The Effect Of Wnt Signal Derived From Hepatocytes On The Progression Of Schistosomiasis Liver Fibrosis

Objective:The infection of Schistosoma japonicum can lead to the deposition of eggs in liver,which then result in liver fibrosis characterized by the formation of egg granuloma as a consequence of the pathological repair.During this process,various inflammatory cells are recruited and then drive the physiological microenvironment into a pro-fibrogenic status.which is usually modulated by inflammatory cytokines and contributes to the activation of hepatic stellate cells(HSCs).The activation of HSCs and excessive accumulation of extracellular matrix are the important aspects in the liver fibrosis formation.Liver parenchymal cells(hepatocytes)are the major cell population associated with the architecture and functions of liver.However,little was known about the contribution of hepatocytes in the development of schistosomiasis liver fibrosis.Wnt signal pathway has been demonstrated to be crucial in the maintenance of liver homeostasis and regulation of liver pathological processes.It has been identified that hepatocytes can express many Wnt signaling molecules,but it remains unclear about the effect of hepatocyte derived Wnt signal on the liver fibrosis in schistosomiasis.Our current studies investigated the hepatocellular expression of Wnt signaling molecules and the mechanism underlying the liver fibrosis caused by the schistosomiasis in mice.Methods:Mice were percutaneously infected with 25 cercariae of S.japonicum.Primary hepatocytes were isolated from normal mice and mice infected with S.japonicum for 6 weeks and 12 weeks.The Wnt related genes(Wntl,Wnt2,Wnt3,Wnt3a,Wnt5a,Fz1,Fz5.?-catenin,Sox9)and fibrotic genes(CTGF,TGF-?)were measured by Real-time PCR,the protein expression of Wnt3/3a,?-catenin and Sox9 was also analyzed by Western blot and immumohistochemical staining.Mice infected with S.japonicum for 4 weeks or 6 weeks were treated with Lv-DKK1-EGFP,Lv-Wnt3a-EGFP or Lv-EGFP by tail intravenous injection.Two weeks later,the fibrosis of liver tissues was measured by Masson trichrome staining,and the expression of Collagen al,Desmin and ?-SMA was detected by Western blot and immumohistochemical staining.The total RNA of liver tissues and hepatocyte from Lv-DKK1-EGFP and Lv-EGFP treated mice were analyzed for DKK1,CTGF,TGF-? and Sox9 by real-time PCR,the protein level of CTGF,TGF-? and ?-catenin were also detected using Western blot.The primary hepatocytes freshly isolated from normal mice and S.japonicum infected mice with or without Lv-DKK1-EGFP administration were co-cultured with normal primary HSCs in Trans-well system.The total mRNA were extracted from HSCs to detect the expression of Collagen ?1,?-SMA,TGF-? and Wnt targets(Axin2,Myc,Sox9 and Twistl)by real-time PCR,and the protein expression of Collagen al and a-SMA were also detected using Western blot.Results:The egg granuloma formation was observed after Schistosomiasis infection.Expression of Wnt genes(Wntl,Wnt2,Wnt3,Wnt3a,Wnt5a,Fzl,Fz5,Sox9)and fibrosis-related genes(CTGF and TGF-(3)were increased in hepatocytes isolated from mice infected with Schistosoma japonicum for 6weeks or 12weeks,no significant difference was observed at the mRNA level of ?-catenin.However,there was an elevation at the protein levels of Wnt3/3a,?-catenin and Sox9.The liver fibrosis was much more serious in Wnt3a-transfected mice and the expression of Collagen al,Desmin and a-SMA were upregulated.The DKK1-transfection alleviated fibrosis accompanied by a lower expression of Collagen al,Desmin and a-SMA in live.In addition,the expression of CTGF and TGF-? were downregulated in liver and hepatocyte as a result of DKK1-transfection.Compared to HSCs cultured alone or co-cultured with normal hepatocytes,mRNA expression of Collagen al,?-SMA,TGF-? and Wnt targets(Axin2,Myc,Sox9 and Twistl)were upregulated under the condition of co-culture with hepatocytes isolated from S.japonicum infected mice,and the higher protein expression of collagen al and a-SMA were also detected.However,co-culture with the hepatocytes from Lv-DKK1-EGFP-treated S.japonicum infected mice did not display the similar effect on primary HSCs.Conclusion:Our studis demonstrated that schistosome infection could promote the expression of Wnt ligands,receptors and downstream molecules in hepatocytes,suggesting that Wnt signal of hepatocyte is activated and the hepatocyte is an important source of Wnt signal during the development of liver fibrosis.The enhancement or suppression of-intrahepatic Wnt signal could exacerbate or alleviate liver fibrosis,these results demonstrate that Wnt signal derived from hepatocytes might contribute to the formation of schistosomal liver fibrosis.Furthermore,we also showed that the expression of fibrotic factors CTGF and TGF-P in hepatocytes increased after schistosome infection,which indicated that hepatocyte is a critical source of pro-fibrogenic signals.Co-culture experiment demonstrated that hepatocytes from S.japonicum infected mice accelerate the activation and collagen expression of HSCs relying on Wnt signal in vitro.Additionally,blocking of Wnt signal suppressed the expression of CTGF and TGF-(3 in hepatocytes,which imply that the Wnt signal derived from hepatocytes might contribute to the schistosomiasis liver fibrosis through the unregulated expression of CTGF and TGF-?.