| Objectives To investigate the effect of glycyrrhizin arginine salt(GLY-ARG)on cholestatic liver fibrosis in rats.Methods 1 For in vivo experiments were as follows: Forty male SD rats were randomly divided into sham-operated group,model group,arginine group,high-dose or low-dose GLY-ARG group.The model group,arginine group,and arginine glycyrrhizic acid group were administered to hepatic duct ligation;arginine glycyrrhizic acid was orally administered by gavage to the rats at dosages of 0.15 and 0.075 g/kg/d on the second day after hepatic duct ligation;the arginine group was given intragastric administration of arginine(0.15 g/kg.d);the sham-operated group and model group were given intragastric administration of the same dose of saline.Then the general state of health of the rats was examined daily and the body weight was measured once every day for 14 days.Rats were sacrificed at the end of the study.Livers were removed and weighed.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),8-isopropanol,malondialdehyde(MDA),and liver tissue hydroxyproline levels were measured.HE staining was used to observe the pathological changes of the liver.Masson staining was used to observe the degree of hepatic fibrosis.Immunohistochemical staining was used to observe the expression of transforming growth factor-?1(TGF-?1)in liver tissue.The expression of ?-smooth muscle actin(?-SMA)in liver tissue was detected by immunofluorescence staining.In order to investigate the effect of glycyrrhizin arginine salt on hepatic fibrosis in cholestasis rats,we checked the matrix metalloproteinases-2 and 9(MMP-2 and MMP-9),nuclear factor-KB(p-NF-kB,NF-kB),tumor necrosis factor-?(TNF-?),TGF-?1 and ?-SMA protein expression levels in liver tissue using western blot analysis.2 For in vitro experiments were as follows: Cultured HepG2,LX-2 cell lines,rat primary hepatocytes and hepatic stellate cells were used.HepG2,LX-2,primary cultured hepatocytes and primary hepatic stellate cells were divided into control group,model group,arginine group,and GLY-ARG group.The effect of glycyrrhizin arginine salt on proliferation of HepG2 cells and primary hepatocytes after bile acid stimulation was detected by using the MTT assay.We next measured the arginine glycyrrhizin salt on TGF-?1 stimulated LX-2 cells,and expression of TGF-?1,?-SMA,MMP-2,and MMP-9 proteins in primary rat hepatic stellate cells(HSC)were evaluated by Western blot analyses.Results Compared with the model group,the rats in the glycyrrhizin arginine salt group grew faster and the liver weight and body weight ratio were significantly decreased.HE staining and Masson's staining showed that liver fibrosis was significantly reduced,and liver hydroxyproline content,bile salt pool were decreased in GLY-ARG group.In addition,serum total bilirubin,ALT,AST,8-isopropanol and the MDA content showed noticeable decrease in GLY-ARG group as well.Moreover,immunohistochemistry and immunofluorescence demonstrated that arginine glycyrrhizin can significantly reduce the expression of TGF-?1 and ?-SMA in liver tissue of cholestasis-induced fibrosis rats.Results from western blot indicated that MMP-2,MMP-9,p-NF-kB,NF-kB,TNF-?,TGF-?1 and ?-SMA protein expression were downregulated in the liver tissue of GLYARG group.Our in vitro experiment results showed that arginine glycyrrhizin salt could significantly improve the bile acid in HepG2 cells and primary hepatocyte in a dosedependent manner.Western blot analysis also indicated that GLY-ARG t group could down-regulate TGF-?1-induced LX-2 cells and rat primary hepatic stellate cell fibrosis related proteins,such as TGF-?1,?-SMA,MMP-2 and MMP-9 protein expression.Conclusions: Arginine glycyrrhizin significantly reduce the degree of hepatic fibrosis in rats with cholestasis.Its mechanism might be contributed to its inhibition of the activation of hepatic stellate cells,reduce oxidative stress and inflammatory response,which can promote the degradation of collagen fibers to inhibit liver fibrosis. |
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