Selenoprotein O Targeting JAK1 Inducing Macrophage Extracellular Traps Release To Regulate The Development Of Ulcerative Colitis In Mice

Ulcerative colitis(UC)is an idiopathic inflammatory bowel disease that has become a great challenge for public health worldwide due to its long-term incurability and easy recurrence.Selenium(Se)is an essential micronutrient for the body,which plays a series of biological functions mainly through binding to selenoproteins.Although the pathogenesis of UC is regulated by multiple factors,dietary Se or selenoprotein deficiency exacerbates the disease process by affecting multiple signaling pathways involved in oxidative stress and inflammation.Macrophages play an important role in selenoprotein-mediated gastrointestinal inflammation and activation of host immune responses,and the release of macrophage extracellular traps(METs)has emerged as an important effector mechanism involved in the development and deterioration of the organism’s disease.Although there are few reports about selenoprotein O(SelO),SelO has been confirmed to have important antioxidant activity.The role played by SelO as well as METs release in UC has also not been reported.In this study,8-10 week old wild-type and SelO knockout C57BL/6 male mice were used to replicate the UC model by administering 2.5% dextran sulfate sodium(DSS)solution for 7days.The proteins interacting with SelO were screened and verified using co-immunoprecipitation(CO-IP)combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)detection,bioinformatics analysis,western blot(WB),fluorescence co-localization,and protein docking simulation;the histopathological changes of colon in mice were observed,and the susceptibility of SelO knockout mice to UC was discussed by disease activity index(DAI)recording,hematoxylin and eosin staining,histopathological analysis,and myeloperoxidase(MPO)activity detection;the changes in oxidative stress level,pyroptosis,macrophage recruitment,METs release,inflammatory cytokine secretion-related regulatory factor expressions and intestinal mucosal barrier damage in mice colon were detected by immunofluorescence staining,antioxidant function assay,alcian blueperiodic acid Schiff’s(AB/PAS)staining,reverse transcription quantitative real-time PCR(RTq PCR),WB and other experimental techniques.In addition,an model of mouse colonic mucosal epithelial cells co-treated with SelO silencing and protein Janus kinase 1(JAK1)inhibitor upadacitinib or oxidative stress inhibitor NAC was replicated in vitro,and a co-culture system with mouse macrophages RAW264.7 was constructed to investigate the effects of SelO silencing on mouse colonic mucosal epithelial cells,and the regulation of SelO-mediated mouse colonic mucosal epithelial cells on macrophages.The purpose of this study was to investigate the effects of SelO on the occurrence of UC in mice and the related regulatory mechanisms,and whether METs release is involved in driving the UC process.The results show as follows:(1)SelO knockout had no significant effect on changes in body weight and colon length in wild-type mice.In the DSS-induced UC model,SelO knockout reduced the survival rate of mice,significantly decreased body weight,shortened colon length,and led to a significant increase in DAI score.Additionally,SelO knockout resulted in partial loss of goblet cells and a small amount of inflammatory cell infiltration in colon of mice.In the DSS-induced UC model,the colonic mucosa was defective,the glandular structure was disordered,and a large number of goblet cells were lost along with a large number of inflammatory cells infiltration in wild-type mice;the colonic damage of SelO knockout mice was more serious,with almost no intact mucosal structure,a large number of epithelial exfoliation,deeper inflammatory cells infiltration,and the histopathological score and MPO activity were significantly higher than those of wild-type mice.These results suggested that SelO knockout increased the susceptibility of mice to UC and exacerbated colon damage of UC mice.(2)CO-IP combined with LC-MS/MS analysis identified JAK1 as a potential interaction protein of SelO,and WB,fluorescence co-localization and protein docking simulations confirmed that the two proteins interacted and formed stable interaction complexes on the cell membrane surface and in the cytoplasm.Compared with wild-type UC mice,the expression and phosphorylation levels of JAK1 and STAT3 were significantly increased in the colon of SelO knockout mice.In mouse colonic mucosal epithelial cells in vitro,low expression of SelO also significantly activated the JAK1/STAT3 pathway,while inhibition of JAK1 activity significantly hindered the activation of the pathway.These results suggested that SelO knockout targeted the activation of the JAK1/STAT3 cascade signaling to induce UC in mice.(3)SelO knockout significantly inhibited the activities of catalase(CAT),superoxide dismutase(SOD)and GPx,significantly decreased the content of GSH,increased the content of MDA,and decreased the total antioxidant capacity(T-AOC)of colon.In the DSS-induced UC model,SelO knockout exacerbated the imbalance of redox homeostasis in colon.In mouse colonic mucosal epithelial cells in vitro,low expression of SelO significantly inhibited the transcription of CAT,SODs,and GPxs genes and stimulated ROS production,while inhibition of JAK1 activity significantly alleviated oxidative stress induced by SelO knockout.These results suggested that SelO knockout aggravated the level of oxidative stress in UC mouse colon by targeting JAK1/STAT3 signal transduction.(4)SelO knockout significantly increased gene transcription and protein expression of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 pyroptosis-related regulators in colon.In the DSSinduced UC model,SelO knockout also significantly stimulated the assembly of NLRP3 inflammasome,the cleavage of GSDMD and the maturation of IL-1β and IL-18 cytokines in colon.In vitro,similar results to the above were obtained for SelO low expression in an mouse colonic mucosal epithelial cell assay,while inhibition of JAK1 effectively impeded the activation of pyroptosis.These results suggested that in UC mice,SelO knockout promoted colonic pyroptosis and inflammatory cytokines releases,and exacerbated colonic inflammatory injury,and the JAK1/STAT3 signaling was involved in the regulation of pyroptosis by SelO.(5)SelO knockout significantly stimulated the gene transcription of macrophage-associated chemokines of CCL2,CCL7,CX3CL1,CCL3 and CCL5 in mouse colon,but had no significant effect on the gene expression of CCL20.In the DSS-induced UC model,the expression of macrophage recruitment chemokines was more significantly increased in the colon of SelO knockout mice.In mouse colonic mucosal epithelial cells in vitro,low expression of SelO significantly stimulated the gene transcription of other chemokines except for CCL3,while co-treatment with JAK1 inhibitor significantly inhibited the upregulation of chemokine gene expression.These results suggested that SelO knockout targeted regulation of the JAK1/STAT3 axis to induce macrophage recruitment to the site of colon injury in UC mice.(6)SelO knockout significantly increased the expression of METs release regulators NOX2,PAD2 and PAD4,METs formation markers H3(cit H3),NE and MPO,and pro-cytokines TNF-α,IL-6 and IL-12,and significantly suppressed the expression of anti-inflammatory cytokine IL-10 in mouse colon.In the DSS-induced UC model,the SelO deficiency further stimulated the release of METs and the secretion of pro-inflammatory cytokines.SelO knockdown of mouse colonic mucosal epithelial cells significantly stimulated the release of METs and the secretion of pro-inflammatory cytokines from macrophages in the cell co-culture system,which was effectively alleviated by JAK1 inhibitor or NAC co-treatment.These results suggested that SelO knockout targeted the JAK1/STAT3 axis to regulate cellular oxidative stress and thus mediated the stimulation of colonic mucosal epithelial cells to macrophages,induced macrophages to release METs and secrete inflammatory cytokines,further amplified the inflammatory signals in colon and caused more severe inflammatory injury in the colon of UC mice.(7)SelO knockout significantly inhibited the expression of Mucin2,ZO-1,Occludin and Claudin1 in mouse colon.In the DSS-induced UC model,SelO deficiency promoted the loss of mucin and tight junction proteins.AB/PAS staining results further confirmed that the mucus on the colonic mucosal surface was disappeared,goblet cells were severely depleted,and mucin secretion in the tissues was significantly reduced in SelO knockout UC mice.The low expression of SelO significantly inhibited the synthesis of mucins and tight junction proteins in mouse colonic mucosal epithelial cells in vitro,while inhibition of JAK1 activity significantly restored their expression.These results suggested that SelO knockout targeted the JAK1/STAT3 pathway to inhibit mucin secretion in colon cells and disrupt the tight junctions between cells,and caused severe defects of the colonic mucus barrier in UC mice.In conclusion,SelO deletion targeted the activation of JAK1/STAT3 signal transduction,increased the level of oxidative stress in colon,promoted colonic pyroptosis,and induced macrophage recruitment to the site of tissue injury,stimulated macrophages to release METs and secrete inflammatory cytokines,amplified inflammatory signals,disrupted the colonic mucosal barrier,and aggravated inflammatory damage in colon of UC mice.This study enriched the physiopathological functions of SelO and provided an important theoretical basis for finding new therapeutic strategies and potential targets for UC,which is of great significance for safeguarding human health.