Expression And Mechanism Of Glutamine Synthetase In Hepatic Progenitor Cells During Progression Of Fibrosis

Background:Most types of chronic liver damage(eg.viral,alcoholic liver disease or non-alcoholic fatty liver disease)would impair the replicative capacity of most remnant hepatocytes,and induce an alternate regenerative process from hepatic progenitor cells(HPC).The activated HPC proliferates in the portal area and migrates to the hepatic parenchyma,where they further differentiate into hepatocytes or biliary cells to repopulate the liver parenchyma.Glutamine synthetase(GS)expression is also increased in early HPCs prior to morphological evidence of hepatocellular differentiation.GS-positive cells derived from HPC is closely related to the severity of liver fibrosis,and distribute around the cirrhotic nodules or wrapped in collagen fibers.Objective:We established a reliable and reproducible fibrosis model to observe the relation between HPC and hepatic fibrosis.Preliminary study of the expression and mechanism of glutamine synthetase in liver fibrosis progression,in order to provide a new target for the development of anti-fibrotic research.Method:1.Using the hepatotoxic compound thioacetamide(TAA)300 mg/L in drinking water to establish hepatic fibrosis model of mice.H&E,immunohistochemistry and transmission electron microscopy were used to observe the spatial distribution and quantitative changes of HPC and hepatic fibrosis in the liver of TAA model.At the same time,GS,CK19 and Ki67 immunohistochemical staining were combined to analyze the source of GS positive cells.2.In vitro,using RT-PCR and automatic protein analyzer,HepG2 cell line and LX-2 cell line were used in vitro experiments,to detect the expression changes of fibrosis-related genes and proteins by inhibiting GS activity.3.In vivo,the GS activity inhibitor MSO was used to inhibit GS activity in TAA model,and the changes of collagen fiber deposition,fibrosis related serum markers and gene expression were observed to preliminary study the effect of GS in the development of liver fibrosis.Results:1.In the TAA hepatic fibrosis model,with the increase of TAA induction time,the severity of liver fibrosis and the intensity of HPC increased simultaneously.Transmission electron microscopy showed that HPC were adjacent to activated hepatic stellate cells and formed direct cell-to-cell contact.2.The proliferating HPC in the liver parenchyma are concentrated in the pricentral area."intermediate hepatocytes" differentiated by HPC strongly express GS protein,and are located in the collagen fiber.There is a clear correlation between GS intensity and hepatic fibrosis.3.Inhibition of GS activity in HepG2 and LX-2 cell line in vitro,the expression of fibrosis related genes and protein decreased,such as TGF-β,Acta2 and CTGF.Consistent with in vitro results,in the experiment of the TAA model in vivo,liver fibrosis progresses slowly and the degree is significantly reduced after inhibiting GS activity.4.The possibility of GS-positive cells in the TAA model is that HPC differentiates under the regulation of the Hippo-YAP pathway.Conclusions:Chronic liver damage impaired replication and proliferation capacity of hepatocytes,and induced an alternate regenerative process from HPC.The activated HPC proliferates and migrates to the hepatic parenchyma and differentiates into hepatocytes under the control of Hippo-YAP pathway.Glutamine synthetase expression is increased in early HPCs prior to morphological evidence of hepatocellular differentiation which promote the occurrence and development of liver fibrosis to a certain extent.