| 【Objective】We established an in vivo model of NAFLD liver fibrosis by feeding SD rats with high fat and high sugar for 24 weeks and an in vitro myofibroblast-like cell model induced by TGF-β1 signaling pathway activator,and treated with different concentrations of scutellarin(SCU)to investigate whether SCU can ameliorate NAFLD liver fibrosis by inhibiting the expressions of related factors in TGF-β1/Smad signaling pathway.This study will provide a scientific and theoretical basis for the treatment of NAFLD liver fibrosis with SCU.【Methods】:1.Network pharmacology target prediction: the target prediction of SCU was performed on Swiss Target Prediction database,Pharm Mapper database and herb database;the disease target prediction of NAFLD liver fibrosis was performed on Gene Cards database and OMIM database,after integrating the targets to exclude duplicate values Venny diagram was plotted to obtain the intersection targets of SCU for NAFLD liver fibrosis.PPI network maps were constructed and KEGG and GO enrichment analyses were performed for the intersection targets of SCU for NAFLD liver fibrosis.2.Experiments in vivo: after 63 male SD rats of 180-220 g were adaptively fed for 2 weeks,10 SD rats were selected as control group by random method,and the others were given high fat and high sugar feeding;control group was given normal feed and high fat and high sugar feeding group was given high fat and high sugar feed,after 24 weeks of feeding,three rats were randomly selected and dead in the model group,and liver tissues were taken for HE,Masson,oil red "O" staining to determine whether the NAFLD liver fibrosis model was successfully established,and after the animal model was successfully established.The high fat and high sugar feeding group was randomly divided equally into model group,scutellarin low,medium and high dose [25/50/100 mg/(kg?d)] treatment group and curcumin(CUR)positive drug[200mg/(kg?d)] treatment group;each group was given gastric therapy for 8 weeks,and high fat and high sugar feeding was continued during the treatment period of gastric gavage.After 8 weeks of treatment,the livers of the rats in each group were weighed under 1% pentobarbital sodium anesthesia,and liver weight/body weight was calculated.Each group was tested for lipids(TC,TG,FFA)in serum,liver function(ALT,AST,AKP)and indexes of oxidative stress-related indicators(ROS,GSH,MDA,SOD,Se Gpx)in serum and liver by enzyme labeling method;HE staining in each group was used to detect the degree of hepatocyte damage,and oil red "O " staining to detect the deposition of fat in the liver and Masson staining to detect the degree of liver fibrosis.Western blotting and immunohistochemistry were used to detect key factors inducing ROS production(NOX2,NOX4),key factors of TGF-β1/Smad signaling pathway(TGF-β1,Smad2/3,P-Smad2/3,Smad4,Smad7),key factors of downstream fibrosis(COL1,COL3,α-SMA),and protein expression levels of factors regulating hepatic extracellular matrix production(MMP2,MMP9,TIMP-1).3.Experiments in vitro(post-processing): cells were divided into control group and model group,the activated myofibroblast cell was constructed by adding TGF-βprotein(10 μg/l)to the model group cells for 24 h induction,and the success of the model was judged by detecting the expression of intracellular TGF-β1 and α-SMA proteins by Western blotting,and after the success,adding TGF-β1 signaling pathway activator(5 μmol/L)for 48 h,then the model group were divided into the model group,low/medium/high dose of scutellarin(200/400/600 μmol/L)treatment group and CUR positive drug(20 μmol/L)treatment group,and after 24 h treatment,Western blotting and immunofluorescence chemistry were used to detect the effect of SCU on key factors of TGF-β1/Smad signaling pathway(TGF-β1,Smad2/3,PSmad2/3,Smad4)in myofibroblast-like cell models constructed by TGF-β1 signaling pathway activator and the protein expression levels of downstream fibrosis key factor(α-SMA).4.Experiments in vitro(SCU pre-processing): Cells were divided into control group,model group,scutellarin low/medium/high dose(200/400/600 μmol/L)treatment group and curcumin-positive drug(20 μmol/L)treatment group.After SCU and CUR treated 24 h,TGF-β protein(10 μg/l)was added for 24 h.After adding TGF-β1 signaling pathway activator(5 μmol/L)for 48 h,Western blotting and immunofluorescence chemistry were used to detect the key factors of TGF-β1/Smad signaling pathway(TGF-β1,Smad2/3,P-Smad2/3,Smad4)in the myofibroblast cell model constructed by the activator of TGF-β1 signaling pathway induced by SCUs and the protein expression levels of downstream fibrosis key factor(α-SMA).【Results】:1.Results of network pharmacology: We obtained 34 targets of SCU in the treatment of NAFLD liver fibrosis in Swiss Target Prediction database,Pharm Mapper database,herb database,Gene Cards database and OMIM database.After GO and KEGG enrichment analysis of the 34 targets,We obtained 478 biological processes,28 cell compositions,38 molecular functions and 57 pathways.The results show that,scutellarin can alleviate NAFLD liver fibrosis through TGF-β signaling pathway.2.Experiments in vivo2.1 After 24 weeks of high fat and high sugar feeding,three rats were randomly selected and executed.HE staining showed that hepatocytes were severely damaged,with a large number of fat vacuoles and inflammatory cell infiltration,and red "O " staining showed a large number of orange-red lipid droplets in hepatocytes,and Masson staining showed blue collagen fibers in the confluent area,and the above results confirmed that the NAFLD liver fibrosis model was successfully established.2.2 Compared with the control group,the liver weight and body weight of rats in the model group increased(P<0.01)and liver weight/body weight increased;compared with the model group,the liver weight and body weight of rats decreased after different doses of SCU and CUR treatment,and the middle and high doses SCU treatment were statistically significant(P<0.05);liver weight/body weight decreased;compared with the control group,the serum levels of lipids(TC,TG,FFA)in the model group increased(P<0.01)and decreased after SCU and CUR treatment(P<0.05);liver function indexes(ALT,AST,AKP)in serum increased in the model group(P<0.01)and decreased after SCU and CUR treatment,and the high dose was statistically significant(P<0.05);oxidative stress indexes(ROS,MDA)in serum and tissues increased in the model group(P<0.01).)were increased(P<0.01)and anti-oxidative stress indicators(GSH、SOD、Se Gpx)were decreased(P<0.05),and oxidative stress indicators were decreased(P<0.05)and anti-oxidative stress indicators were increased after SCU and CUR treatment.2.3 The results of HE staining showed that: compared with the control group,hepatocytes in the model group were disorganized,with cell edema,inflammatory cell infiltration and a large number of fat vacuoles;compared with the model group,cell disorganization was reduced after SCU and CUR treatment,and fat vacuoles were reduced;oil red "O" staining showed that: compared with the control group,a large number of orange-red lipid droplets were observed in hepatocytes in the model group,the deposition of lipid droplets was reduced after SCU and CUR treatment.Masson staining showed that a large number of collagen fibers were deposited in the confluent area of the liver of the model group compared with the control group,and the collagen fibrosis deposition was reduced after SCU and CUR treatment.2.4 Immunohistochemistry and Western blotting results showed that key factors regulating ROS production(NOX2,NOX4),key factors of TGF-β1/Smad signaling pathway(TGF-β1,Smad2/3,P-Smad2/3,Smad4),key factors of downstream fibrosis(COL1,COL3,α-SMA),key factor that increases ECM formation(TIMP-1)expression increased(P<0.05),factor that decreases ECM formation(MMP2,MMP9)expression decreased(P<0.05),key factor that regulates ROS production after SCU and CUR treatment(NOX2,NOX4),key factor of TGF-β1/Smad signaling pathway(TGF-β1,Smad2/3,P-Smad2/3,Smad4),key downstream fibrosis factors(COL1,COL3,α-SMA),key factor that increases ECM formation(TIMP-1)expression was decreased(P<0.05),and factor that decreases ECM formation(MMP2,MMP9)expression was increased(P<0.05).3.Experiment in vitro(SCU post-processing): The results of immunofluorescence chemistry and Western blotting showed that NOX4,TGF-β1,Smad2/3,P-Smad2/3,Smad4,α-SMA were increased in MFB cells after 48 h administration of TGF-β1signaling pathway activator(P<0.05).NOX4,TGF-β1,Smad2/3,P-Smad2/3,Smad4,α-SMA expression decreased after treatment with CUR(P<0.05).4.Experiment in vitro(SCU pre-processing): The results of immunofluorescence chemistry and Western blotting results showed that NOX4,TGF-β1,Smad2/3,P-Smad2/3,Smad4,α-SMA expression was reduced in MFB cells after 48 h of TGF-β1 signaling pathway activator effect in SCU preincubated with CUR for 24 h(P<0.05),while NOX4,TGF-β1,Smad2/3,P-Smad2/3,Smad4,α-SMA expression increased in MFB cells after 48 h of TGF-β1 signaling pathway activator action(P<0.05).【Conclusions】:1.Network pharmacological analysis indicate that Scutellarin can alleviate NAFLD liver fibrosis through TGF-β signaling pathway.2.Scutellarin can improve the abnormalities of lipid metabolism in NAFLD liver fibrosis rats,alleviate oxidative stress and liver function damage,and thus inhibit the development of NAFLD liver fibrosis.3.Scutellarin can ameliorate NAFLD induced liver fibrosis by inhibiting the TGF-β1/Smad signaling pathway. |
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