| Objective:The rat model of early liver fibrosis of Nonalcoholic fatty liver disease(NAFLD)were established by high fat diet and high sugar diet and the fibroblast model induced by the activator of TGF-β1 signaling pathway.The model set was treated with different concentrations of ginsenoside Rgl to investigate the positive effect of it on early liver fibrosis in NAFLD,and explore whether it can actively regulate the TGF-β1/Smad signaling pathway to improve the degree of liver fibrosis.Explore new drugs for the treatment of NAFLD anti-liver fibrosis and related drug targets,and provide a new scientific theoretical basis for the treatment of ginsenoside Rg1 for the disease.Methods:We used an adaptive feed method to feed the male SD rats during the two weeks.These rats were 6weeks old and their weight were 180-200g.Next,adopt the method of random sampling to select ten rats fed normal fodder for 18 weeks and made them as the normal group.The rest of the rats were all fed the fodder with high sugar and high fat for 18 weeks.5 rats were put to death randomly and used to define if built the models successfully.We conducted oil red "O" staining from liver and then found severe fat deposition.These were found dispersive fibrosis in central hepatic portal area by Masson staining.NAFLD early fibrosis models were built successfully by these phenomena.The rest of the SD rats fed the fodder with high sugar and high fat were randomly divided into model control group,the treatment group treated by low-dose,medium-dose and high-dose ginsenoside Rg1(25/50/100mg/kg/d),the positive drug treatment control group treated by curcumin(100mg/kg/d).These groups went on being fed by the fodder with high sugar and high fat for 8 weeks,and the drug treatment group was treated by gavage for 8 weeks.After gavage for 8 weeks,the rats were weighed,and the rats were sacrificed under anesthesia and the materials were taken.Weigh the liver weight of each group of rats,and calculate the ratio of liver weight to body weight.Each group used automatic biochemical analyzer,microplate method and enzyme-linked immunoassay to detect blood lipids(TC/TG/HDL),liver function(ALT/AST/y-GGT),and oxidative stress(SOD/GSH),inflammatory factors(IL-1/IL-6)and fibrosis-related factors(PC-3/COL-4)indicators.Take out the liver tissue of each group,and conduct pathological section experiments on it.Firstly,we used the HE staining method to test and look into the pathological changes of the liver.And then,we used the oil red "O" staining method to observe the condition and extent of lipid deposition.Finally,use masson staining method to view that the early liver fibrosis degree.Western blotting and immunohistochemical methods were used to test fibrosis-related factors(ⅵα-SMA,COL-1,COL-3,MMP2,MMP9)and TGF-β1/Smad signaling pathway related factors(TGF-β1,Smad7,Smad2/3,P-Smad2/3)protein content in rat liver tissue.Western blotting was used to detect the protein expression levels of TGF-β1/Smad signaling pathway related factors(TGF-β1,Smad2/3,P-Smad2/3,Smad7)in the fibroblast model induced by TGF-β1 signaling pathway activator.Results:1.Five rats,given high-sugar and high-fat food about 18 weeks,were sacrificed randomly.To stain the oil red "O" of liver tissue,the staining displayed that the liver tissue presented the serious deposits;Masson staining showed that liver pools were found after staining.Diffuse fibrosis in the tube area is a sign of early liver fibrosis.This confirms the success of the NAFLD early liver fibrosis model.2.Comparing the model group with the normal control group,it show that the body weight and liver weight of rats increased markedly(P<0.01).although liver weight and body weight ratio are meaningless analysis from a statistical perspective,but the liver-to-body ratio has increased;The concentration of TC,TG,ALT,AST,γ-GGT,IL-1,IL-6,PC-3 and COL-4 in serum increased(P<0.05);the concentration of HDL SOD and GSH significantly reduce compared with the model group(P<0.01).Beside after give the different concentrations of ginsenoside Rgl and curcumin,the weight and body weight of rat decreased(P<0.05),and the liver weight/weight ratio decreased,but it was not statistically significant(P>0.05),serum TC,TG,ALT,AST,y-GGT,IL-1,IL-6,PC-3,COL-4 concentrations all decreased(P<0.05);HDL,SOD and GSH concentrations increased High(P<0.05).3.(1)The outcome of hematoxylin-eosin staining indicated that,in the model group,cells were disordered,edema and vacuoles were present,and a large number of pathological changes were observed compared with the normal group;and compared with the model group,treated with different concentrations of ginsenoside Rgl and curcumin Later,the pathological changes of the liver gradually improved.(2)The oil red "O" staining results suggest that:Compared with the normal control group,the model group has a large amount of lipid deposits in the liver;after different concentrations of drug intervention,the Rg1 dose group and the curcumin group are compared with the model group,The accumulation of lipids is significantly reduced.(3)Masson staining was performed.Compared with the normal control group,the model group showed early fibrosis in the portal area,and there was a tendency to bridge fibrosis.After treatment with different concentrations of ginsenoside Rgl and curcumin,liver tissue The degree of fibrosis was significantly reduced.4.Immunohistochemistry and Western blotting results demonstrated that in animal models,protein expression of key factors in fibrosis was α-SMA,COL-1,COL-3,MMP2 compared to the comparison group,and MMP9 was also included.And TGF-β1,Smad2/3 and P-Smad2/3 enhanced(P<0.05);compared with the model control group,after treatment with different concentrations of ginsenoside Rgl and curcumin,the expression of the above factors decreased(P<0.05).The expression of Smad7 in the model control group was lower than that in the normal control group(P<0.05).After treatment with different concentrations of ginsenoside Rgl and curcumin,the expression of Smad7 increased significantly(P<0.05).4.In the cell model,after treatment with different concentrations of ginsenoside Rgl and curcumin,compared with the model group,the expression of TGF-β1,Smad2/3,P-Smad2/3 decreased,and the expression of the negative regulatory factor Smad7 Compared with the model group,the amount showed a significantly higher trend,which was basically consistent with the results of the animal model.Conclusions:Ginsenoside Rg1 may increase the protein expression of Smad7,inhibit the activation of Smad2/3 to P-Smad2/3,affect the TGF-β1/Smad signaling pathway,and reduce the early liver fibrosis of NAFLD.In addition,ginsenoside Rg1 may reduce liver fibrosis mainly by inhibiting the production of collagen fibers rather than promoting its degradation in the early liver fibrosis of NAFLD. |
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