Preparation Of Recombinant Mouse IL-36 Mature Peptide And Its Preliminary Study On The Role In IBD

Objective:Inflammatory bowel disease(IBD)is generally believed to be caused by the interaction of various factors,such as infection,heredity and environment.IL-36 is one of the cytokines in the IL-1 family,which has four members,IL-36?,IL-36? and IL-36? are three agonizing ligands of IL-36 Receptor(IL-36R),Il-36 Receptor antagonist(IL-36Ra)can inhibit IL-36 R signal transmission,and IL-38 can also bind to IL-36 R.IL-36 excited after ligands with IL-36 R can raise IL-1 receptor related protein(IL-1 receptor accessory protein,IL-1RAcP)to form dimers downstream signal,in order to study on IL-36 to adjust the action of the intestinal mucosal immune mechanism,We detected the expression of IL-36 family cytokines,and found that the expressions of IL-36?,IL-36? and IL-36? were significantly increased in DSS intestinal inflammation in mice.To further explore the role of IL-36,we successfully prepared recombinant mice IL-36?,IL-36? and IL-36? mature peptide.In previous literature,IL-36? and IL-36? play a dual role in intestinal mucosal immunity in mice,and the specific mechanism is unknown,and IL-36? regulates intestinal mucosal immunity in mice is still unknow.After using IL-36? mature peptide to intervene the intestinal inflammation induced by DSS in mice,we found that IL-36? can aggravate the intestinal inflammation in mice through the innate immunity and adaptive immunity,laying a theoretical foundation for clinical trials and drug development.Methods: 1.Expression of IL-36?,IL-36? and IL-36? was detected after induction of 2.5% DSS intestinal inflammation in mice.2.The codon optimization and chemical synthesis of IL-36?,IL-36? and IL-36? ripened peptide genes were carried out,which were inserted into the prokaryotic expression vector pET28a(+),respectively to construct pET28a-IL-36?,pET28a-IL-36? and pET28a-IL-36? ripened peptide plasmids,which were transformed into escherichia coli BL21(DE3)and purified and prepared by IPTG-induced expression and nickel affinity chromatography.3.Recombinant IL-36? mature peptide was used to intervene 2.5% DSS-induced mouse IBD model,and body weight,survival rate,disease activity index,intestinal length and histopathology of the model mice were detected.4.On the fifth day after 2.5%DSS induced intestinal inflammation in mice,lamina propria lymphocyte(LPL)was isolated and extracted.It was cultured in vitro under the stimulation of PMA/ionomycin/BFA for 5h.Flow cytometer was applied to detect the proportion and absolute number of cells related to intestinal mucosal immune response.5.Place LPL in a 96-well plate with or without TCR stimulation;After 48 hours in vitro culture,the cells in each well were cultured with the supernatant,and the secretion of cytokines in the culture supernatant was detected by ELISA kit according to the instructions.Results: 1.The experimental colitis model of mice induced by DSS was successfully established.After intestinal inflammation was induced in mice,the expression levels of IL-36?,IL-36? and IL-36? were significantly increased,especially the IL-36? was positively correlated with the induction time.2.Mature peptides of recombinant mice IL-36?,IL-36? and IL-36? with a purity of up to 95% were successfully obtained.3.Compared with the control mice,The body weight and survival rate in the group of recombinant mice treated with IL-36? mature peptide decreased,and the disease activity index increased.Histopathology showed that the intestinal mucosal glands disappeared,and a large number of inflammatory cells were infiltrated,suggesting that IL-36? could aggravated the intestinal inflammation.4.Compared with the control mice,the responses of neutrophils and Th2 cells were enhanced and Treg cells was inhibited in the group of recombinant mice treated with IL-36? mature peptide.At the same time,the expression level of proinflammatory cytokines IL-6 and TNF-? in LPLculture superscript were increased,the IL-5 secreted by Th2 cells was increased,and IL-10 secreted by Treg cells was decreased.This indicated that IL-36? could affect the differentiation of innate immune cells and adaptive immune cells and the secretion of cytokines,leading to the increased susceptibility to DSS.Conclusion: 1.The expression of IL-36??IL-36? and IL-36? in DSS colitis was significantly increased,and the expression of IL-36? was positively correlated with the induction time of DSS.2.IL-36? can induce the secretion of pro-inflammatory cytokines,regulate the response of neutrophils and Th cells in the intestinal mucosa propria,and thus aggravate the intestinal inflammation in mice.