Molecular Mechanism Of IL-1Ra Protecting Hepatocytes Against ConA-induced Cell Apoptosis

Background and objectLiver injury refers to the phenomenon of inflammation or death of liver cells caused by toxic drugs,viruses,autoimmune defects,etc.The main forms of liver cell death are apoptosis and cell necrosis.ConA(Concanavalin A,ConA)is a polyclonal mitogen that can directly induce hepatocyte apoptosis.rhIL-1Ra has been shown to protect ConA-induced liver injury in animal models.Phosphorylation is one of the most widely studied and most concerned post-translational modifications.It can coordinate a variety of cellular functions,such as cell growth,differentiation,and apoptosis.It also plays an important role in liver cell proliferation and regeneration.FGL1(fibrinogen like 1,FGL1)is a member of the fibrinogen family and is secreted specifically by hepatocytes.Studies have shown that FGL1 is involved in the repair of hepatocyte damage by promoting the regeneration of damaged liver tissue and down regulating the expression of pro-apoptotic factors.This study uses ConA to induce AML12(alpha mouse liver 12,AML12)cell injury to establish a phosphorylated proteomics model,and to study the protective effect and mechanism of rhIL-1Ra on ConA-induced hepatocyte apoptosis.MethodsPhosphorylation was used to detect the changes in phosphorylationrelated proteins that were localized and quantified in the case of ConAinduced AML12 cell injury,and to determine the information of the phosphorylation modification sites.Constructing ConA-induced AML12 cell and HepG2 cell injury models;by using different concentrations of ConA,observing the morphological changes of the two cells and detecting cell survival rate and hepatocyte apoptosis,to examine rhIL-1Ra’s effect on ConA-induced liver cell apoptosis.Using the Dual Luciferase Reporter Gene Assay Kit,Real Time Polymerase Chain Reaction(Real Time PCR),and western blot to detect the transcription level,mRNA level,and protein level of FGL1 under the stimulation of rhIL-1Ra.FGL1 overexpression plasmid was constructed,transiently transfected into AML12 cells and to test the effect of FGL1 overexpression under ConA-induced injury conditions.ResultsThis study successfully constructed a phosphorylated proteomics model of ConA-induced AML12 cell injury,and quantitatively detected 82 phosphorylated modified peptides and 77 phosphorylated modified proteins.The biological processes,molecular functions,GO analysis of cellular components,and protein interaction network construction were performed on them,and specific phosphorylation modification site information was obtained.In the injury model of ConA to AML12 cells and HepG2 cells,it can be seen that ConA has a concentration-dependent killing inhibitory effect on both cells.rhIL-1Ra can significantly reduce the apoptosis rate of liver injury cells induced by ConA,improve the survival rate of liver cells,and effectively protect liver cells injury.Real Time PCR results showed that rhIL-1Ra can increase the level of FGL1 mRNA;dual luciferase reporter gene results showed that rhIL-1Ra increased the transcription level of FGL1 gene in AML12 cells.Western blot results showed that the expression of FGL1 in model cells increased with the increase of antagonist concentration.The over-expressed FGL1 plasmid also protected liver cells in injury models.These results show that both rhIL-1Ra and FGL1 can protect ConA-induced liver cell injury,and rhIL-1Ra can increase FGL1 levels in liver cells.