Studies On Themolecular Immunological Mechanism Of NLRP6 Inflammasome Activation Regulated By Lactobacillus Plantarum

Inflammasome is a novel discovered intracellular polyprotein complex which consist of pattern-recognition receptor(PRR),caspase-1,and apoptosis associated speck-like protein cotaining a CARD.NOD-like receptors(NLRs)are activated by agonists and assemble into a macromolecular complex via receptor oligotrophy and homophilic interactions.Caspase-1 precursors were recruited to the macromolecular complex,which were activated to cleave IL-1?and IL-18 precursors into mature IL-1?and IL-18 to achieve their stable and active state,promoting their secretion to the extracellular and exerting biological effects.One functions of the probiotic is to prevent intestinal inflammatory reaction to prevent disease by regulating the balance of pro-inflammatory and anti-inflammatory cytokines.Lactobacillus plantarum can regulate the composition of the host intestinal microecology,adjust the relationship between bacterial flora,form a biological barrier and exert mucosal immune function.Enormous experimental data indicate that L.plantarum may play an important role in inflammation,and participate in host anti-infection and anti-tumor immune response by enhancing phagocyte function,inducing the release of cytokines and nitric oxide and other immune mediators.Previous studies have confirmed that members of the Nod-like receptor(NLR)family play an important role in relieving the host of chemical-induced colitis.In this study,a model of colitis disease was modelled with dextran sodium sulfate(DSS),then L.plantarum was used to intervene in the above colitis model to further elucidate the mechanism whether L.plantarum were involved in regulating the release of Pro-IL-1?/Pro-IL-18,induction of inflammation signaling,shearing,maturation and release to regulate the inflammatory response.The main research contents and results are as follows:(1)Effect of NLRP6 on L.plantarum intervention in mice colitisIn this experiment,C57B/L6 mice were free to drink 4%DSS to establish an acute colitis model,to explore the immunomodulatory function of L.plantarum on mouse colitis,and to evaluate the prevention and treatment effects of L.plantarum in DSS model mice.The experimental results showed that compared with the DSS model group,body weight of mouse in the L.plantarum prophylaxis and treatment groups was significantly higher,DAI was significantly decreased,the length of the colon was longer,and the microscopic pathological changes of the colon tissue were significantly relieved.Levels of CD3~+CD4~+IL-4~+and CD3~+CD4~+IL-10~+T cells in mesenteric lymph nodes(MLNs)increased.IL-18 in the colon tissue was increased,and IL-1?was decreased.NLRP6 protein expression in the colon tissue increased during treatment of L.plantarum.The above results showed that L.plantarum can activate NLRP6 inflammasome in the intestinal tract to inhibit IL-1?secretion and promote IL-18 secretion,which indicated that L.plantarum plays an important role in acute colitis model.(2)L.plantarum-induced NLRP6 inflammasome activation against colitisTo reveal the biological function of NLRP6 protein regulated by L.plantarum,In this experiment,the primary C57B/L6 mouse bone marrow-derived macrophages were used as a research model to analyze the effect of L.plantarum on NF-?B signaling pathway.Whether L.plantarum was involved in regulation of NLRP6inflammasome after LPS activated the first signal of an inflammasome and the effects of autophagy signal and nitric oxide synthase(iNOS)on inflammasome signal.The experimental results of fluorescence quantitative PCR,immunoblotting and immunofluorescence analysis showed that L.plantarum could activate LC3?and nitric oxide synthase,and NLRP6 was strongly expressed in colonic tissues and mainly expressed in inflammatory cells after L.plantarum treatment.Experiments showed that L.plantarum could induce NLRP6 expression,inhibit the phosphorylation of I?B?and thereby inhibit the expression of NF-?B.L.plantarum can activate NLRP6 inflammasome after the first signal of NLRP6 inflammasome activated by LPS,which may inhibit the maturation of IL-1?,cleave and release mature IL-18,and the whole process rest with NLRP6 signaling.The results showed that the peptidoglycan,a component of L.plantarum cytodrem,may be the active component of inflammasome.In summary,this study found that L.plantarum can induce the activation of NLRP6 inflammasome thereby inhibit the secretion of IL-1?and enhance the secretion of IL-18,and relieve DSS-induced colitis inflammation.The experimental results of this paper preliminarily revealed the molecular immune mechanism of L.plantarum to prevent and treat colitis in mice,which has important theoretical value and application value.