Study of the roles of caspase-3 and nuclear factor kappa B in myenteric neurons in a P2X7 receptor knockout mouse model of ulcerative colitis

作     者：Henrique Inhauser Riceti Magalhães Felipe Alexandre Machado Roberta Figueiroa Souza Marcos Antônio Ferreira Caetano Vanessa Ribeiro Figliuolo Robson Coutinho-Silva Patricia Castelucci 

作者机构：Department of SurgerySchool of Veterinary Medicine and Animal SciencesUniversity of São PauloSão Paulo 05508-270Brazil Department of AnatomyUniversity of São PauloSão Paulo 05508-000Brazil Biophysics Institute Carlos Chagas FilhoFederal University of Rio de JaneiroRio de Janeiro 21941-902Brazil 

出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)

年 卷 期：2023年第29卷第22期

页      面：3440-3468页

核心收录：

学科分类：1002[医学-临床医学] 10[医学] 

基　　金：Supported by the National Council for Scientific and Technological Development,No.168015/2018-8 the São Paulo Research Foundation,No.2014/25927-2 and No.2018/07862-1. 

主　　题：Cell death Enteric nervous system Gastroenterology Inflammatory bowel diseases P2X7 receptor Purinergic signaling 

摘      要：BACKGROUND The literature indicates that the enteric nervous system is affected in inflammatory bowel diseases(IBDs)and that the P2X7 receptor triggers neuronal death.However,the mechanism by which enteric neurons are lost in IBDs is unknown.AIM To study the role of the caspase-3 and nuclear factor kappa B(NF-κB)pathways in myenteric neurons in a P2X7 receptor knockout(KO)mouse model of IBDs.METHODS Forty male wild-type(WT)C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid(colitis group).Mice in the sham groups were injected with vehicle.The mice were divided into eight groups(n=5):The WT sham 24 h and 4 d groups,the WT colitis 24 h and 4 d groups,the KO sham 24 h and 4 d groups,and the KO colitis 24 h and 4 d groups.The disease activity index(DAI)was analyzed,the distal colon was collected for immunohistochemistry analyses,and immunofluorescence was performed to identify neurons immunoreactive(ir)for calretinin,P2X7 receptor,cleaved caspase-3,total caspase-3,phospho-NF-κB,and total NF-κB.We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion,the neuronal profile area(μm^(2)),and corrected total cell fluorescence(CTCF).RESULTS Cells double labeled for calretinin and P2X7 receptor,cleaved caspase-3,total caspase-3,phospho-NF-κB,or total NF-κB were observed in the WT colitis 24 h and 4 d groups.The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(2.10±0.13 vs 3.33±0.17,P0.001;2.92±0.12 vs 3.70±0.11,P0.05),but was not significantly different between the KO groups.The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group(312.60±7.85 vs 278.41±6.65,P0.05),and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group(104.63±2.49 vs 117.41±1.14,P0.01).The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(19.49±0.35 vs 22.21±0.18,P0.001;20.35±0.14 vs 22.75±0.51,P0.001),and no P2X7 receptor-ir neurons were observed in the KO groups.Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group.The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(485949±14140 vs 371371±16426,P0.001;480381±11336 vs 378365±4053,P0.001),but was not significantly different between the KO groups.The total caspase-3 CTCF,phospho-NF-κB CTCF,and total NF-κB CTCF were not significantly different among the groups.The DAI was recovered in the KO groups.Furthermore,we demonstrated that the absence of the P2X7 receptor attenuated inflammatory infiltration,tissue damage,collagen deposition,and the decrease in the number of goblet cells in the distal colon.CONCLUSION Ulcerative colitis affects myenteric neurons in WT mice but has a weaker effect in P2X7 receptor KO mice,and neuronal death may be associated with P2X7 receptor-mediated caspase-3 activation.The P2X7 receptor can be a therapeutic target for IBDs.