| Background:Crohn’s disease（CD）is a chronic,non-specific gastrointestinal disease with unknown etiology and pathogenesis,characterized by a variety of symptoms and relapses.At present,it is believed that the pathogenesis of CD is multifactorial.The interplay between genetic,environmental,microbial,and immune factors can cause the degeneration,necrosis,and loss of epithelial cells,which in turn leads to the destruction of intestinal barrier function.This damage stimulates the infiltration of inflammatory cells and the secretion of various pro-inflammatory cytokines,ultimately resulting in abnormal intestinal immunity and inflammatory responses.Further research on the pathogenesis of CD is of great significance for the early diagnosis and treatment of the disease.Damage-associated molecular patterns（DAMPs）are endogenous danger signal molecules that are released by tissue cells and activate inflammatory responses,including proteins,DNA,and metabolic molecules.The transmission of inflammatory signals mediated by DAMPs plays a crucial role in the development of CD.The initial injury factors acting on the intestinal tract cause the degeneration and necrosis of intestinal epithelial cells.These damaged cells release a large amount of DAMPs,which activate multiple innate immune signaling pathways by acting on specific pattern recognition receptors of inflammatory cells,such as macrophages,and subsequently induce the activation of inflammatory cells.This activation leads to the upregulation of pro-inflammatory genes,triggering intestinal inflammation and tissue damage.The NLRP3 inflammasome,as a crucial component of the innate immune system,is critical for the recognition of DAMPs.The investigation of DAMPs has the potential to reveal novel mechanisms underlying the occurrence and progression of CD and may aid in the identification of previously unknown therapeutic targets.Peroxiredoxin 1（Prdx1）is an intracellular antioxidant protein.Recent studies have demonstrated that Prdx1 can also be released outside of the cell and act as a DAMP,promoting inflammatory responses.Our previous study has shown that Prdx1 was released into the serum by damaged renal tubular epithelial cells during the early stage of acute kidney injury,which induced the infiltration and activation of inflammatory cells,leading to the secretion of a large number of pro-inflammatory cytokines.This ultimately promoted renal inflammatory responses,further aggravating the acute kidney injury.We also found that Prdx1 could act as a DAMP in acute liver injury and aggravate liver injury by promoting liver inflammation.However,the role of Prdx1 in intestinal inflammation in CD remains unknown.Objective:In this study,we conducted experiments on clinical samples collected from active CD patients and acute intestinal inflammation mouse models established using Prdx1 gene knockout(Prdx1-/-)mice.Additionally,in vitro experiments were performed on intestinal epithelial cells.We investigated the expression of Prdx1 in the intestinal tissues and serum of CD patients and mice with acute intestinal inflammation.In addition,we examined Prdx1 expression outside of the intestinal epithelial cells.Our study aimed to clarify the role of Prdx1 in the acute intestinal inflammation mouse model and explore its mechanism of action.This could potentially offer novel insights for the early diagnosis and clinical treatment of intestinal inflammation in CD.Methods:1.Investigating the Prdx1 expression in the intestinal tissues and serum of active CD patients and mice with acute intestinal inflammation,and examining the expression of Prdx1 outside of the intestinal epithelial cells.（1）Serum was collected from both healthy controls and active CD patients,and intestinal tissue samples from surgically resected lesions of active CD patients and adjacent normal intestinal tissue samples from patients with colon cancer were also obtained.The expression of Prdx1 in the intestinal tissues and serum of active CD patients was detected using ELISA,immunohistochemical（IHC）staining,Western blot,and RT-q PCR.（2）Acute intestinal inflammation mouse models were established using dextran sodium sulfate（DSS）and 2,4,6-trinitrobenzene sulfonic acid（TNBS）respectively.The expression of Prdx1 in the colon tissues and serum of mice with acute intestinal inflammation was assessed by IHC staining,Western blot,RT-q PCR and ELISA.（3）After stimulation with TNF-α,the expression of Prdx1 in the supernatants from intestinal epithelial cells was detected.2.Clarifying the role of Prdx1 in the acute intestinal inflammation mouse model.（1）Investigating the effect of Prdx1 knockout on acute intestinal inflammation in mice.Prdx1-/-mice were generated and used to examine the effect of Prdx1 deletion on acute intestinal inflammation in mice induced by DSS and TNBS.（2）Investigating the effect of exogenous supplementation of Prdx1 on acute intestinal inflammation in mice.In order to further validate the effect of Prdx1 on intestinal inflammation,recombinant Prdx1（r Prdx1）was administered to supplement Prdx1 in Prdx1-/-mice.3.The mechanism of Prdx1 in the acute intestinal inflammation mouse model.（1）Proteins were extracted from colon tissues of mice with acute intestinal inflammation induced by DSS and subjected to Western blot and ELISA analysis to investigate the effect of Prdx1 knockout on NLRP3 inflammasome activation.（2）To further validate the effect of Prdx1 on NLRP3 inflammasome activation and its possible activation mechanism,mouse primary peritoneal macrophages（PPMs）were isolated and stimulated with r Prdx1.Immunofluorescence staining,Western blot,and ELISA were used to analyze the results.Results:1.Prdx1 protein levels were significantly reduced in damaged intestinal tissue,while the concentration of Prdx1 was significantly increased in serum.（1）The protein levels of Prdx1 were found to decrease in the intestinal tissues of active CD patients,while its m RNA levels were increased.Additionally,the concentration of Prdx1 in serum was significantly increased and positively correlated with the degree of inflammation caused by the disease.（2）The protein levels of Prdx1 were found to decrease in the colon tissues of mice with acute intestinal inflammation,while its m RNA levels were increased.In addition,the concentration of Prdx1 in serum was significantly increased and positively correlated with the disease activity index.（3）After TNF-αstimulation,Prdx1 expression was detected in the supernatants of intestinal epithelial cells.2.Prdx1 promoted acute intestinal inflammation in mice.（1）Prdx1knockout resulted in a significant reduction in acute intestinal inflammation induced by both DSS and TNBS in mice,as evidenced by reduced body weight loss,decreased disease activity index,reduced colonic damage,and reduced mortality.This was accompanied by decreased macrophage infiltration and the expression of pro-inflammatory cytokines in colon tissue.（2）Conversely,exogenous supplementation of Prdx1 was found to significantly aggravate DSS-induced acute intestinal inflammation in Prdx1-/-mice,resulting in significant weight loss,worsened disease activity,increased colonic damage,as well as elevated expression levels of pro-inflammatory cytokines.3.Prdx1 triggered lysosomal rupture,leading to the activation of the NLRP3 inflammasome,which in turn promoted inflammatory responses.（1）Prdx1 knockout resulted in a significant reduction of NLRP3,a key component of the inflammasome,and IL-1β,which is an indicator of NLRP3 inflammasome activation,in the colon tissues of mice with acute intestinal inflammation induced by DSS.（2）r Prdx1 stimulation resulted in a significant increase in NLRP3 and IL-1βexpression in mouse PPMs,and inhibiting NLRP3 was found to significantly reduce the expression of IL-1βinduced by r Prdx1.Additionally,r Prdx1 treatment induced the rupture of lysosomes in PPMs,leading to the release of the hydrolase cathepsin D into the cytoplasm,which subsequently resulted in IL-1βexpression.Inhibiting lysosomal rupture was found to significantly reduce r Prdx1-induced IL-1βexpression.Conclusions:（1）In active CD patients and acute intestinal inflammation mouse models,Prdx1 protein levels were significantly reduced in damaged intestinal tissue,while the concentration of Prdx1was significantly increased in serum.Importantly,the elevated Prdx1 in serum likely originates from damaged intestinal epithelial cells;（2）Prdx1in serum may act as a DAMP in facilitating the inflammatory response and aggravating intestinal injury in acute intestinal inflammation;（3）Prdx1 may activate the NLRP3 inflammasome via the pathway of lysosomal rupture,thereby contributing to the promotion of inflammation. |
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