Regulation Of Gasdermin D On Dextran Sulfate Sodium-induced Colitis In Mice

Inflammatory bowel disease is a chronic recurrent disease affecting the gastrointestinal tract.It has gradual and destructive characteristics and seriously threatens the health of animals and humans.Fully understanding the molecular regulatory mechanism of the occurrence and development of enteritis is beneficial to the development of drugs for intervention.Gasdermin D?GSDMD?,also known as GSDMDC1 or DFNA5L,is mainly expressed on the surface of immune cells and intestinal mucosal epithelial cells.GSDMD protein-mediated pyroptosis was first reported in 2015.Pyroptosis is a programmed cell death dependent on activation of the inflammasome signal.The role of GSDMD in bowel diseases such as enteritis and bowel cancer is unclear.In this paper,the colitis induced by Dextran sulfate sodium?DSS?in mice to explore the regulation and mechanism of GSDMD on the occurrence of enteritis.Wild-type?WT?and GSDMD gene-deficient(GSDMD^-/-)mice received oral DSS to induce colitis.The results showed no significant difference in susceptibility to enteritis in the two genotype mice.The specific manifestations are the same trend in weight change,and there is no difference in survival?P?0.05?,pathological damage of the colon,secretion of inflammatory factors,inflammatory cell infiltration and intestinal mucosal damage.The intestinal microbiota plays an important role in the physiological and pathological activities of the local intestinal mucosa and distal organs.Under the influence of the host's endogenous signals or food and other exogenous signals,the intestinal microbiota structure succession to maintain microbiota homeostasis and compensate for internal and external signals.GSDMD as the executor of pyroptosis,is widely involved in inflammatory diseases,but why is there no difference in colon inflammation compared with wild-type mice after GSDMD gene-deficient.Is it related to changes in intestinal microbiota structure after GSDMD gene deletion?In order to determine the effect of GSDMD gene deletion on intestinal microbiota,we aseptically collected fresh feces from two genotypes of mice and 16S rRNA gene sequencing.The results showed that the GSDMD gene deletion caused changes in the intestinal microbial structure of mice.In order to further verify hypothesis,intestinal microbial clearance experiments and co-housing experiments were performed to investigate the effect of GSDMD gene and intestinal microbial changes on the occurrence of enteritis.The results showed that after commensal depleted?CD?,CD-GSDMD^-/-mice had more severe enteritis than CD-WT mice,which was reflected in a significant weight loss and a significant shortening of the colon?P?0.01?,and after co-housing the intestinal microbial of the two groups of mice converged(WT-co-h mice,GSDMD^-/-co-h mice),compared with GSDMD^-/-co-h mice the WT-co-h mice enteritis was significantly reduced.The specific manifestation is that there is almost no weight loss,extremely low mortality,colonic pathological damage is minor,less infiltration of inflammatory cells,and basically complete intestinal mucosal barrier,there is no difference in cytokine secretion in both colon tissues except KC?KC secretion is less in WT-co-h mice?P?0.05??.At the same time,it was observed that colitis in WT-co-h mice was significantly reduced compared to WT without co-housing,while colitis in GSDMD^-/-mice had no significant change before and after co-housing.The above results indicate that WT mice are less susceptible to DSS-induced enteritis after co-housing with GSDMD^-/-mice.Based on this phenomenon,we speculate that the special intestinal microbial formed by the GSDMD gene deletion will affect the original intestinal microbial structure of WT mice through co-housing,forming a new intestinal microbial environment.This new intestinal microbial may improve enteritis by inhibiting KC.To verify this conjecture,we performed the following group of mouse fecal microbial transplantation?FMT?experiments?WT-co-h mice fecal microbial transplanted into CD-WT mice:WT-co-h.B?CD-WT;WT mice fecal microbial transplanted into CD-WT mice:WT.B?CD-WT?.The results showed that WT.B?CD-WT mice were more susceptible to DSS-induced colitis,and WT-co-h intestinal bacteria showed an effect of inhibiting KC secretion.The above results show that compared with WT mice intestinal microbial the WT-co-h mice intestinal microbial can inhibit KC secretion and inhibit DSS-induced colitis.16S rRNA test results showed that intestinal microbial of WT mice and GSDMD^-/-mice converged after co-housing,but GSDMD^-/-co-h mice were more susceptible to DSS-induced colitis.This suggests that the inhibition of enteritis by WT-co-h mice intestinal microbial may require the coordination of GSDMD.Therefore,the following grouped FMT experiments were performed(WT-co-h.B?CD-WT;WT-co-h.B?CD-GSDMD^-/-)to investigate the anti-inflammatory effects of GSDMD signals on WT-co-h mice intestinal microbial influences.The results showed that after DSS-induced colitis,WT-co-h.B?CD-WT group mice showed less inflammation and less KC secretion than WT-co-h.B?CD-GSDMD^-/-group mice?P?0.001?.At the same time,Compared with WT.B?CD-GSDMD^-/-mice the WT-co-h.B?CD-GSDMD^-/-mice had less enteritis,and it was found that mice intestinal microbial still showed the effect of inhibiting KC to improve enteritis in GSDMD^-/-mice,but the final anti-inflammatory effect is weaker than when GSDMD signal is present.The above results indicate that the effect of intestinal microbial of WT-co-h mice on inhibiting KC to improve enteritis requires the GSDMD signal,but it does not depend entirely on this signal.In order to further explore the role of GSDMD in the anti-inflammatory process of intestinal bacteria in WT-co-h mice,Bone marrow-derived macrophages?BMDM?were infected with mice intestinal bacteria to detect KC secretion and GSDMD signal activation.Consistent with in vivo results,WT-co-h mice's intestinal bacteria significantly inhibited KC secretion compared with WT mice's intestinal bacteria?P?0.05?,and the inhibitory effect was more significant in the presence of GSDMD signals?P?0.01?.Western Blot test found that compared with WT mice's intestinal bacteria,WT-co-h mice's intestinal bacteria significantly activated the GSDMD signal,released the active N-terminus,and found the same phenomenon in vivo experiments.This indicates that WT-co-h mice's intestinal bacteria may inhibit KC secretion by activating GSDMD signal.The change of intestinal microbial structure is one of the important causes of IBD.Studies have found that the release of TNF-?causes extensive destruction of glycosaminoglycans in patients with IBD.Glycosaminoglycans usually attach to mucins and help form a protective barrier that separates bacteria from intestinal epithelium.The N-acetylglucosamine?NAG?subunit of the bacterial peptidoglycan?PGN?sugar backbone is a naturally occurring amino sugar precursor of synthetic epithelial glycosaminoglycans.Compared with other glycosaminoglycan precursors,IBD patients lack NAG.In vivo experiments have found that NAG can significantly reduce indomethacin-induced intestinal inflammation in mice,and that human peritoneal mesothelial cells have been treated with NAG and a dose-dependent increase in glycosaminoglycan synthesis has been found.At present,NAG has been used in the clinical treatment of IBD and has achieved significant results.Therefore,we speculate whether the inhibitory effect of WT-co-h intestinal microbial on enteritis is related to an increase in the abundance of Gram-positive bacteria in the intestine and an increase in NAG content?For this purpose,we tested the content of lipopolysaccharide?LPS?and peptidoglycan?PGN?in the intestines of mice in 4groups:WT,GSDMD^-/-,WT-co-h,and GSDMD^-/-co-h to determine changes in the abundance of Gram-negative and Gram-positive bacteria in the intestine.LPS is a surface marker of Gram-negative bacteria,PGN is the main component of the cell wall of Gram-positive bacteria,accounting for 80%of the dry weight of positive bacteria.The results showed that the PGN content in the intestine of WT-co-h mice was significantly higher than that in WT mice?P?0.001?,and there was no significant change in LPS content.LPS and NAG were used to stimulate WT and GSDMD^-/-mice BMDMs to detect KC secretion.It was found that NAG can inhibit KC secretion,and the inhibitory effect is more significant in the presence of GSDMD signal.Further testing revealed that NAG can significantly activate GSDMD signal compared with LPS,and the degree of activation was positively correlated with NAG concentration.At the same time,KC secretion was negatively correlated with the degree of GSDMD signal activation.The above results show that NAG can inhibit KC secretion and activate GSDMD signal.In summary,this study reveals that the GSDMD gene deletion causes changes in the intestinal microbial structure of mice,can affects the original intestinal microbial structure of WT mice by co-housing.Make WT mice form a new intestinal microbial structure with high abundance of Gram-positive bacteria.This new intestinal microbial may activate GSDMD signal through NAG,inhibit KC secretion and improve enteritis.