,and then subjected to histopathological HE staining,Masson staining,and Sirius red staining after 5μm sectioning.Under a microscope,the morphological changes of mouse liver tissue were observed and analyzed,and the degree of liver fibrosis in mice was examined.In order to evaluate the correlation between Tan ⅡA ameliorating CCl₄-induced liver fibrosis and oxidative stress in mice,the SOD activity,GSH and MDA content of oxidative stress-related indicators were measured in the liver tissue of mice.The expression levels of KRAS,PI3K,p-PI3K,Akt,p-Akt,Nrf2,HO-1proteins in mouse liver tissue were detected by immunohistochemistry and Western blot（WB）.3.In vitro experiment,we established the proliferation model of hepatic stellate cells（HSCs）stimulated by H₂O₂,and simultaneously used Tan ⅡA for preventive drug intervention.The effects of different concentrations of H₂O₂ on activation and proliferation of HSCs were observed.Furthermore,the effects of different concentrations of Tan ⅡA on the proliferation of activated HSCs at different duration were observed.The proliferation of HSCs was detected by CCK-8 assay and cell colony formation assay.The expression levels of KRAS,PI3K,p-PI3K,Akt,p-Akt,Nrf2,HO-1 proteins in HSCs were detected by WB.Results:1.Network pharmacological analysis results:（1）There are 317 and 6693 genes related to Tan ⅡA and liver fibrosis respectively,of which 246 are differentially expressed.These differentially expressed genes can be regarded as the core potential target of Tan ⅡA anti-liver fibrosis.（2）The results of GO enrichment analysis and KEGG pathway analysis show that the action pathways and signaling pathways of Tan ⅡA intervention in liver fibrosis were complex and diverse.From the GO enrichment analysis results,at MF level,Tan ⅡA intervention in liver fibrosis is mainly related to steroid hormone receptor activity,drug binding,enzyme binding,protein tyrosine kinase activity,protein binding,etc.At CC level,it is mainly related to cytosol,nucleoplasm,extracellular exosome,etc.At BP level,it is mainly related to steroid hormone mediated signaling pathway,negative regulation of apoptosis process,cell proliferation,protein autophosphorylation,signal transduction,etc.The results of pathway enrichment analysis of KEGG show that Tan ⅡA can play a role in the intervention of liver fibrosis from multiple aspects,such as pathways in cancer,PI3K/Akt signaling pathway,Fox O signaling pathway,etc.（3）Through the construction of“Tan ⅡA-liver fibrosis”common target PPI network,it can be found that Tan ⅡA is associated with many liver fibrosis related proteins,among which KRAS protein,PI3K/Akt and Nrf2/HO-1 signaling pathway related proteins are most closely related to liver fibrosis.2.The establishment of CCl₄-induced mouse liver fibrosis model and the results of Tan ⅡA intervention:（1）Compared with the Control group,the liver index of the Model group was significantly higher（P<0.01）,and that of the drug group decreased after drug intervention（P<0.01）.（2）The detection results of four serological indicators（AST,ALT,LDH,andγ-GT）showed that the indicators in the Model group were significantly higher than those in the Control group（P<0.01）,and the indicators in the drug group were lower than those in the Model group（P<0.01）.（3）The results of mouse liver biopsy showed that the liver surface of the Control group was smooth,dark red in color,soft in texture,with sharp and clear edges,showing normal liver performance.The liver surface of the Model group mice was rough and slightly swollen,brown and hard.Compared with the Model group,the roughness of the liver surface of mice was relieved after drug treatment,the liver edge was sharper and the color was redder.（4）The HE staining results of mouse liver tissue showed that the liver lobules in the Control group showed normal liver lobule structure,without abnormal collagen fiber proliferation and inflammatory cell infiltration.In the Model group,the structure of hepatic lobule was seriously damaged,collagen fibers were deposited in large quantities,and pseudolobules were formed.Compared with the Model group,the inflammatory cell infiltration,collagen fiber deposition,steatosis and cell necrosis of mice in the drug group were alleviated.（5）The results of Masson staining and Sirius red staining of mouse liver tissue showed that there was only a small amount of collagen deposition in the central vein region in the liver tissue of the Control group mice.Compared with the Control group mice,the collagen fibers in the liver of the Model group mice were significantly deposited（P<0.01）,and the structure of hepatic lobule was destroyed.Compared with the Model group,the drug group reduced collagen deposition in the liver of mice in different degrees（P<0.01）.（6）The detection results of SOD,GSH and MDA in liver tissue of mice showed that compared with the Control group,the SOD activity and GSH content in the Model group decreased significantly（P<0.01）,and the MDA content increased significantly（P<0.01）.Compared with the Model group,the SOD activity and GSH content were restored after drug intervention（P<0.01）,and the MDA content was reduced（P<0.01）.（7）The results of immunohistochemistry showed that the expression levels of KRAS,p-PI3K,p-Akt,Nrf2 and HO-1 proteins in liver tissue of the Model group mice were significantly higher than those of the Control group（P<0.01）.Compared with the Model group,the expression levels of corresponding proteins in the drug group decreased（P<0.01）.（8）The results of WB experiment showed that the expression levels of KRAS,p-PI3K,p-Akt,Nrf2 and HO-1 proteins in liver tissue of the Model group mice were higher than those of the Control group（P<0.01）.Compared with the Model group,the expression levels of corresponding proteins in liver tissue of mice in drug group were significantly lower（P<0.05）.This is consistent with the results of immunohistochemistry.3.Establishment of H₂O₂ stimulated hepatic stellate cell proliferation model and Tan ⅡA intervention results:（1）The results of the exploratory experiment of different concentrations of H₂O₂on activation and proliferation of HSCs showed that the activation and proliferation of LX-2and HSC-T6 cells reached the highest when the concentration of H₂O₂was 5μmol/L.（2）The results of the exploratory experiment of the effect of different concentrations of Tan ⅡA on the proliferation of activated HSCs at different duration showed that with the increasing concentration of Tan ⅡA,the proliferation of activated HSCs was significantly inhibited（P<0.05）,and the inhibitory effect reached a peak near the concentration of Tan ⅡA in 80μmol/L.Meanwhile,the proliferation of LX-2 and HSC-T6 cells was most significantly inhibited under the condition of 48-hour drug intervention.（3）CCK-8 test results showed that compared with the Control group,the cell viability of the H₂O₂ group was significantly increased（P<0.01）.Compared with the H₂O₂ group,the drug group significantly inhibited the viability of LX-2 and HSC-T6 cells（P<0.01）.（4）The results of cell cloning experiment showed that compared with the Control group,the clonogenic ability of cells in the H₂O₂ group was significantly enhanced.Compared with the H₂O₂ group,the drug group significantly inhibited the cloning of LX-2 and HSC-T6 cells.（5）The results of WB experiment showed that the expression levels of KRAS,p-PI3K,p-Akt,Nrf2 and HO-1 proteins in LX-2 and HSC-T6 cells were significantly increased under the stimulation of H₂O₂ compared with the Control group（P<0.01）.However,after the simultaneous administration of drugs,the corresponding protein expression levels decreased（P<0.05）.Conclusions:1.Network pharmacology predicts that Tan ⅡA can exert its anti-liver fibrosis effect by regulating multiple targets and pathways.KRAS protein,PI3K/Akt and Nrf2/HO-1signaling pathways may be the key factors to its anti-liver fibrosis effect.2.Tan ⅡA has the effect of anti-CCl₄-induced liver fibrosis in mice,can significantly improve the liver function of mice and slow down the liver fibrosis lesions in mice.3.Tan ⅡA can significantly inhibit the proliferation of HSCs,such as LX-2 and HSC-T6cells,stimulated by H₂O₂.4.The anti liver fibrosis mechanism of Tan ⅡA may be related to the PI3K/Akt Nrf2/HO-1signaling pathway dependent antioxidant pathway,which effectively inhibits the activation and proliferation of HSCs.In addition,Tan ⅡA may promote its anti liver fibrosis effect to some extent by downregulating the expression level of KRAS protein.