The Mechanism Of OSGEP Regulating Ferroptosis In Hepatic Ischemia-reperfusion Injury

Background:Hepatic ischemia-reperfusion injury(HIRI)refers to that when the blood supply to the ischemic liver is restored,the functional metabolic disorders and structural damage caused by reperfusion will be worsen.HIRI is a common surgical complication,which is common in partial hepatectomy,liver transplantation,hypovolemic shock and trauma.It is also the main cause of early liver dysfunction,acute liver failure and death after liver surgery or liver transplantation,seriously affecting the prognosis of patients,and it also brings a huge economic burden.As a new concept of cell death,more and more evidence indicated that ferroptosis may be involved in HIRI.Ferroptosis is an iron-dependent regulated form of cell death.Excessive lipid peroxidation causes the accumulation of a large amount of toxic reactive oxygen species and leads to cell death.It is different from other methods of cell death in gene,morphology and biochemistry.The OSGEP gene encodes O-sialoglycoprotein endopeptidase,which exists in a variety of organisms and has a high degree of homology.It is one of the subunits of the KEOPS(Kinase,Endopeptidase and Other Proteins of small Size)complex.Its expression in the liver is high,and a large number of evidences have shown that OSGEP is involved in the regulation of cell proliferation and mitochondrial metabolism,we speculate that it may be involved in the mechanism of HIRI.Extracellular signal-regulated kinase(ERK1/2)is a classical member of mitogen-activated protein kinase(MAPKs),and it participates in the regulation of cell growth,differentiation and death.Current research has confirmed that mitogen extracellular kinase(MEK1/2)is the upstream regulator.MAPKs are involved in the regulation of liver metabolism,and ERK1/2 plays a more important role in liver.Studies have shown that after liver-specific knockout of ERK1/2 in mice,lipid metabolism and glucose metabolism will be affected,leading to endoplasmic reticulum stress and insulin resistance.Purposes:Currently,the treatment for HIRI includes surgery,drug therapy,and gene therapy.However,because the pathological mechanism of HIRI is very complex,the above methods can’t effectively treat HIRI,and further research is urgently needed on the mechanism of HIRI.The purpose of this study was to explore whether OSGEP could regulate ferroptosis in HIRI,and whether this process was regulated by members of the MEK1/2-ERK1/2 pathway.It is hoped that this study will provide a new direction for the treatment of HIRI and effectively control the HIRI by controlling or monitoring OSGEP.Methods:1.An oxygen glucose deprivation/reperfusion(OGD/R)model was established in HepG2 cell line to simulate HIRI.The cell viability was measured using CCK8 kit,and the dynamic changes of OSGEP content in each group were detected by Western Blot.We select appropriate processing time for subsequent modeling when the survival rate was about 50%.2.HepG2 cell line was used for OGD/R treatment.Western Blot was used to detect the changes of expression levels and phosphorylation levels of members of MEK1/2-ERK1/2 pathway between different groups.The concentrations of ferrous ion and malondialdehyde between groups were detected by the kit to verify whether this pathway or ferroptosis was involved in the OGD/R model in HepG2.3.HepG2 cells with overexpression and knock-down of OSGEP gene and their negative control cells were used for OGD/R treatment,the cell viability of each group was measured by CCK8 kit,and the concentration of ferrous ion and malondialdehyde(MDA)among different groups were detected by the kit to verify whether OSGEP participated in the damage of OGD/R by regulating ferroptosis.4.HIRI model was established in C57BL/6 male mice.The automatic biochemical analyzer was used to detect the changes of ALT and AST concentration in serum.HE staining was used to determine the histopathological changes of the liver and to verify whether HIRI occurred.Western Blot was used to detect the dynamic changes of OSGEP content.We selected the time with the most severe injury for subsequent modeling.5.The kit was used to detect the changes in the concentration of ferrous ion and MDA in the liver respectively.Western Blot was used to detect the changes in the expression of MEK1/2-ERK1/2 pathway,to verify whether the pathway or ferroptosis was involved in HIRI.6.Mice with specific knockout of OSGEP gene in the liver were cultured for modeling.First,the changes of ALT and AST in serum were detected by automatic biochemical analyzer to determine whether OSGEP gene regulated HIRI.The kits were used to detect the changes in the concentration of ferrous ion and MDA in the liver.Western Blot was used to determine the changes in the expression of MEK1/2-ERK1/2pathway members.To investigate whether OSGEP mediate ferroptosis in HIRI by regulating the MEK1/2-ERK1/2 pathway.Results:1.OSGEP regulated OGD/R injury in HepG2 cells and HIRI in mouse,and the expression of OSGEP was negatively correlated with the severity of the injury.2.OSGEP inhibited ferroptosis which induced by model in vitro and in vivo.3.OSGEP participated in HIRI by regulating the phosphorylation of the MEK1/2-ERK1/2 signaling pathway.Conclusions:OSGEP inhibited ferroptosis in HIRI,which may be related to the regulation of phosphorylation in the MEK1/2-ERK1/2 signaling pathway.