Exploring The Molecular Mechanism Of Ursolic Acid Against Liver Fibrosis Based On Notch3/NOX4 Signaling Pathway

Background:Liver fibrosis is a repair response of the liver to various chronic injury stimuli.Continued progression of liver fibrosis can lead to cirrhosis and liver cancer,which is a serious threat to human health.The pathogenesis of liver fibrosis is complex and has not been fully elucidated.There is still a lack of effective anti-fibrotic drugs clinically.Hepatic stellate cell(HSC)activation is a central event in liver fibrosis.Notch signaling pathway(especially Notch3 signaling pathway)and NOX4 signaling pathway are closely related to the occurrence of liver fibrosis,but the specific mechanisms by which both promote liver fibrosis remain to be further investigated.Recent studies have found that Notch and NOX4 signaling pathways are mutually regulated in a variety of diseases.Considering the important role of Notch3 in the development of liver fibrosis,is there a mutual regulation between Notch3 and NOX4 signaling pathways in the development of liver fibrosis? It is still unclear.Ursolic acid(UA),a herbal monomer widely found in many plants,has unique anti-fibrotic effects.The specific mechanism of UA against liver fibrosis remains to be further elucidated.Our preliminary study showed that UA can inhibit HSC activation and liver fibrosis through down-regulation of NOX4 signaling pathway.UA has been shown to inhibit Notch signaling pathway activation in human colon cancer cells.Meanwhile,our pre-experiments revealed that the Notch signaling pathway may act as an upstream regulator of NOX4 during HSC activation.Therefore,we hypothesized that UA may inhibit HSC activation and liver fibrosis by regulating the Notch3/NOX4 signaling pathway.Objective:1.To clarify the specific regulatory relationship between Notch3 and NOX4 in the development of liver fibrosis.2.To clarify whether UA inhibits HSC activation and liver fibrosis by regulating the Notch3/NOX4 signaling pathway.Methods:Ⅰ The effect of ursolic acid on liver fibrosis in mice1.Experimental grouping and drug treatment: 30 C57BL/6J mice were randomly divided into 3 groups: Control group,CCl4 group,and UA treatment group(UA group).Mice in the Control group were injected intraperitoneally with 1ml/kg olive oil twice a week for a total of 6 weeks.Mice in the CCl4 group were injected intraperitoneally with 1ml/kg CCl4(diluted in olive oil to a 20% solution)twice a week for a total of 6 weeks.Mice in the UA group were injected intraperitoneally with 1ml/kg CCl4 twice a week for a total of 6 weeks,and then orally treated with50mg/kg UA once a day for the last 4 weeks.After modeling,Ocular serum and liver specimens were obtained for subsequent molecular biology experiments and pathological examinations.2.Liver samples were evaluated for structural damage and collagen deposition through HE staining,Sirius Red staining,Masson staining,as well as hydroxyproline(HYP)content detection.Liver function was evaluated through serum transaminases,albumin(ALB),and total bilirubin(TBil).Hepatic inflammation was assessed by measuring m RNA expression of TNF-α,IL-1β,and F4/80.Hepatic oxidative stress was assessed through malondialdehyde(MDA)and total antioxidant capacity(T-AOC).Western blotting and q PCR were used to detect expression of genes and proteins related to hepatic fibrosis.Immunohistochemistry was used to detect expression of liver Col1α1 and α-SMA.Ⅱ The effect of UA on the activation of HSC.1.Experimental grouping and drug treatment: Human HSC cell line LX2 in logarithmic growth phase was divided into Control group,TGF-β1 group,and UA intervention group(UA group).The Control group cells were cultured in DMEM medium containing 2% FBS for 48 hours,the TGF-β1 group cells were cultured in DMEM medium containing 10ng/ml TGF-β1 for 48 hours,and the UA group cells were pre-treated with DMEM medium containing 50μmol/L UA for 30 minutes,and then cultured in DMEM medium containing 10ng/ml TGF-β1 for 48 hours.2.WB and q PCR were used to detect the expression of HSC α-SMA.MTT assay was used to measure HSC proliferation.Flow cytometry was used to detect HSC apoptosis.Transwell assay was used to assess HSC migration and invasion.WB and q PCR were used to detect the expression of fibrotic-related proteins and genes in HSCs.Ⅲ Exploring the effect of UA on HSC activation based on the Notch3/NOX4 signaling pathway.1.The effect of UA on the expression of Notch3,Hes1,and NOX4 in activated HSCs.Experimental grouping: LX2 was divided into Control group,TGF-β1 group,and UA group.According to the experimental grouping,cells were treated with TGF-β1 or UA.2.Silencing of Notch3 experimental grouping: After transfection of cells with Si-NC or Si-Notch3,they were divided into Si-NC group,TGF-β1+Si-NC group,TGF-β1+Si-NC+UA group,TGF-β1+Si-Notch3 group and TGF-β1+Si-Notch3+UA group.According to the experimental grouping,cells were treated with TGF-β1 or TGF-β1+UA.3.Silencing of NOX4 experimental grouping: Cells were transfected with Si-NC or Si-NOX4 and divided into Si-NC group,TGF-β1+Si-NC group,TGF-β1+Si-NC+UA group,TGF-β1+Si-NOX4 group,and TGF-β1+Si-NOX4+UA group.According to the experimental grouping,cells were treated with TGF-β1 or TGF-β1+UA.4.WB and q PCR were used to detect the expression of HSC Notch3,Hes1,and NOX4.WB was used to detect the expression of fibrosis-related proteins in HSC.MTT assay was performed to assess HSC proliferation.Flow cytometry was used to measure HSC apoptosis.Transwell assay was employed to evaluate HSC migration and invasion.Ⅳ Exploring the effect of UA on liver fibrosis based on the Notch3/NOX4 signaling pathway.1.The effect of UA on the expression of Notch3,Hes1,and NOX4 in mice with liver fibrosis.Experimental grouping: Mice were randomly assigned to the Control group,CCl4 group,and UA group.According to the experimental grouping,modeling was induced by olive oil or CCl4.UA was used for treatment.After modeling,serum and liver samples were collected for subsequent molecular biology experiments and pathological examination.2.Experimental grouping of Notch3 inhibition: Mice were randomly divided into the Control group,CCl4 group,UA group,Notch3 inhibitor(DAPT)group,and DAPT+UA group.According to the experimental grouping,modeling was induced by olive oil or CCl4,and intervened with UA,DAPT,or DAPT+UA.After modeling,serum and liver samples were collected for subsequent molecular biology experiments and pathological analysis.3.Experimental grouping of NOX4 inhibition: Mice were randomly divided into the Control group,CCl4 group,UA group,NOX4 inhibitor(GKT)group,and GKT+UA group.According to the experimental grouping,modeling was induced by olive oil or CCl4,and intervened with UA,DAPT,or DAPT+UA.After modeling,serum and liver samples were collected for subsequent molecular biology experiments and pathological analysis.4.Liver HE staining,Sirius Red staining,Masson staining,and hydroxyproline(HYP)content in serum and liver samples were performed to assess liver structural damage and collagen deposition.Liver function was evaluated by measuring serum transaminase,albumin(ALB),and total bilirubin(TBil).q PCR was used to detect the m RNA expression of TNF-α,IL-1β,and F4/80 in the liver to evaluate liver inflammation.Malondialdehyde(MDA)and total antioxidant capacity(T-AOC)were detected to evaluate liver oxidative stress.WB and q PCR were used to detect the expression of Notch3,Hes1,NOX4,and liver fibrosis-related genes and proteins in the liver.Immunohistochemistry was used to detect the expression of Col1α1 andα-SMA in the liver.Results:Ⅰ The effect of ursolic acid on liver fibrosis in mice1.Establishment of liver fibrosis mouse modelCompared with Control mice,the results of HE,Sirius Red,and Masson staining of the liver in CCl4 mice suggested disruption of liver lobular structure,infiltration of a large number of inflammatory cells,collagen deposition,and scar formation.The hydroxyproline content in the serum and liver of CCl4 mice was significantly increased compared with Control mice(P < 0.05).2.UA improves liver lobule structure disruption and collagen deposition in liver fibrosis miceCompared with the CCl4 group,the UA group showed an improvement in liver lobule structure based on HE,Sirius Red and Masson staining,with a reduction in inflammatory cell infiltration,collagen deposition,and scar formation in the liver.The content of hydroxyproline in the serum and liver of the UA group mice was significantly lower than that in the CCl4 group mice(P < 0.05).3.The effect of UA on liver function in mice with liver fibrosisCompared with the CCl4 group,the UA group mice showed a significant decrease in serum ALT,AST,and TBil levels and an increase in serum ALB level,with statistical significance.4.Effects of UA on liver inflammation and oxidative stress in liver fibrosis miceCompared to the CCl4 group mice,the results of HE,Sirius Red,and Masson staining of the liver in the UA group showed improved hepatic lobular structure,decreased infiltration of inflammatory cells,reduced collagen deposition and scar formation(P<0.05).The levels of TNF-α,IL-1β,and F4/80 m RNA expression,and MDA levels in the liver of the UA group mice were significantly decreased(P<0.05),but there was no significant difference in T-AOC levels(P>0.05).5.Effects of UA on liver fibrosis-related genes and proteins in mice with liver fibrosisCompared to the CCl4 group mice,the UA group mice showed a significant decrease in the protein and gene expression of Col1α1,α-SMA,and TIMP1 in the liver(P<0.05),but an increase in MMP1 protein and gene expression(P<0.05).Immunohistochemical analysis showed that the expression of Col1α1 and α-SMA in the liver of the UA group mice was significantly reduced compared to the CCl4 group mice(P<0.05).Ⅱ The effect of UA on the activation of HSC.1.The effect of UA on the expression of α-SMA in HSCCompared with the TGF-β1 group,the HSC in UA group showed a significant decrease in α-SMA protein and gene expression(P<0.05).2.The effect of UA on the biological behavior of HSCCompared with the TGF-β1 group,the HSC in UA group showed a significant decrease in proliferation,migration,and invasion ability,as well as an increase in apoptosis,with statistical significance.3.The effect of UA on the expression of fibrosis-related proteins and genes in HSCCompared with the TGF-β1 group,the HSC in UA group showed a significant decrease in the protein and gene expression of Col1α1 and TIMP1(P<0.05),while the protein and gene expression of MMP1 increased(P<0.05).Ⅲ Exploring the effect of UA on HSC activation based on the Notch3/NOX4 signaling pathway.1.The effect of UA on the expression of Notch3,Hes1,and NOX4 in activated HSC.Compared with the TGF-β1 group,the HSC in UA group showed a significant decrease in the expression of Notch3,Hes1,and NOX4 proteins and genes(P<0.05).2.Screening of small interfering RNAs(si RNAs)targeting Notch3Compared with the Si-NC group,Western blotting and q PCR results showed that Si-Notch3-3 had the best silencing effect on Notch3,and it was selected for subsequent experiments targeting Notch3.3.Screening of si RNAs targeting NOX4Compared with the Si-NC group,Western blotting and q PCR results showed that Si-NOX4-1 had the best silencing effect on NOX4,and it was selected for subsequent experiments targeting NOX4.4.Exploring the regulatory relationship between Notch3 and NOX4 signaling pathways by silencing Notch3 or NOX4 in HSC4.1 Exploring the regulatory relationship between Notch3 and NOX4 signaling pathways by silencing Notch3 in HSCCompared to the Si-NC group,TGF-β1+Si-NC group showed significant increases in the protein expression of Notch3,Hes1 and NOX4(P < 0.05).After silencing Notch3,the protein expression of Notch3,Hes1 and NOX4 in the TGF-β1+Si-Notch3 group was significantly decreased compared to the TGF-β1+Si-NC group(P < 0.05).There was no significant difference in the expression of Notch3 and NOX4 between the TGF-β1+Si-Notch3+UA group and the TGF-β1+Si-Notch3 group(P>0.05).The protein expression of Notch3,Hes1 and NOX4 in the TGF-β1+Si-NC+UA group was significantly decreased compared to the TGF-β1+Si-NC group(P<0.05).4.2 Exploring the regulatory relationship between Notch3 and NOX4 signaling pathways by silencing NOX4 in HSCCompared with the Si-NC group,TGF-β1+Si-NC group had significantly increased protein expression of Notch3,Hes1,and NOX4(P<0.05).After silencing NOX4,the TGF-β1+Si-NOX4 group had a significant decrease in NOX4 protein expression compared to the TGF-β1+Si-NC group(P < 0.05),but there was no significant difference in Notch3 and Hes1 protein expression(P>0.05).Compared with the TGF-β1+Si-NOX4 group,the TGF-β1+Si-NOX4+UA group had no significant difference in NOX4 protein expression(P > 0.05),but there was a significant decrease in Notch3 and Hes1 protein expression(P < 0.05).The TGF-β1+Si-NC+UA group had a significant decrease in protein expression of Notch3,Hes1,and NOX4 compared to the TGF-β1+Si-NC group(P<0.05).5.UA inhibits HSC activation via the Notch3/NOX4 signaling pathway5.1 The effect of silencing Notch3 on HSC activationAfter silencing Notch3,the protein expression of Col1α1,α-SMA,and TIMP1 in the TGF-β1+Si-Notch3 group was significantly decreased compared to the TGF-β1+Si-NC group(P < 0.05),while the protein expression of MMP1 was significantly increased(P<0.05).Compared to the TGF-β1+Si-NC group,the HSC in the TGF-β1+Si-Notch3 group showed significantly increased apoptosis and decreased migration,invasion,and proliferation abilities.5.2 The effect of silencing NOX4 on HSC activationAfter silencing NOX4,the TGF-β1+Si-NOX4 group showed a significant decrease in the protein expression of Col1α1,α-SMA,and TIMP1 compared to the TGF-β1+Si-NC group(P<0.05),while the protein expression of MMP1 increased significantly(P < 0.05).Compared to the TGF-β1+Si-NC group,after silencing NOX4,the HSC in the TGF-β1+Si-NOX4 group showed significantly increased apoptosis and decreased migration,invasion,and proliferation abilities.5.3 The effect of UA on HSC activation was investigated by silencing Notch3 or NOX4.Compared with the TGF-β1+Si-Notch3 group,there was no significant difference in HSC α-SMA,TIMP1,and MMP1 protein expression,as well as apoptosis,migration,invasion,and proliferation in the TGF-β1+Si-Notch3+UA group(P > 0.05).Similarly,compared with the TGF-β1+Si-NOX4 group,there was no significant difference in HSC Col1α1,α-SMA,TIMP1,and MMP1 protein expression,as well as migration,invasion,and proliferation in the TGF-β1+Si-NOX4+UA group(P > 0.05).In the TGF-β1+Si-NC+UA group,the expression of fibrotic-related proteins(Col1α1,α-SMA,and TIMP1),cell migration,invasion,and proliferation were significantly decreased compared with the TGF-β1+Si-NC group(P < 0.05),while MMP1 protein expression and cell apoptosis were significantly increased(P<0.05).Ⅳ Exploring the effect of UA on liver fibrosis based on the Notch3/NOX4 signaling pathway.1.The effect of UA on the expression of liver Notch3,Hes1,and NOX4 in mice with liver fibrosisCompared with the CCl4 group,the protein and gene expression of liver Notch3,Hes1,and NOX4 in the UA group was significantly decreased(P<0.05).2.Investigating the regulatory relationship between Notch3 and NOX4 signaling pathways in the liver by inhibiting Notch3 or NOX42.1 Inhibiting Notch3 to explore the regulatory relationship between Notch3 and NOX4 signaling pathways in the liverCompared with the Control group,the protein and gene expression of liver Notch3,Hes1,and NOX4 in the CCl4 group was significantly increased(P<0.05).After inhibiting Notch3,the protein and gene expression of liver Notch3,Hes1,and NOX4 in the DAPT group was significantly decreased compared with the CCl4 group(P<0.05).Compared with the DAPT group,there was no significant difference in the protein and m RNA expression of liver Notch3,Hes1,and NOX4 in the DAPT+UA group(P > 0.05).The protein and m RNA expression of liver Notch3,Hes1,and NOX4 in the UA group was significantly decreased compared with the CCl4 group(P<0.05).2.2 Inhibiting NOX4 to explore the regulatory relationship between Notch3 and NOX4 signaling pathways in the liverCompared with the Control group,the protein and gene expression of liver Notch3,Hes1,and NOX4 in the CCl4 group was significantly increased(P<0.05).After inhibiting NOX4,the protein and gene expression of NOX4 in the GKT group were significantly decreased compared to the CCl4 group(P<0.05),but there was no significant difference in the protein and gene expression of Notch3 and Hes1(P>0.05).Compared to the GKT group,there was no statistically significant difference in the protein and m RNA expression of NOX4 in the GKT+UA group(P>0.05),but there was a significant decrease in the protein and m RNA expression of Notch3 and Hes1(P<0.05).The protein and m RNA expression of Notch3,Hes1,and NOX4 in the UA group were significantly decreased compared to the CCl4 group(P<0.05).3.UA improves liver function,liver inflammation,and oxidative stress in mice with hepatic fibrosis through the Notch3/NOX4 signaling pathway.3.1 The effects of inhibiting Notch3 on liver function,liver inflammation,and oxidative stress in mice with liver fibrosisThe serum levels of ALT,AST,and TBil in CCl4 group mice were significantly increased compared to those in Control group mice(P<0.05),while the serum ALB level was significantly decreased(P<0.05).After inhibiting Notch3 with DAPT,the serum levels of ALT,AST,and TBil in the DAPT group were significantly decreased compared to those in the CCl4 group(P < 0.05),and the serum ALB level was significantly increased(P < 0.05).Liver inflammation(TNF-α,IL-1β,and F4/80 m RNA)and oxidative stress(MDA)were significantly increased in the CCl4 group compared to the Control group(P < 0.05),but were significantly relieved in the DAPT group after inhibiting Notch3(P<0.05).There was no significant difference in T-AOC in the liver of mice among the Control group,CCl4 group,and DAPT group.3.2 The effects of inhibiting NOX4 on liver function,liver inflammation,and oxidative stress in mice with liver fibrosisCompared to the Control group mice,the serum levels of ALT,AST,and TBil in the CCl4 group mice were significantly increased(P<0.05),while the serum ALB level was significantly decreased(P<0.05).After inhibiting NOX4 with GKT137831,the serum levels of ALT,AST,and TBil in the GKT group were significantly decreased compared to those in the CCl4 group(P<0.05),and the serum ALB level was significantly increased(P<0.05).Liver inflammation(TNF-α,IL-1β,and F4/80 m RNA)and oxidative stress(MDA)were significantly increased in the CCl4 group compared to the Control group(P<0.05),but were significantly relieved in the GKT group after inhibiting NOX4(P < 0.05).There was no significant difference in T-AOC in the liver of mice among the Control group,CCl4 group,and GKT group.3.3 Inhibition of Notch3 or NOX4 to explore the effects of UA on liver function,liver inflammation,and oxidative stress in mice with liver fibrosisCompared to the DAPT group,there was no significant difference in the serum ALT,AST,TBil,and ALB levels in the DAPT+UA group(P>0.05).There was also no significant difference in liver inflammation(TNF-α,IL-1β,and F4/80 m RNA)and oxidative stress(MDA)between the DAPT+UA and DAPT groups(P > 0.05).Additionally,there was no significant difference in the serum ALT,AST,TBil,and ALB levels between the GKT+UA and GKT groups(P>0.05).There was also no significant difference in liver inflammation and oxidative stress between the GKT+UA and GKT groups(P > 0.05).Compared to the Control group,the CCl4 group of mice showed a significant increase in serum transaminases and bilirubin levels,liver inflammation,and oxidative stress(P<0.05),and a significant decrease in serum ALB levels(P<0.05).After UA intervention,the mice in the UA group showed a significant decrease in serum transaminases and bilirubin levels,liver inflammation,and oxidative stress,and a significant increase in serum ALB level(P<0.05).4.UA improves liver fibrosis in mice through the Notch3/NOX4 signaling pathway4.1 Inhibition of Notch3 on liver fibrosis in miceCompared to the CCl4 group,the DAPT group showed improvements in liver lobule structure,reduced collagen deposition and fibrous septum formation,as indicated by the results of liver pathological analysis(P < 0.05).Compared to the CCl4 group,the DAPT group also had reduced serum and liver hydroxyproline levels,decreased protein and gene expression of Col1α1,α-SMA,and TIMP1 in the liver(P<0.05),and increased protein and gene expression of MMP1 in the liver(P<0.05).4.2 Inhibition of NOX4 on liver fibrosis in miceCompared to the CCl4 group,the GKT group showed improvements in liver lobule structure,reduced collagen deposition and fibrous septum formation,as indicated by the results of liver pathological analysis(P < 0.05).Compared to the CCl4 group,the GKT group also had reduced serum and liver hydroxyproline levels,decreased protein and gene expression of Col1α1,α-SMA,and TIMP1 in the liver(P<0.05),and increased protein and gene expression of MMP1 in the liver(P<0.05).4.3 Inhibition of Notch3 or NOX4 to explore the effect of UA on liver fibrosisCompared with the DAPT group,there was no significant difference in liver lobule structure,liver collagen deposition and fibrosis interval formation,and serum and liver hydroxyproline content in the DAPT+UA group(P>0.05).The expression of liver fibrosis-related proteins and genes in the DAPT+UA group was also not significantly different from that in the DAPT group(P>0.05).Meanwhile,there was no significant difference in liver lobule structure,liver collagen deposition and fibrosis interval formation,and serum and liver hydroxyproline content between the GKT+UA group and the GKT group(P > 0.05).The expression of liver fibrosis-related proteins and genes in the GKT+UA group was not significantly different from that in the GKT group(P>0.05).Compared with the Control group,the CCl4 group mice showed significant disruption of liver lobular structure,significant increase in serum and liver hydroxyproline content,significant increase in the expression of liver Col1α1,α-SMA,and TIMP1,and significant decrease in the expression of MMP1.After treatment with UA,the above indicators were significantly improved in the UA group mice.Conclusion:1.During the development of liver fibrosis,Notch3 acts as an upstream regulator of NOX4.Notch3/NOX4 signaling pathway is one of the pathogenic mechanisms of liver fibrosis.2.UA can inhibit HSC activation and liver fibrosis by regulating the Notch3/NOX4 signaling pathway.