Mechanism Of TAK-242 Inhibition Of Septic Peritonitis In Mice Through Sele Regulation Of NF-κB Pathway

ObjectiveSeptic peritonitis is a common and severe disease in clinic,which can cause multiple organ failure and even death.Despite advances in antibiotic development,intensive care techniques and nutritional therapy in recent years,the mortality rate of septic peritonitis is still very high,so there is an urgent need to find new treatments.The aim of this study was to investigate the therapeutic effect of Resartovir(TAK-242)in septic peritonitis,screened and validated the core differentially expressed genes(DEGs)by transcriptomics,and performed structural prediction and molecular dynamics analysis of the core proteins.Subsequently,in vivo and in vitro peritonitis models were constructed,Sele was knocked down and overexpressed using adenoviral plasmid transfection,respectively,and the mechanism of TAK-242 inhibiting septic peritonitis in mice through Sele-regulated NF-κB pathway was revealed using flow cytometry and other methods.aiming to provide new ideas and means for the prevention and treatment of septic peritonitis.MethodsLPS-induced mouse peritonitis model was established,which were divided into Control group,a septic peritonitis model group(LPS group)and a TAK-242 intervention septic peritonitis model group(LPS+TAK-242 group),and the clinical symptom scores of mice in each group were analyzed,and the routine blood tests for leukocyte and neutrophil changes,the total number of leukocytes in the peritoneal lavage fluid of the mice were detected,the bacterial load in the peritoneal lavage fluid was observed by bacterial culture,and the changes of inflammatory cytokines in the serum and peritoneal lavage fluid were detected by the Enzyme Linked Immunosorbent Assay(ELISA)method.Subsequently,peritoneal tissues were isolated and transcriptome sequencing was performed.Differential genes were analyzed and obtained using DESeq2 R package(1.16.1);enrichment analysis was performed using KEGG and Metascape.Finally,10 core genes were screened in the TAK-242 intervention group,and the expression of the core genes was verified using the fluorescent q PCR method.And the three-dimensional structures of the core proteins were constructed by homology modeling.The three-dimensional structure of the core protein was optimized and obtained by molecular dynamics(300 ns).Finally,the binding mode between TAK-242 and the core protein was investigated by molecular docking,and the relative binding energy was calculated based on the binding site information.The expression of signaling pathway proteins clustered by the enriched pathway was analyzed by protein immunoblotting(Western blot,WB).Next,the molecular mechanism of TAK-242 inhibition of peritonitis was investigated.Firstly,peritonitis cell model was constructed in vitro,and adenoviral plasmid was used to transfect Sele for knockdown and overexpression,respectively;cell proliferation and apoptosis were detected,and expression of inflammatory factors in cells were performed in the Sele knockdown group,and inhibition of septic peritonitis in mice through the regulation of the NF-κB pathway by Sele was detected by Western blot.Cell proliferation and apoptosis,expression of inflammatory factors in cells were detected after TAK-242 intervention in the Sele overexpression group,and the inhibitory effect of TAK-242 on murine septic peritonitis through the regulation of NF-κB pathway by Sele was detected by Western blot.A septic peritonitis model was constructed in vivo,and the mice were divided into Control,LPS,LPS+TAK-242 and LPS+TAK-242+OE-Sele groups,and the proliferation and apoptosis of peritoneal tissues of the mice in each group were detected by immunofluorescence and TUNEL assays.Based on the specificity of Sele targets,we investigated inhibitor miRNA-like molecules other than small molecule compounds.Bioinformatics screening was utilized to obtain miR-31 that can regulate Sele,and a dual luciferase reporter gene assay was used to find that Sele may be a target gene of miR-31,and to study that exosomes produced by endothelial progenitor cells(EPCs-Exos)inhibit septic peritonitis by delivering miR-31.Firstly,a septic peritonitis model was constructed in vivo,and the mice were divided into a sham operation group,a septic peritonitis model group,an EPCs-ExosmiR-NC group,and an EPCs-ExosmiR-31 group,and the expression of inflammatory factors in the intraperitoneal lavage,the survival time of the mice,and the bacterial loads of the mice in the intraperitoneal lavage and peripheral blood were examined,respectively.Finally,peritonitis cell model was constructed in vitro,miR-31 was transfected using adenoviral plasmid,cell proliferation and apoptosis were detected after LPS stimulation,and the expression levels of Sele,Caspase-3,Bax and Bcl-2 were detected by Western blot.Results1.LPS-induced peritonitis model in mice was established.Compared with the control group,mice in the LPS-induced peritonitis model group showed significantly higher clinical symptom scores,significantly upregulated blood leukocyte count,neutrophil count and inflammatory cytokines with leukocyte count and bacterial load and etc.in the peritoneal lavage fluid;compared with the peritonitis model group,mice in the TAK-424-treated group showed significantly lower indexes than the peritonitis model group.2.Transcriptomic clues were obtained for the treatment of septic peritonitis in mice with TAK-242.A total of 4201 DEGs were obtained.30 DEGs were altered by TAK-242.The expression of the 10 core genes was verified by q PCR.The results showed that the gene expression changes of the 10 core genes were consistent with the sequencing results.3.Enrichment to obtain TAK-242 inhibits signaling pathways involved in septic peritonitis in mice.The analysis showed that the inhibition of LPS-induced peritonitis by TAK-242 mainly involved different pathways,and the inhibition of DEGs by TAK-242 mainly involved NF-κB signaling pathway and JAK-STAT signaling pathway.Western blot validation revealed that the experimental results were consistent with the above analysis.4.The analysis obtained the sites of interaction between TAK-242 and core proteins.The molecular docking of TAK-242 with Sele,Gzmb and eight other core proteins was performed to derive the three-dimensional structural map of the TAK-242-core protein complex,and the relative binding energy was calculated based on the binding site information.5.TAK-242 was found to inhibit septic peritonitis through the Sele regulation of NF-κB pathway.TAK-242 was found to act through the sele regulation of NF-κB pathway through in vivo experiments and in vitro experiments.6.Try to develop miRNA-like regulators(miR-31)targeting Sele.Further attempts to alleviate septic peritonitis using some biologic means of molecular targeting therapy.It was found that EPCs-Exos alleviated the symptoms of septic peritonitis by delivering miR-31,and Sele was found to be a target gene of miR-31 by experiments such as CCK-8 assay and Dual-luciferase reporter gene.Conclusions1.TAK-242 attenuates LPS-induced septic peritonitis in mice.2.Sele,a core gene of murine septic peritonitis,was obtained,and TAK-242 inhibited septic peritonitis by regulating the NF-κB pathway through Sele.3.Sele may be a target protein of TAK-242 and miR-31 has a therapeutic effect on septic peritonitis by targeting Sele.