Effect And Mechanism Of Purinergic Receptor P2X4 On Inflammatory Bowel Disease

Inflammatory bowel disease（IBD）is a nonspecific chronic inflammatory disease of the intestine,and its incidence has gradually increased in recent years with the development of society.The exact mechanism of IBD has not been elucidated,but it is believed to be the result of multiple factors such as inflammation,immune microenvironment and gut microbiota disturbance.ATP is the main energy source of life and an immune mediator in the process of inflammation.It can trigger a wide range of pathophysiological reactions in cells expressing various purinergic receptors.The P2 receptor family,which includes the ionotropic receptor P2X and the G protein-coupled receptor P2Y,is the family of ATP-activated purinergic receptors.Currently,seven P2X receptors have been cloned as P2X1-P2X7.P2X4,as a member of the purinergic receptor family,plays an important role in preventing infection,inflammation and organ damage.However,the relationship between the P2X4 receptor and the development of IBD,particularly regarding the changes in gut microbiota,is still unknown and requires further investigation.Therefore,in this study,colitis mouse model induced by Dextran Sulfate Sodium Salt（DSS）was used to investigate the potential role of P2X4 receptor in the occurrence and development of IBD and the possible mechanism of action.PurposeThe purpose of this study was to investigate the effect of P2X4 receptor alteration on acute inflammatory bowel disease and its possible mechanism.Method1.The effect of P2X4 receptor deletion on IBDThe expression difference of the P2RX4（P2rx4）gene between normal and IBD group was analyzed using the GEO database.Based on this,WT and P2rx4 knockout(P2rx4^-/-)mice were given3%DSS solution for 6 days to induce colitis.Thereafter,they were provided with regular drinking water to observe their survival rate over 21 days.Based on these results,DSS was freely available to mice for 6 days,then replaced with regular drinking water,and mice were killed on the 7th day.Body weight,DAI score and colon length were recorded during the period.In this study,H&E and PAS staining were used to evaluate the pathological changes of colorectal tissue.The levels of inflammatory factors in mouse plasma and colorectal tissue were determined by ELISA.The expression of MAPK signal pathway related proteins was detected by Western blot.The intestinal mucosal permeability of mice was detected by Evans Blue.Bacterial translocation was evaluated by plate coating method.16S r DNA sequencing was used to evaluate the changes of gut microbiota in cecum contents of mice.Meanwhile,we explored the interaction between gut microbiota changes and IBD in P2rx4^-/-mice using antibiotic mixture consumption,cohousing and fecal microbiota transplantation experiments.2.To explore the influence of Ivermectin（IVM）,an allosteric regulator of P2X4 receptor,on IBDC57BL/6 female WT mice were randomly divided into 4 groups:Control,DSS,DSS+2mg IVM group and DSS+5mg IVM group.During DSS induction,mice in the corresponding groups were given the dose of 2 mg/kg or 5 mg/kg IVM every other day.The changes of body weight,DAI score and colon length were observed.In this study,H&E and PAS staining were used to evaluate the pathological changes of colorectal tissue.The levels of inflammatory factors in mouse plasma and colorectal tissue were determined by ELISA.The expression of MAPK signal pathway related proteins was detected by Western blot.16S r DNA sequencing was used to evaluate the changes of gut microbiota in cecum contents of mice.Finally,P2rx4^-/-mice were given IVM at the dose of 2 mg/kg or 5 mg/kg to assess body weight,DAI score and colon length.Results1.GEO database analysis confirmed that the m RNA expression of P2RX4（P2rx4）was down-regulated in the colorectal tissues of IBD patients or DSS-induced IBD mice.However,this study found that the expression of P2X4 receptor protein increased first and then decreased with the extension of DSS induction time.2.P2X4 receptor deletion resulted in decreased survival in DSS-induced IBD mice and deterioration of related clinical markers,including body weight,DAI score,colon length,pathological inflammatory cell infiltration,and goblet cell number changes.Meanwhile,P2X4 receptor deletion increases intestinal permeability,bacterial migration,and the production of systemic and local inflammatory cytokines（IL-1β,IL-6,and TNF-α）.Further mechanism studies confirmed that this effect may be mediated by activating MAPK signaling pathway and gut microbiota imbalance.3.Ivermectin treatment was capable of reducing weight loss and DAI scores in IBD mice,improving the shortening of colon length induced by inflammation,enhancing the pathological score of colon tissue,increasing the number of calulet cells,and reducing the production of inflammatory factors.These effects may be mediated by inhibiting the MAPK signaling pathway and correcting the imbalance of gut microbiota.4.After P2X4 receptor deletion,ivermectin still plays a protective role in DSS-induced IBD.ConclusionThis study confirmed that P2X4 receptor deficiency exacerbates the development of inflammatory bowel disease in mice.Ivermectin,an allosteric regulator of P2X4 receptor,improved IBD symptoms in wild-type mice,but the protective effect was not through P2X4 receptor.The protective effects of both may be closely related to inflammation and changes in gut microbiota.