Study On The Relationship Between The Spred-2 Deficiency And Exacerbates Carbon Tetrachloride-induced Chronic Liver Injury And Fibrosis

Background:Liver fibrosis can be caused by kinds of factors, including hepatitis virus, alcoholic liver, fatty liver disease,autoimmune disease in clinic.Liver fibrosis is the abnormal proliferation of connective tissue in the liver, resulting in pathological process of diffuse extracellular cell matrix excessive deposition.At present, hepatic stellate cells are the main source of extracellular cell matrix.In the normal liver the hepatic stellate cells located in lacuna and main storage vitamin A. Accompanied by chronic injury, hepatic stellate cells were induced into fibroblast-like cells with the function of promote inflammation and fibrogenesis.Activated hepatic stellate cells migrated to the tissue repair and started proliferation, secreting large amounts of extracellular dell matrix and regulating the crack of ECM.Platelet derived growth factor (PDGF)produced by Kupffer cells is the main stimulation factor of hepatic stellate cell. MAPKs are involved in chronic liver injury and fibrosis.Mitogen activated protein kinase (MAPK)has a key role of transferring the extracellular signaling to the nucleus, so leading to many cellular responses including proliferation, differentiation, specific metabolism regulation pathway. Spred-2 can hinder the Ras protein dependent extracellular signal regulated kinase signal pathway. However, the regulatory mechanism remains unknown.Purpose:We want to detect the mRNA level of kinds of cytokines and chemokines such as TGF-b、IFN-g、CXCL2、CXCL9、CXCL10 and the fibrosis marker a-SMA、 Collagen-1 in the liver of Wild type mice and Speed-read KO mice treated by carbon tetrachloride and Olive oil. Western blot was used to detect the protein level of a-SMA and the Sircol was used to detect the protein level of Collagen-1. In order to explore the regulatory role of Spred-2 gene in CC14- induced chronic liver injury and fibrosis of in the Ras/ERK signaling pathway.Method:Spred-2 knockout mice and wild type mice littermate were used in the chronic liver injury model. Spred-2 KO mice and WT mice were treated with CC14 intraperitoneal injection three times a week at a dose of 2ml/kg (CC14:olive oil,1:4, [v:v]) according to body weight for 6 weeks. Control groups of both Spred-2 KO mice and C57BL/6J mice were treated with equal Olive oil. At 48 hours after last injection, mice were sacrificed, liver and blood were collected. The liver was devided into three parts. One part embeded in paraffin for dyeing, one part put into liquid nitrogen quickly for Western blot, and the third part put into RNA later reagent for Rt-PCR. Liver pathology was investigated by performing histology, alpha smooth muscle actin (a-SMA) immunoblotting, and real-time polymerase chain reaction (PCR).Results:The liver exposure to carbon tetrachloride for long time, and the liver was injury with large mounts of inflammation cells recruitment leading to hepatic cells injury、necrosis, and the destroy of liver structure.Compared with the wild type mice, Spred-2 KOmice showed significantly severe CCL4-induced chronic liver injury and fibrosis.Conclusion:Shown by histological and immunoblotting results, the deficiency of Spred-2gene increased themice liver injury and inflammation and Spred-2 gene deficiency exacerbates liver fibrosis. Spred-2 KO mice showed increased liver injury and fibrosis following CCL4 treatment. These results suggested that Spred-2 negatively regulates CCL4-induced chronic liver injury and fibrosis.