AS-Ⅳ Regulates PSmad3C/3L Against Hepatic Fibrosis In Mice

Backgrounds and Aim:We have previously researched and developed compound Qishen extract（CASE）and found that its regulation of Smad2/3 link region and C-terminal phosphorylation can significantly inhibit the deposition of liver fibrosis.However,CASE is a traditional Chinese medicine compound guided by the theory of Chinese medicine compatibility.The ingredients are complex and it is difficult to confirm which or those ingredients play the leading role of CASE.Current research shows that AS-Ⅳ has a variety of pharmacological effects,which suggests that AS-Ⅳ may be the dominant component of CASE.Therefore,this study is to clarify that 1.AS-Ⅳ regulates the expression of link region and C-terminal phosphorylation of Smad2/3 to inhibit DEN/CCl₄/C₂H₅OH-induced liver fibrosis in mice through,2.Regulation of p Smad3gene C terminal phosphorylation site mutation in the occurrence of liver fibrosis.Method:1.the effect of AS-Ⅳ against DEN/CCl₄/C₂H₅OH-induced liver fibrosis and the mechanism of TGF-β/Smad pathway in vivoDEN/CCl₄/C₂H₅OH-induced mouse liver fibrosis model was established:DEN 100mg/kg,intraperitoneal injection（once in the first week of modeling:the second time in the second week）;20%CCl₄ olive oil solution（0.05ml/10g,2 times/week;start from the third week of modeling to the end of week 7）,gavage;give drinking water containing 10%ethanol（change every other day,start from the third week of modeling to the end of week12）,mice are free Drink;20%CCl₄ olive oil solution(0.06ml/10g,2 times/week,from the 8^th week of the modeling to the end of the 12^th week).At the 12^th week（liver fibrosis stage）,the samples were collected in batches.A total of 36 healthy male C57BL/6J mice,6-8 weeks old,weighing 18-23g,SPF grade;randomly divided into 6 groups:normal control group;model group,low astragaloside Ⅳ,medium,high-dose group（20,40,80mg/kg/d;ig）positive drug control group colchicine group（0.1 mg/kg/d;ig）,6 rats in each group,continuously administered for 12 weeks.At the beginning of modeling,the three AS-Ⅳ dose groups and the colchicine group were given corresponding doses of drugs.The control group and the model group were given corresponding solvents for continuous administration,and the samples were taken during the 12-week liver fibrosis period.Liver biopsy,hematoxylin,and eosin（HE）staining were performed to observe the pathological changes of the liver.Masson staining was used to observe the degree of liver fibrosis in each group.Immunohistochemical staining was used to detect the distribution ofα-SMA,and Western blotting was used to detect TGF-β₁.Changes in the levels of p Smad2C,p Smad2L,p Smad3C,p Smad3L,PAI-1,α-SMA,and immunofluorescence staining to detect the expression and distribution of TGF-β₁,p Smad3C,and p Smad3L.2.In vitro experiments verify that AS-Ⅳ regulates TGF-β₁ to stimulate P-Smad2/3in HSC-T6 cellsCultivate HSC cells to the exponential growth phase and inoculate them evenly in each experimental group:vehicle control group,TGF-β₁stimulation group,and AS-Ⅳ low,medium,and high dose（5,10,20μmol/L）groups（density 1.0×10⁶/L）.Cells were routinely cultured for 24 hours,replaced with serum-free medium,and cultured for 24hours after adding various doses of AS-Ⅳ drugs.Before the end of the culture,100μmol/L TGF-β₁ was added to the TGF-β₁ stimulation group and the TGF-β₁ group,the blank group Add an equal volume of solvent,and after the completion of the culture,extract the total protein of the experimental group cells.Immunoblotting was used to detect the changes in the levels of p Smad2C,p Smad2L,p Smad3C,p Smad3L,PAI-1,andα-SMA.Immunofluorescence staining was used to detect the expression and distribution of p Smad3C.3.Establishment of CCl₄-induced liver fibrosis model in mice with mutations in the C-terminal phosphorylation site of Smad3 geneThe successful construction of C57BL/6J mouse Smad3 C-terminal phosphorylation site mutation heterozygous mice were expanded and identified,and male 6-week-old heterozygous mice(genotype:p Smad3C^+/-)and their homologous wild-type mice were screened out Mouse(genotype:p Smad3C^+/+).Smad3 gene C-terminal phosphorylation site mutation mice 6 weeks old,males,a total of 24,were randomly divided into p Smad3C^+/+control group（WT-Control）,p Smad3C^+/+model group（WT-CCl₄）,p Smad3C^+/-control group（HT-Control）,p Smad3C^+/-model group（HT-CCl₄）four groups with 6 animals in each group.15%CCl₄（2ml/kg）（solvent corn oil）was injected intraperitoneally twice a week for six consecutive weeks,and the material was taken at the end of the sixth week.The body weight,liver,and liver index were measured for different groups of mice.The mouse serum was collected to detect ALT,AST,HE,and Masson staining to observe the changes in histopathological characteristics of the mice and to grade the liver fibrosis of the mice.Western blotting test Changes in levels of p Smad2C,p Smad2L,p Smad3C,PAI-1,TGF-β₁.Results1.AS-Ⅳ exerts anti-DEN/CCl₄/C₂H₅OH-induced liver fibrosis effect by regulating TGF-β₁/P-Smad2/3Liver biopsy,HE,Masson showed that AS-Ⅳ significantly reduced DEN/CCl₄/C₂H₅OH-induced liver fibrosis.The results of immunoblotting experiments showed that AS-Ⅳ significantly inhibited the expression of TGF-β₁,p Smad2C,p Smad2L,p Smad3L,PAI-1,andα-SMA in the liver,and significantly promoted the expression of p Smad3C.Immunofluorescence staining results showed that AS-Ⅳ significantly inhibited the expression of TGF-β₁ and p Smad3L,and at the same time promoted the expression of p Smad3C and promoted its nuclear entry.2.AS-Ⅳ regulates TGF-β₁ to stimulate the Smad2/3 pathway in HSC-T6 cellsThe results of immunoblotting experiments showed that AS-Ⅳ significantly promoted the phosphorylation of Smad3C,inhibited the phosphorylation of Smad2C,Smad2L,and Smad3L;and inhibited the expression of PAI-1 andα-SMA in TGF-β₁stimulated HSC-T6 cells.Immunofluorescence staining results showed that AS-Ⅳ significantly promoted the expression of p Smad3C.3.Mutations in the C-terminal phosphorylation site of the Smad3 gene enhance CCl₄-induced liver fibrosisLiver index,HE,Masson,liver fibrosis classification results showed that the mutation of the C-terminal phosphorylation site of the Smad3 gene significantly promoted the further deposition and deterioration of CCl₄-induced liver fibrosis.The results of western blotting showed that the expression of p Smad3C in the liver of mice with mutations in the C-terminal phosphorylation site of Smad3 gene was significantly suppressed;at the same time,mutations in the C-terminal phosphorylation site of the Smad3 gene significantly promoted the expression of TGF-β₁,p Smad2C,p Smad2L,PAI-1 to promote liver fibrosis induced by CCl₄.Conclusion:1.AS-Ⅳ exerts an anti-DEN/CCl₄/C₂H₅OH-induced liver fibrosis effect by regulating the Smad2/3 link region and C-terminal phosphorylation.2.Smad3 gene C-terminal phosphorylation site mutation significantly inhibited the expression of p Smad3C and significantly promoted the expression of TGF-β₁,p Smad2C,p Smad2L,and PAI-1 to promote the development of liver fibrosis.