Mechanism Of Selenoprotein S Targeting Uba52 Regulates YAP-Induced M1 Polarization In The Pathogenesis Of Ulcerative Colitis In Mice

As an essential trace element in the body,selenium realizes its biological function mainly through binding to selenoproteins.Selenoprotein S（SelS）is one of the important members of the selenoprotein family,which has received extensive attention for its functions in antioxidation,regulation of endoplasmic reticulum stress,and maintenance of glucose metabolism homeostasis.SelS is a selenoproteins highly expressed in colon tissue,plays a crucial role in regulating inflammatory responses,but its specific role in ulcerative colitis（UC）remains unclear.UC is one of the main types of inflammatory bowel disease（IBD）,characterized by recurrent intestinal inflammation,which is prone to occur in various animals and humans,seriously affecting the health status and quality of life,and is becoming a major global public health problem.Studies indicate that macrophage polarization is an important link involved in the inflammatory response of UC,and that oxidative stress,intestinal epithelial cell death,and impaired barrier function can promote the development of UC.In vivo and in vitro studies have demonstrated that SelS knockdown exacerbates inflammatory responses.However,the mechanism of SelS regulating macrophage polarization and its role in the necroptosis of colonic epithelial cells in UC remains unknown.Therefore,this study determined the interaction between SelS and ubiquitin protein A52 residue ribosomal protein fusion product 1（Uba52）by co-immunoprecipitation combined with mass spectrometry（Co IP-MS）,immunofluorescence co-localization and protein-protein molecular docking.The GEO database was used to explore the correlation of SelS,Uba52 and Yes-associated protein（YAP）with immune cells such as macrophages in IBD.In the construction of 3.5%dextran sulfate（DSS）-induced SelS knockout（KO）mice acute UC model,2 mg/kg selenium intervention wild-type mouse UC model,interleukin-1β（IL-1β）stimulated SelS knockdown J774.1 macrophage model,the co-culture model of J774.1 macrophage and MCEC colonic epithelial cell.H&E staining,transmission electron microscopy,enzyme-linked immunosorbent assay（ELISA）,real-time fluorescence quantitative PCR（q RT-PCR）,Western blot,immunofluorescence and other technologies were applied to observe the pathological and morphological changes of colon,evaluate the level of oxidative stress,and detect the mRNA and protein levels of macrophage polarization,inflammatory cytokines,necriptosis and tight junction-related markers,aiming to clarify the effects of SelS targeted Uba52 regulating the expression of YAP on macrophage polarization and colonic epithelial cell injury,and reveal the mechanism of SelS in the process of UC.The main results are as follows:（1）The detection results of selenoprotein expression showed that the overall expression level of selenoprotein in the colon tissue of UC mice was higher than that of Ctrl mice,and SelS was the selenoprotein with the most obvious increase in expression.The results of clinical symptom observation showed that SelS KO decreased the feed intake,water intake and body weight of UC mice,and increased the scores of fecal properties,fecal occult blood and DAI（P<0.05）.The detection results of histopathological examination showed that the scores of inflammatory cell infiltration,infiltration range,hyperplasia,goblet cells loss,ulceration,granulation tissue,and glandular atrophy,as well as the damage scores of microvilli,intestinal epithelial cells,and tight junction in UC mice were higher than those of Ctrl mice（P<0.05）.SelS KO further increased the aforementioned injury scores in UC mice,and these results suggest that SelS KO aggravates colon injury.（2）Co IP-MS screening of interacting proteins of SelS showed that Uba52 was one of the interacting proteins of SelS;immunofluorescence colocalization results revealed that SelS and Uba52 co-localized in the cytoplasm;molecular docking results indicated that there were interaction sites between SelS and Uba52;the protein levels of SelS and Uba52 in the colon of UC mice and IL-1β-treated J774.1 cells were simultaneously increased（P<0.05）,and the protein levels of SelS and Uba52 in SelS KO UC mice and SelS knockdown J774.1 cells were significantly decreased（P<0.05）.These results suggest that SelS has a targeted regulatory effect on Uba52.（3）The detection results of YAP expression showed that compared with mice and J774.1 cells of Ctrl group,the mRNA and phosphorylated protein levels of MST1,LATS1 and YAP did not show significant changes in the colon of UC mice and IL-1β-treated J774.1 cells,but the YAP protein level increased significantly（P<0.05）,indicating that the expression of YAP protein is not regulated at the transcriptional level,nor does it depend on the negative regulation of a series of kinase cascade reactions upstream of the traditional Hippo signaling pathway,which may involve post-translational modification regulation.The increased level of YAP protein in SelS KO UC mice and SelS knockdown J774.1 cells was accompanied by the decrease of Uba52 protein level,and Uba52 knockdown in J774.1 cells down-regulated the ubiquitination of YAP protein and decreased the degradation rate of YAP protein,increased the protein stability of YAP（P<0.05）.These results suggest that targeting Uba52 by SelS promotes the ubiquitination and degradation of YAP protein in a proteasome-dependent manner and reduce the stability of YAP protein.（4）The detection results of macrophage polarization showed that SelS KO increased the number of CD86-labeled M1 macrophages and the mRNA levels of M1 macrophage markers（i NOS,TNF-α,IL-6,IL-12 and MCP-1）,while reducing the number of CD163-labeled M2 macrophages and the mRNA levels of M2 macrophage markers（CCL24,MRC1,Arg1,IL-4,IL-10 and Fizz1）（P<0.05）.Under IL-1βtreatment,the M1-type J774.1 cells were increased,while the M2-type J774.1 cells were decreased in the si SelS group compared with si NC group,but pretreatment with YAP inhibitor Verteporfin（VP）effectively reversed the polarization state of J774.1 cells（P<0.05）.These results suggest that SelS KO up-regulates YAP expression and promote the polarization of macrophages to M1 in the colon of UC mice.（5）The results of GEO data mining of IBD showed that the expression levels of SelS,Uba52,and YAP were higher in UC patients than normal subjects（P<0.05）,but had no significant change in CD patients.Pearson correlation analysis showed that SelS and Uba52 were closely related to a variety of immune cell infiltration in patients with UC and CD,including M1 and M2 macrophages.YAP was only correlated with immune cell infiltration in UC patients,but not with the level of immune cell infiltration in CD patients.The results of GO enrichment analysis showed that the gene aggregation composed of SelS,Uba52,YAP,and macrophage polarization markers was mainly involved in biological processes such as cytokine production involved in immune response,regulation of inflammatory response,cell response to oxidative stress,macrophage activation,and IκB kinase/NF-κB signaling.The results of KEGG enrichment analysis showed that the above gene set were significantly enriched in inflammatory bowel disease,Toll-like receptor signal pathway,and NOD-like receptor signaling pathway.（6）The detection results of oxidative stress level showed that SelS KO increased the content of malondialdehyde（MDA）,decreased the content of glutathione（GSH）,and the activities of glutathione peroxidase（GPX）,catalase（CAT）,total superoxide dismutase（T-SOD）and total antioxidant capacity（T-AOC）in the colon of UC mice（P<0.05）.Under IL-1βtreatment,co-culture with J774.1 cells increased the ROS level in MCEC cells,and decreased the mRNA levels of SOD1,SOD2,GST and CAT（P<0.05）.Co-culture with SelS knockdown J774.1 cells further exacerbated the changes of the aforementioned mRNA in MCEC cells（P<0.05）.However,VP pretreatment of J774.1 cells and antioxidant N-acetylcysteine（NAC）pretreatment of MCEC cells effectively reversed the mRNA changes of antioxidant genes in MCEC cells（P<0.05）.These results suggest that SelS KO up-regulates YAP expression,which promotes the polarization of M1 macrophages,and increases the level of oxidative stress in colonic epithelial cells.（7）The detection results of necroptosis showed that SelS KO increased the mRNA and protein levels of FADD,RIPK1,RIPK3,and MLKL in colon of SelS KO UC mice were increased（P<0.05）.Under IL-1βtreatment,co-culture with J774.1 cells increased the number of programmed necrotic cells in MCEC cells,and up-regulated the mRNA and protein expression of FADD,RIPK1,RIPK3,and MLKL（P<0.05）.Co-culture with SelS knockdown J774.1 cells further exacerbated the increase in the number of necroptosis cells and the changes of the above-mentioned gene expression in MCEC cells（P<0.05）.However,VP pretreatment of J774.1 cells and NAC pretreatment of MCEC cells effectively reversed the changes of necroptosis-related gene expression in MCEC cells（P<0.05）.These results suggest that SelS KO up-regulates YAP expression,which promotes the polarization of M1 macrophages,and induces necropyosis by increasing the level of oxidative stress in colonic epithelial cells.（8）The detection results of inflammatory reaction showed that the mRNA and protein expression of genes（NLRP3,ASC,Caspase-1,IL-1β,and IL-18）related to NF-κB/NLRP3 inflammatory signal pathway were significantly increased in the colon of SelS KO UC mice and IL-1β-treated SelS knockdown J774.1 cells（P<0.05）.The mRNA level of NF-κB p65 and the protein level of p-NF-κB p65was also significantly increased（P<0.05）.In addition,SelS KO increased the concentrations of TNF-α,IL-1β,IFN-γ,IL-6,and IL-17,and decreased the concentration of IL-10 in the colon of UC mice（P<0.05）.Meanwhile,increased the mRNA and protein expression levels of TNF-α,IL-1β,and IL-6,and decreased IL-10 expression level in the colon of UC mice（P<0.05）.Under the treatment of IL-1β,co-culture with J774.1 cells increased the mRNA and protein expression levels of TNF-α,IL-1β,and IL-6 in MCEC cells（P<0.05）,while decreasing the expression level of IL-10（P<0.05）.Co-culture with SelS knockdown J774.1 cells further aggravated the changes in inflammatory cytokine expression in MCEC cells（P<0.05）.However,VP pretreatment of J774.1 cells and NAC pretreatment of MCEC cells effectively reversed the changes in inflammatory cytokine expression in MCEC cells（P<0.05）.These results suggest that SelS KO up-regulates YAP expression,which promotes the polarization of M1macrophages and activates NF-κB/NLRP3 inflammatory signal pathway,thereby inducing inflammation through increasing the level of oxidative stress in colonic epithelial cells.（9）The detection results of tight junction showed that SelS KO aggravated the damage of tight junction structure in colon of UC mice,and decreased the mRNA and protein expression levels of Claudin1,Occludin,and ZO-1（P<0.05）.Under the treatment of IL-1β,the mRNA and protein expression levels of Claudin1,Occludin and ZO-1 in MCEC cells were down-regulated by co-culture with J774.1 cells（P<0.05）.Co-culture with SelS knockdown of J774.1 cells further aggravated the changes of the above gene expression in MCEC cells（P<0.05）.However,VP pretreatment of J774.1cells and NAC pretreatment of MCEC cells effectively reversed the changes of gene related to tight junction in MCEC cells（P<0.05）.These results suggest that SelS KO up-regulates the expression of YAP,promotes the polarization of M1 macrophages,and destroys the tight junction structure of colonic epithelial cells by increasing the level of oxidative stress.（10）The effects of different selenium sources on colon damage in UC mice showed that selenium supplementation supplementation increased feed intake,water intake,and body weight of UC mice（P<0.05）;decreased the fecal trait score,fecal occult blood score,DAI score,the damage score of microstructure and ultrastructure of colon（P<0.05）;increased the protein level of SelS and Uba52,decreased the protein level of YAP（P<0.05）;downregulated the expression of M1 macrophage markers（i NOS,TNF-α,IL-6,IL-12,MCP-1,and MIG）,and upregulated the expression of M2 macrophage markers（CCL24,MRC1,Arg1,IL-4,IL-10,and Fizz1）at mRNA level（P<0.05）;decreased MDA content,increased GSH content,enhanced GPX,CAT,T-SOD and T-AOC activity（P<0.05）;down-regulated the protein levels of p-NF-κB p65,NLRP3,ASC,Caspase-1,IL-1β,IL-18,TNF-α,and IL-6,and up-regulated the protein level of IL-10（P<0.05）;decreased the protein levels of FADD,RIPK1,RIPK3,and MLKL（P<0.05）;increased the protein levels of Claudin1,Occludin,and ZO-1（P<0.05）.These results suggest that selenium supplementation improved colonic damage in UC mice,and the order of different selenium sources from strong to weak was Nano-Se,Se Met,Se-Car and Na₂SeO₃.In summary,SelS KO promotes M1 macrophage polarization by targeting down regulation of Uba52to inhibit ubiquitin degradation of YAP protein,thereby enhancing oxidative stress,necroptosis,inflammatory factor release,and tight junction damage in colonic epithelial cells,and ultimately aggravating colon injury in UC.Selenium supplementation suppresses the M1 macrophages mediated necroptosis of colonic epithelial cells by up-regulating the expression of SelS,thus attenuating colon injury in UC.Therefore,SelS targeting Uba52 to regulate YAP-mediated M1 polarization has an important effect on colonic epithelial injury in UC.The results of this study reveal the new biological function of SelS,and provide evidence that SelS regulates macrophage polarization to reduce the injury of colonic epithelial cells,which provides a theoretical basis for the treatment of livestock UC and provides a reference for comparative medicine.