Effect Of TLR4-NLRP3-IL-1? Pathway On Cronobacter Sakazakii Induced Necrotizing Enterocolitis

Background and ObjectCronobacter sakazakii belongs to Enterobacteriaceae which is Gram-negative.Cronobacter sakazakii mainly cause bacterial meningitis and necrotizing enterocolitis in premature infants and low birth weight neonates,which are seriously endangering the life safety of newborns.The contaminated milk powder is the main route of infection.The research mainly focused on for the detection of Cronobacter sakazakii,for the cause of necrotizing enterocolitis without further detailed research report.NEC is an inflammatory disease and NLRs is involved in many kinds of intestinal inflammation.Therefore,it is speculated that NEC induced by CS is correlated with NLRs.And NLRP3 is the main form of action in NLRs.After the cell is stimulated,NLRP3 and Caspase-1 and ASC form a trimer.On the one hand,it stimulates the maturation of inflammatory factors and on the other hand,it promotes the activation of GSDMD,which mediates intestinal inflammation.In addition,TLRs,an adaptive immune response,is also closely related to inflammation.Especially,the combination of TLR4 and LPS mediates the downstream NF-?B,while NF-?B activates the expression of NLRP3,IL-1? and IL-18,thus regulating inflammsome.Therefore,there is a close relationship between inflammasome and TLR4,which may mediate the occurrence of inflammation.Therefore,through necrotizing enterocolitis caused by C.sakazakii in vitro and in vivo,this experiment study whether NLRP3 play role in necrotizing enterocolitis induced by C.sakazakii.At the same time,we studied the role of TLR4 in the regulation of NLRP3 inflammasome and provided the basis for further study on the pathogenesis of C.sakazakii.Methods(1)The C.sakazakii model group and the PBS control group were set up,the human colon cancer cell HT-29 and the neonatal rats of SD were used to construct the C.sakazakii induced model of NEC in vivo and in vitro.The pathogenicity of C.sakazakii was determined by trypan blue staining,PI staining,HE staining and cell cycle detection.(2)Western blot and Q-PCR were used to detect the NLRP3 related proteins and downstream inflammatory factors in the NEC model in vitro and in vivo and to determine the role of NLRP3 in C.sakazakii induced NEC.(3)Western blot and immunohistochemistry was used to detect TLR4 related molecules,to explore the relationship between TLR4 and NLRP3 in C.sakazakii induced NEC,and to determine whether the inflammasome NLRP3 could mediate NEC through the regulation of TLR4/NF-?B.Result1.In vitro,C.sakazakii could cause damage to HT-29 cells,and there was a significant difference between the control group and the control group.At the same time,the cell cycle results showed that C.sakazakii could inhibit the proliferation of cells.In intestinal tissue of model group,the intestinal cavity inflated and the intestinal tube expanded obviously,and the HE staining showed that the intestinal tissue structure was incomplete and the microvilli were thin.There was partial detachment and necrosis,and pathological damage was obvious.No obvious pathological changes were found in the control group.2.Western Blot results showed that the expression of NLRP3 related protein(NLRP3,ASC,Caspase-1),downstream inflammatory factor IL-lp and pyroprotein GSDMD-NT were up regulated in the model group,and there was a significant difference between the control group and the control group.At the same time,the transcriptional level of IL-1? mRNA was downregulated in the blockage group after blocking the NLRP3 in the model group.3.The results of Western Blot and immunohistochemistry showed that the expression of TLR4 and MyD88 in the model group was up regulated,and there was a significant difference between the control group and the control group.At the same time,after blocking the TLR4,the expression of Caspase-1 protein and the expression of IL-1 PmRNA in the blockage group decreased compared with the model group.ConclusionC.sakazakii and its bacterial related components stimulate the expression of TLR4 on the surface of intestinal epithelial cells,activate the MyD88 pathway to regulate the downstream NF-?B p65 into the nucleus,and induce the expression of the NLRP3 inflammatory proteins,promotes IL-1? maturation,activates GSDMD and induces cell pyroptosis,leading to NEC.