| ObjectiveIn this paper,a mouse model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride(CCl4).The progression of liver fibrosis in mice after intervention with different concentrations of ergothioneine and the effect of ergothioneine on TGF-β/Smad signaling pathway were observed.Provide the basis for the treatment of liver fibrosis.MethodsC57/BL6 J mice were randomly divided into Oil control group(control),CCl4 model group(Mod),CCl4+EGT-L(CCl4+ergothioneine low-dose group,5mg/kg),CCl4+EGT-H(CCl4+ergothioneine high-dose group,10 mg /kg).Six weeks later,liver tissue and blood samples were taken from mice.The contents of aspartate aminotransferase(AST),alanine aminotransferase(ALT)and total bilirubin(TBIL)was detected,the changes of liver and tissue structure of mice were observed by pathological examination such as he staining and Sirius red staining,and liver tissue was detected by Masson staining.The deposition of α-smooth muscle actin(α-SMA),collagen type I(Col-I)and collagen type III(Col-III)were detected by immunohistochemistry.The degree of hepatic fibrosis and activation of hepatic stellate cells(HSC)were observed.The contents of laminin(LN)and hydroxyproline(Hyp)in mouse serum were detected by ELISA.mRNA was used to detect α-SMA,transforming growth factor(TGF-β1),tumor necrosis factor(TNF-α),collagen type Ⅰ(Col-Ⅰ),collagen type Ⅲ(The expression levels of collagen type III,Col-III),interleukin beta(IL-beta),interleukin-6(IL-6),Beclin-1and Caspase-3.Western blot was used to analyze the content of α-SMA,TGF-β1,Col-Ⅰ,Col-Ⅲ,Smad3,p-Smad3(phosphorylated Smad3),Smad2,p-Smad2.Results1.Compared with the control group,the serum ALT,AST,TBIL,LN and Hyp of the liver fibrosis in the model group was significantly increased(P<0.05).Comparison of ALT,AST,TBIL,LN and CCl4+EGT-L(CCl4+ergothioneine lowdose group,5mg/kg)and CCl4+EGT-H(CCl4+ergothioneine high-dose group,10mg/kg)and CCl4 model group Hyp was significantly decreased,with statistical significance(P<0.05).2.Through he staining,it can be found that there was no pathological changes in Contorl group,and the tissue cells are intact.In the CCl4 model group,degeneration and necrosis of hepatocytes,fatty degeneration,different sizes of hepatic lobules,and inflammatory infiltration of hepatocytes in the portal area were found.Cell necrosis was alleviated,and inflammatory infiltration was the mainstay.In Masson staining and Sirius red staining,a large amount of collagen fiber deposition was observed in the CCl4 model group,while the collagen deposition in the CCl4+EGT-L group and CCl4+EGT-H group was relatively less.3.RT-PCR detection of CCl4 model group mice liver tissue Col-Ⅰ,Col-Ⅲ,IL-β,IL-6,α-SMA,TGF-β1,TNF-α significantly increased compared with the control group(P<0.05),the CCl4+EGT-L group and CCl4+EGT-H group were significantly higher than the CCl4 model group in Col-Ⅰ,Col-Ⅲ,IL-β,IL-6,α-SMA,TGF-β,TNF-α were decreased(P<0.05).4.Western blot analysis showed that the expressions of Col-Ⅰ,Col-Ⅲ,α-SMA,TGF-β1,Smad3,p-Smad3,Smad2,p-Smad2 in the liver tissue of the CCl4 model group were significantly higher than those of the control group(P<0.05).The expressions of Col-Ⅰ,Col-Ⅲ,α-SMA,TGF-β1,Smad3,p-Smad3,Smad2,p-Smad2 in CCl4+EGT-L group and CCl4+EGT-H group were decreased compared with CCl4 model group(P<0.05).5.The expression levels of collagen α-SMA,Col-I and Col-III in liver tissues of Control group,Mod group,CCl4 + EGT-L(5mg / kg)group and CCl4 + EGT-H(10mg / kg)group mice were detected by immunohistochemical staining.EGT can reduce the positive expression of these collagens.Conclusions1.Ergothioneine has a protective effect on CCl4-induced liver fibrosis in mice,and can effectively reduce liver damage.2.Ergothioneine can inhibit the production of pro-inflammatory factors IL-β,IL-6,TGF-β1,and TNF-α,thereby inhibiting liver inflammation and reducing liver fibrosis.3.Ergothioneine exerts anti-fibrotic effect by down-regulating TGF/Smad signaling pathway. |
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