Bioactive Compound C498-0670(C498) Alleviates Sepsis Via JAK/STAT And NFκB Signaling Pathways

Objective:Inflammation is an innate immune response to infection,noxious stimuli,tissue damage and trauma,especially in acute inflammation.However,if inflammation is overactivated,it can lead to organismal damage or even death.JAK/STAT and NFκB signaling pathways are two major inflammatory signaling pathways that are usually involved simultaneously in the body’s inflammatory response to bacterial or viral infections.The side effects of classical drugs such as NSAIDs and COX1/2 inhibitors and the limitations of the therapeutic effects of single-target drugs have made new multitargeted anti-inflammatory drugs a hot research topic for future new drug development.Over-activation of the prominent JAK/STAT and NFκB signaling pathways is associated with various immune-related diseases and mortality,suggesting an urgent need for the development of JAK/STAT and/or NFκB signaling pathway drugs.To this end,this study hopes to find bioactive drugs that can inhibit both JAK/STAT and NFκB signaling,leading to the development of drugs for the treatment of major inflammatory diseases.Methods:In this study,we pre-screened 18,840 compounds of the biologically active compound library,clinical compound library,approved drug library,and mini scaffold compound library from Target Mol.by SKA-Ⅱ containing STAT3 and NFκB dual-signal responses based high-throughput screening system and identified one bioactive compound,C498-0670(C498).At the molecular level,this study determined that C498 inhibited both JAK/STAT and NFκB signaling by WB and RT-PCR.In addition,in this study,mouse peritoneal macrophages of four groups,the negative control group,LPS positive control group,LPS+ C498 treatment group,and C498 treatment group,were subjected to transcriptome sequencing by extracting RNA samples.By transcriptome sequencing and bioinformatics methods,including KEGG analysis,GSEA,trend analysis,IPA and PPI network analysis,as well as multiple comparative analyses based on IPA involving canonical pathways,upstream regulators,biological functions,and disease related,etc.Therefore,we further validated the anti-inflammatory effect of C498 in vivo using an LPS-induced sepsis model in mice.The JAK/STAT and NFκB signaling pathways play important biological effects during septic shock,producing large amounts of proinflammatory cytokines such as IL-1,IL-6 and TNF-α.In LPS-induced septic shock model,The anti-inflammatory effect of C498 was further validated in an LPS-induced sepsis model in vivo in mice.In addition,the putative drug targets were identified using a SPR-HPLC-MS-based target trapping approach.Results:C498 showed better inhibitory effect in multiple screening processes.C498 inhibited STATs phosphorylation and p-IKKα/β induced by TNF-α in immortalized cell lines in vitro.And C498 could inhibit IL-6/IFN-β/IFN-γ induced STAT1/2/3 activation and the TNF-α and LPS induced IKK phosphorylation in mouse primary peritoneal macrophages in a dose dependent manner,while inhibiting LPS-induced inflammation-related factors gene expression such as IL-6,IL-1β,TNF-α,and CXCL1 detected by RT-PCR.All bioinformatics methods proved that the C498 has the general anti-inflammatory effects in depth.And in the disease/functional analysis,it predicted that C498 could alleviate sepsis/septic shock.The C498 ameliorated the decrease in body temperature,reduced liver and kidney injury and the release of inflammatory factors in blood,and inhibited the systemic inflammatory response induced by LPS,and the high affinity of JAK2(JAK/STAT signaling),NFKBIA(NFκB signaling)and IL-1β and NLRP1b(inflammasome signaling)for C498 was verified by a molecular docking approach.Conclusion:C498 inhibits the activation of STATs and p-IKKα/β in both the immortalized cell lines and primary peritoneal macrophages,while suppressing the expression of LPSinduced inflammatory mediators in vitro.In addition,the overall anti-inflammatory effects of C498 were thoroughly investigated.C498 was predicted to alleviate sepsis/septic shock,which was further verified in the LPS-induced mouse sepsis model in vivo.C498 reduced LPS-induced liver and kidney damages,myeloid cell infiltration,and pro-inflammatory cytokine and chemokine production in vivo.Furthermore,the high affinities of JAK2(JAK/STAT signaling),NFKBIA(NFκB signaling),and IL-1β,NLRP1b(inflammasome signaling)for C498 were verified.These results suggest that C498 can be used as a dual-target inhibitor of JAK/STAT and NFκB signaling pathways for the treatment of various inflammatory diseases,especially septic shock.