CpG-Oligodeoxynucleotides Alleviate Tert-butyl Hydroperoxide-Induced Macrophage Apoptosis By Regulating Mitochondrial Function And Suppressing ROS Production

BackgroundsSepsis is a systemic inflammatory response syndrome caused by infection and a life-threatening multiple organ dysfunction caused by the maladjustment of the body s own response to infection.The clinical mortality of sepsis is very high,which can develop into septic shock in severe cases.The potential circulatory disorders,cell and metabolic abnormalities brought by sepsis greatly increase the risk of death,which is still one of the main causes of global death.Oxidative stress caused by sepsis is closely related to mitochondrial dysfunction.Oligonucleotides containing CpG motifs have been shown to have potential immunotherapeutic efects.ObjectivesThe aim of present work is to explore the underlying mechanism of the cytoprotective function of CpG ODN by employing the oxidative stress modulation in immune cells.MethodsWe used t-BHP to stimulate the mice macrophages of RAW264.7 to simulate the environment of oxidative stress.CpG ODN(500 nm)was used to pretreat the cells.Cell counting kit 8 and apoptosis detection reagent were used in apoptosis assay.The apoptosis induced by t-BHP was observed by Annexin V/PI combined with fluorescence microscopy and image flow cytometry;the content of ROS in cells was detected by DCFHDA fluorescence probe;The protein phosphorylation levels of p38 mitogen activated protein kinase(p38 MAPK),c-jun amino terminal kinase(JNK),extracellular signal regulated kinase(ERK)and protein kinase B(Akt)were detected by western blotting.Meanwhile,to explore the cytoprotective effect mechanism of CpG ODN in oxidative stress-induced apoptosis,cells were pretreated with ERK and PI3K/Akt signal pathway related kinase inhibitors U0126 and LY294002;The change of mitochondrial membrane potential was detected by JC-1;We construct cecal ligation puncture(CLP)model of sepsis animal to further explore the cytoprotective role of CpG ODN in vivo;In addition,the effect of CpG ODN on phagocytosis of macrophages in RAW264.7 cells was detected by constructing and expressing E.coli-EGFP fusion protein.Results1.With the increasing of t-BHP concentration,cell viability gradually decreased,and the number of apoptotic cells were also increased.The expression of cleaved caspase-3,cleaved caspase-9 and cleaved PARP were increased,and the content of ROS in cells was also increased.2.Pretreatment with CpG ODN can reduce the proportion of apoptotic cells,decrease the production of ROS and reverse the change of mitochondrial membrane potential.3.The ERK and Akt signaling pathways are involved in the cytoprotection effect of CpG ODN.CpG ODN increases the phosphory lation level of ERK and Akt signaling pathway kinases protein and decreases the expression of NOX2 protein in cells.The protective effect of CpG ODN in mitigation of t-BHP induced apoptosis is dependent on the reduction of ROS.4.In vivo experiments,results show that CpG ODN can reduce ROS production in peritoneal macrophages of CLP mice,and also reduce AST and ALT content in serum of CLP mice,so as to reduce liver function damage caused by CLP;meanwhile,CpG ODN can improve the phagocytosis of macrophages.ConclusionsT-BHP can induce apoptosis of macrophages in RAW264.7 cells,which is mediated by mitochondrial pathway.CpG ODN can up regulate the phosphorylation of ERK and PIK/Akt signaling pathway kinase,then down regulate the expression of NOX2 protein in cells,reduce the production of ROS in cells,and thus reduce the apoptosis caused by t-BHP.Meanwhile,CpG ODN can reduce the production of ROS in peritoneal macrophages of CLP mice,reduce the liver damage of CLP mice,and enhance the phagocytosis ability of RAW264.7 cells,which shows obvious cytoprotective effect...