| BackgroundUlcerative colitis（UC）is an inflammatory bowel disease.Clinical patients often present with abdominal pain,diarrhea,bloody stools and other symptoms,mainly in the rectum and distal colon.Studies have shown that the occurrence and development of UC are closely related to factors such as abnormal immune response,impaired intestinal mucosal barrier and disorder of intestinal flora.Inhibitor of differentiation 2（ID2）is a member of helix-loophelix（HLH）protein family,which regulates the differentiation and development of a variety of immune cells and bacterial colonization.The increased expression can reduce the differentiation and development of myeloid progenitor cells into neutrophils and promote the maturation of neutrophils.The specific deletion of Id2 gene in mice leads to ulcerative colitis,suggesting that ID2 protein plays an important role in the development of UC.Objective1.Recombinant human ID2（h ID2）protein was prepared to evaluate its effect on UC mice.2.To elucidate the protective mechanism of h ID2 protein on UC mice.Method1.Paraffin samples of colon tissue and the blood routine results of clinically diagnosed UC patients were collected（Ethical Review Approval No.XYLL-2021047）,and the expression of ID2 was detected by immunohistochemistry technology and the changes of immune cells were analyzed.2.Recombinant plasmid was constructed to prepare recombinant h ID2 protein and identified by western blot and mass spectrometry.3.Twenty-four male C57BL/6J mice aged 6-8 weeks were randomly divided into 4groups: healthy control group（CON）,disease model group（DSS）,recombinant h ID2 protein intervention group（DSS+h ID2）,and recombinant h ID2 protein control group（h ID2）.The following methods were used to evaluate the effects of recombinant h ID2 protein on DSS induced UC mice: 1)The body weight was monitored,disease activity index was calculated,colon length was measured,and H＆E staining was used to evaluate the pathological damage of colon tissue;2)RT-q PCR was used to detect the m RNA expression level of proinflammatory cytokines in colon tissues;3)The expression levels of mucin muc-2 and tight junction proteins（Claudin-1 and ZO-1）were detected by RT-q PCR and Western blot respectively;4)The intestinal mucosal barrier function of mice was detected by FITCDextran and attenuated Salmonella invasion assay;5)Flow cytometry was used to detect the number of immune cells in spleen,peripheral blood and lamina propria of colonic mucosa;6)The infiltration of inflammatory cells in colon tissue was detected by immunofluorescence.4.To explore the protective mechanism of recombinant h ID2 protein from three possible aspects of intestinal flora,intestinal epithelial cells and immune cells: 1)The intestinal flora structure of mice in each group was detected by 16 S r DNA high-throughput sequencing;2)In vitro cell culture experiment to evaluate the effect of recombinant h ID2 protein on DSS-induced injury of human clonal colon adenocarcinoma cell（Caco2）;3)Flow cytometry,RT-q PCR,western blot and elimination of neutrophil by anti-αGr-1 antibody were used to explore the role of neutrophil in alleviating UC in mice with recombinant h ID2 protein.Result1.The expression of ID2 was significantly reduced in colon tissues of UC patients and the number of neutrophils in peripheral blood of patients with UC was significantly increased（P<0.001）.2.The relative molecular weight of recombinant human ID2 protein is about 15 k Da,and its nucleotide and amino acid sequences are highly similar to mice,reaching 95% and99%,respectively.3.Recombinant h ID2 protein effectively alleviated the symptoms of UC mice,including increased body weight（P < 0.01）,decreased DAI（P < 0.001）,increased colon length（P < 0.05）and reduced pathological injury of colon tissue（P < 0.001）.4.Recombinant h ID2 protein significantly reduced the secretion of pro-inflammatory cytokines（TNF-α,IL-6,IL-1β,IL-17,IL-2 and IFN-γ）in colon tissues of UC mice.5.Recombinant h ID2 protein protected colonic mucosal barrier function of UC mice by increasing m RNA relative abundance of Muc2（P < 0.05）and the expression of tight junction proteins of Claudin-1 and ZO-1（P < 0.05）,reduced the content of FITC-dextran in serum（P < 0.001）and decreased the number of attenuated Salmonella in colon tissue（P <0.05）.6.Recombinant h ID2 protein increased the number of Treg cells in spleen of UC mice（P < 0.001）,reduced the number of Th17 cells（P < 0.05）to maintain the balance of Treg/Th17 cells.7.Recombinant h ID2 protein reduced the number of neutrophils in lamina propria of colonic mucosa（P < 0.001）and peripheral blood（P < 0.01）in UC mice.8.Intestinal flora of UC mice was disorganized,and DSS+h ID2 group and DSS group formed clusters,which were far away from CON and ID2 groups by principal component analysis,and recombinant h ID2 protein could not change the decrease of α diversity index and the increase of Firmicute/Bacteroidetes ratio（P > 0.05）,indicating that the protective effect of recombinant h ID2 protein on UC mice did not play by affecting intestinal floral community.9.Recombinant h ID2 protein did not protect against DSS damage to human colorectal adenocarcinoma cell（Caco2）（P > 0.05）.10.Recombinant h ID2 protein,alone or in combination with NF-κB inhibitor（PDTC）,inhibited the activation of IKKβ-IκBα-NF-κB pathway in neutrophils and reduced the secretion of pro-inflammatory cytokines,suggesting that recombinant h ID2 protein alleviated symptoms of UC mice mainly by targeting neutrophils.ConclusionRecombinant h ID2 protein inhibited the activation of IKKβ-IκBα-NF-κB signaling pathway and reduced the secretion of pro-inflammatory cytokines in neutrophils,thus effectively alleviating DSS-induced UC mice,suggesting that recombinant h ID2 protein has the potential as a treatment for UC. |
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