The Protective Effect And Mechanism Of IRG1 In Acute Lung Epithelial Cell Injury In Sepsis Mice

Objective:Immune-responsive genel(IRG1)is a metabolism-related protein expressed in the mitochondrial matrix and involved in cellular metabolism.In the early stage,our group first discovered and reported the role of IRG1 in sepsis-mediated endotoxin tolerance.And subsequent studies by other scholars showed that it regulates cellular inflammation response in the process of sepsis and antiviral innate immunity,and has a protective effect on cells.Studies in macrophages have shown that IRG1 is expressed in the mitochondrial matrix and is involved in the regulation of the bactericidal activity of macrophages,the production of pro-inflammatory factors,and the inhibition of inflammasome activation.A study on the effect of IRG1 on hepatic ischemia-reperfusion injury found that primary hepatocytes from IRG1-deficient mice exhibited increased sensitivity to tumor necrosis factor α(TNF-α).The number of cell death increased significantly after TNF-α stimulation.At present,the research on IRG1 is mostly limited to its role in macrophages or hepatocytes,and its specific role and mechanism in epithelial cell injury are still unclear.Combined with multiple previous animal studies showing that itaconic acid,a metabolite of IRG1,can alleviate ischemia-reperfusion injury,we put forward the following hypothesis:IRG1 is up-regulated in acute lung epithelial injury,and mainly by mediating cellular metabolic remodeling,increasing ROS production,promoting itaconic acid synthesis,thereby reducing mortality of lung epithelial cells in response to inflammation.This study will carry out a series of experiments to verify this hypothesis:the research group mainly used IRG1 knockout mice to evaluate the effect of IRG1 on the viability of lung epithelial cells in a TNF-α-induced mouse lung epithelial cell apoptosis model,The effects of IRG1 and its metabolite itaconic acid on apoptosis of lung epithelial cells and related mechanisms were studied in vitro.This study is helpful to understand the pathophysiological process of pulmonary epithelial immune response,and provides ideas for the future development of protective drugs for sepsis-mediated acute pulmonary epithelial injury,which has certain theoretical significance and potential clinical application value.Methods:1、The mouse lung epithelial cell line(MLE-12)was used to stimulate the cells with different concentrations of TNF-α and IL-1β,and the expression level of IRG1 in lung epithelial cells was detected by qPCR.The cells were then stimulated with TNF-α in different phases,and the expression levels and trends of IRG1 in different phases were detected by qPCR and Western Blot analysis.2、IRG1 deficient mice were divided into two groups by radiation myeloablation and transplantation of wild-type mouse bone marrow cells to change the phenotype of immune cells in vivo,and injected TNF-α or PBS intraperitoneally respectively.The apoptosis level of lung epithelial cells was observed by immunofluorescence technique.In vitro,IRG1 small interfering RNA(siRNA)and overexpression plasmid were transfected in vitro(MLE-12),and cell apoptosis under different IRG1 expression levels was detected by CCK-8,flow cytometry(FACS),and Western Blot analysis.Levels and expression changes of apoptosisrelated proteins(caspase-3,PARP).3、MLE-12 was simultaneously transfected with IRG1-siRNA and treated with exogenous itaconic acid derivatives,and the changes of apoptosis level were detected by CCK-8 and flow cytometry(FACS)technology.4、MLE-12 was transfected by IRG1-siRNA,and the expression changes of phosphorylated IKKα/β,phosphorylated P65 and P65 were detected by Western Blot analysis.Results:1、The expression of IRG1 can be induced by TNF-α in lung epithelial cells,but the expression change is not obvious under the induction of IL-1β.Under a single TNF-ainduced lung epithelial cells,the expression of IRG1 gradually decreased after reaching a peak at 3-6 hours.2、The number of TUNEL(+)cells in the lung tissue of mice induced by TNF-α was significantly higher than that of the PBS group.Among the mice induced by TNF-α,the IRG1 gene-deficient mice after bone marrow transplantation The number of TUNEL(+)cells in the lung tissue of the mice was significantly higher than that of the wild-type mouse group.In vitro,the number of apoptotic cells in the IRG1 interference group was significantly increased compared with the control group,and treatment with the caspase inhibitor z-VAD could reverse the difference in apoptosis.The expression levels of caspase3 and PARP in the IRG1 overexpression group were significantly lower than those in the control group.3、MLE-12 was pretreated with 4-octyl-itaconic acid(OI),and the number and proportion of apoptotic cells in the IRG1-siRNA group were significantly higher than those in the control group.In normal MLE-12 cells pretreated with OI,the cell viability in the low concentration OI group was significantly increased compared with the solvent(DMSO)control group,but the cell viability in the high concentration OI group was lower than that in the DMSO group.4、MLE-12 was pretreated with CHX,and there was no significant difference in cell viability between the IRG1-siRNA group and the control group.The protein expression levels of phosphorylated IKKα/β and phosphorylated P65 in the IRG1-siRNA group were significantly lower than those in the control group.Conclusions:1、IRG1 is involved in TNF-α-mediated acute lung injury,and is induced by TNF-αand highly expressed in lung epithelial cells,while IL-1β cannot induce IRG1 expression changes.2、IRG1 plays a protective role in lung epithelial cell apoptosis in TNF-α-mediated acute lung injury.The apoptosis is related to the cell pathway mediated by apoptosis family caspase.Pretreatment with apoptosis inhibitor can greatly increase cell viability and reduce cell apoptosis rate.3、IRG1 has an anti-apoptotic effect on lung epithelial cells by participating in the synthesis of itaconic acid.Pretreatment with itaconic acid derivatives can reduce the apoptosis rate.However,high concentrations of itaconic acid derivatives may produce cytotoxic effects.4、IRG1 promotes the activation of NF-κB pathway by participating in the synthesis of endogenous itaconic acid in cells.At the same time,itaconic acid derivatives also have anti-apoptotic protective effects on cells,which may serve as potential targets for the treatment of tissue damage.