Mechanisms And Effects Of CMPK2 In Sepsis-induced Lung Injury By Regulating The Expression Level Of TNF-α

ObjectiveAs a novel monophosphokinase localized on the mitochondrial membrane,cytidine/uridine monophosphate kinase 2（CMPK2）participates in the mitochondrial DNA remediation synthesis pathway.It has been shown to play an important role in the treatment of diseases such as bacterial infections,viral infections,malignant tumors,etc.Nevertheless,in the field of therapeutic research on sepsis,the treatment strategy with CMPK2 as the core is rarely mentioned,weakening the potential therapeutic value of this new target.The objective of this project is to explore the specific effects and potential signaling pathways of CMPK2 on sepsis injury.It aims to provide theoretical support and new drug therapy targets for sepsis organ injury prevention and treatment and sepsis immunotherapy program.Methods1.The sepsis model was established by intraperitoneal injection of LPS.Serum from wild-type and genetically defective mice was obtained and the expression level of different inflammatory factors in the serum were quantitatively analyzed by ELISA.Lung tissue of wild-type(CMPK2^+/+)and gene-deficient(CMPK2^-/-)mice was obtained,HE stain was used to observe lung tissue damage and inflammatory infiltration,and immunohistochemical（IHC）technology was used to observe inflammatory factor infiltration.After injection of lethal dosage of LPS into mice,the survival curve was plotted by recording the survival time of each mouse.2.Peritoneal macrophages（PMs）and bone marrow-derived macrophages（BMDMs）were obtained from CMPK2^+/+and CMPK2^-/-mice,LPS stimulation was given,and TNF-αexpression level was detected by q-PCR and ELISA respectively.Subsequently,the overexpression model was constructed.With the p CMV6-Entry plasmid as the carrier and CMPK2 overexpression amplification primers,CMPK2overexpressed plasmid was constructed.The constructed plasmid was amplified and then transfected to RAW264.7 cells.The stable overexpressed cell lines were screened by genetic mycin G418.The expression levels of LPS-induced TNF-αwere detected.3.Western Blot（WB）was used to detect the activation of signal molecules of JAK/STAT,MAPK-ERK,and NF-κB signaling pathways,and found that CMPK2upregulated the phosphorylation level of STAT1.Mass Spectrometry was used to find CMPK2-related protein（Pyruvate Kinase 2,PKM2）.Co-immunoprecipitation made the interaction between CMPK2 and PKM2 clear.Confocal laser scanning microscope（CLSM）was used to locate PKM2 and STAT1.Western Blot and Co-IP were used to further clarify the effect of CMPK2 on phosphorylation of PKM and STAT1,and the interaction between PKM and STAT1.Results1.Serum inflammatory factors:In the sepsis model constructed by LPS injection,the concentration of TNF-αin the serum of CMPK2^+/+mice was significantly higher than that of CMPK2^-/-mice from 3h to 9h.For IL-6 and IL-10,the difference in concentrations between CMPK2^+/+and CMPK2^-/-mice at the same time did not have a consistent trend in several experiments.2.The pathology of Lung tissue:HE staining results show that the inflammatory cell infiltration and alveolar wall destruction of lung tissue in CMPK2^+/+mice were more severe than those in CMPK2^-/-mice.The IHC results showed that the LPS group had more positive coloration of TNF-αand IL-6 in the lung tissue than in the saline control group.Among them,the positive coloring of TNF-αin CMPK2^+/+mice was more obvious than that in CMPK2^-/-mice.3.Macrophages:After the LPS stimulation,the concentration of TNF-αin PMs and BMDMs derived from CMPK2^+/+mice were significantly higher than those derived from CMPK2^-/-mice in the levels of protein and m RNA.4.CMPK2-FLAG-RAW264.7:After transfection of RAW264.7 cells with p CMV6-CMPK2-FLAG,we obtained stable expression strains by G418.After the LPS stimulation,CMPK2-FLAG-RAW264.7 expressed more TNF-αthan p CMV6-Entry-RAW264.7 in the levels of protein and m RNA.5.STAT1&PKM2:The WB results showed that CMPK2 upregulated the phosphorylation level of STAT1 after LPS stimulation.The Co-IP results showed no interaction between PKM2 and CMPK2.Mass spectrometry detection and analysis identified the relevant protein PKM2,and Co-IP results showed an interaction between PKM2 and CMPK2.CLSM results showed that after CMPK2 deficiency,PKM2 and STAT1 were less localized in the nucleus with or without LPS stimulation.After LPS stimulation,PKM2 and STAT1 are largely nucleated,and after CMPK2defect,PKM2 and STAT1 co-localization are reduced,and STAT1 is less nucleated.Further WB and Co-IP results showed that CMPK2 deficiency reduced phosphorylation and nucleation levels of PKM2 and STAT1 after LPS stimulation,suggesting that CMPK2 could promote the activation of PKM2 and STAT1.There was an interaction between PKM2 and STAT1,and the CMPK2 defect significantly reduced the interaction between PKM2 and STAT1.ConclusionCMPK2 promotes the phosphorylation and nucleation of STAT1 by regulating the phosphorylation of PKM2,initiates the inflammatory amplification effect of STAT1.This may be the mechanism for what we observed in the sepsis model that CMPK is able to upregulate the expression level of TNF-αand affect acute lung injury.