| 1.Purpose Cirrhosis results from different mechanisms of liver injury that lead to liver fibrosis,which is reversible.Then researchers have been stimulated to identify antifibrotic therapies.Pirfenidone(PFD)is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis,and it has been widely used as an oral formulation for systemic treatment of idiopathic pulmonary fibrosis.Our previous research has also shown that PFD can prevent the proliferation and activation of cultured HSC and reduce the synthesis of collagen I.In the present study,pirfenidone is to be perfused to the liver via the portal vein.This method will block collagen secretion,degrade ECM and even reverse fibrosis.This study would provide new insight into the mechanism of liver fibrosis and novel treatment for cirrhosis.2.Materials and methods 2.1 Effects of pirfenidone on cell cycle,proliferation,migration and differentiation of LX-2 cells: LX-2 cells were cultured and divided into two groups: the control group and the pirfenidone treated group.After treatment of LX-2 cells by pirfenidone,Flow cytometry analyzed cell cycle,CCK-8 assay kit evaluated cell proliferation,wound healing assay measured the changes of cell migration,western blot detected the expression of MCP-1 and qrt-PCR detected the expression of collagen type I.2.2 SD fibrotic rats induced by DEN were randomly divided into six groups:(1)Control group(n=5);(2)DEN control group(n=5);(3)DEN+PFD by gavge(n=5);(4)DEN+PFD by tail vein injection(n=5);(5)DEN+PFD infusion by the hepatic artery(HA)(n=5);(6)DEN+PFD infusion by the portal vein(PV)(n=5).At the end of 4 weeks,animals were anaesthetized(phenobarbitalsodium)and killed before the collection of blood samples and livers.Serum was separated from blood samples for liver function tests,such as serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST).The same lobe from each liver was fixed in buffered formalin for histological assessment of liver sections.The rest of the liver was frozen at-80°C until measurement of transforming growth factor-1(TGF-β1),Smad7,MCP-1,matrix metalloproteinase-2(MMP-2),tissue inhibitor of metalloproteinase 1(TIMP-1),heat shock protein47(HSP47),Collagen Type I.3.Results 3.1 After the treatment with pirfenidone,Flow cytometry indicated the percent of cells in G1 decreased compared with the control group(p<0.05).CCK-8 assay indicated that the concentration of pirfenidone presented obvious inhibiting effect(p<0.05).It demonstrated that the inhibition of pirfenidone on LX-2 cells was concentration dependent(p<0.05);Pirfenidone inhibited the migration abilities of LX-2 cells in a dose-dependent manner(p<0.01).Pirfenidone had a dose-dependent effect on the expressions of MCP-1 proteins in LX-2 cells(p<0.05).Compared with the control group,pirfenidone inhibited the expression of Collagen type I m RNA in LX-2 cells(p<0.01)and the inhibition had a dose-dependent trend(p<0.05).3.2.DEN treatment resulted in a significant increase in ALT and AST at the end of the study period(p<0.01);All the pirfenidone administrations significantly reduced both ALT and AST values(p<0.01).HE and Masson’s Staining:Compared with the DEN control group,PFD degraded the liver fibrosis of DEN-induced fibrosis models in rats and the PFD infusion by the portal vein group made the best work;The m RNA expression detection results by qrt-PCR: Compared with the control group,the expression levels of TGF-β1,MCP-1,TIMP-1,MMP-2,Collagen I m RNA were significantly higher in DEN group(p<0.01);Compared with the DEN group,the levels of them weresignificantly lower in the different pirfenidone delivery systems(p<0.05).The expression of MMP-2 was lowest in the PFD gavge group(p<0.01),but there was no significant difference compared with the PFD infusion by the portal vein group;and the expression of TGF-β1,MCP-1,TIMP-1,Collagen I m RNA was lowest in the PFD infusion by the portal vein group(p<0.01);The results detected by Western Blot:(1)Compared with the control group,the expression levels of HSP47 were significantly higher in DEN group(p<0.01);Compared with the DEN group,the levels of HSP47 were significantly lower in the different pirfenidone delivery systems(p<0.05),and the level were lowest both in the PFD infusion by the hepatic artery and portal vein groups(p<0.05);(2)Compared with the control group,the expression levels of Smad7 were significantly lower in DEN group(p<0.01);Compared with the DEN group,the levels of Smad7 were significantly higher in the different pirfenidone delivery systems(p<0.05),and the level were highest in the PFD infusion by the portal vein groups(p<0.05).4.Conclusions(1)After the treatment of pirfenidone on LX-2 cells,pirfenidone modified the cell cycle and inhibited cell proliferation,migration and fibrosis by degrading the extracellular matrix.(2)The infusion of PFD via the portal vein could significantly alleviate the inflammatory reaction,protect the hepatocyte cells and reduce the fibrosis degree in treated rats by down-regulating the expression of TGF-β1,MCP-1,TIMP-1,MMP-2 and HSP47,as well as up-regulateing the Smad7 protein level. |
