The Changes Of NLRP3 And The Effects On NF-?B Signaling Pathway In Porcine Alveolar Macrophages Caused By The Lipopolysaccharide Of Haemophilus Parasuis

Haemophilus parasuis(HPS)is a Gram-negative bacterium that is widespread in the upper respiratory tract of pigs.It is the causative agent of pig Gracer's Disease,which is characterized by cellulosic serosal inflammation,meningitis and multiple arthritis.It mainly affects 5-8 weeks old nursing pigs,causing serious economic losses to the pig industry.Porcine alveolar macrophage(PAM)plays an important role in the natural immunity of the lungs and is the first line of defense against the invasion of foreign pathogenic microorganisms in the respiratory tract of mammals.It can phagocytize and kill pathogens,release cytokines and chemokines and other immune cells to the infection site to play immune functionThe purpose of this project is to study the extraction of LPS from Haemophilus parasuis by the hot phenol water method,and then use the sulfuric acid-phenol method and ultraviolet spectrophotometry to determine the polysaccharide and nucleic acid content in the LPS extract.Identify the extract.The extracted LPS was applied to porcine alveolar macrophages to determine the effect of NLRP3 inhibitors on the expression of IL-1? in Haemophilus parasuis LPS-infected porcine alveolar macrophages,and to explore the activation of TLR4 receptor by Haemophilus parasuis LPS In turn,it induces changes in the NF-?B signaling pathway.The results are as follows:1.Extraction,purification and identification of lipopolysaccharides from haemophilus parasporusThrough hot phenol water extraction pig serum LZ type 5 strains of haemophilus LPS,and using the method of enzymatic hydrolysis and super purified HPS LPS extract,extracted from 2.46 g HPS bacteria precipitation purified LPS 60 mg,the average yield of 2.44%.The results of phenol with sulfuric acid method showed that the LPS extractsdid not contain a reductive monosaccharides,and the concentrations of polysaccharide is 176 mu g/mL.Thecontent of nucleic acid in the LPS extract was 430.1?g/mL by trace ultraviolet spectrophotometer determination.The silver staining identification results showed that the molecular weight of the purified HPS LPS extracts ranged from 35 kDa to 160 kDa.2.Effects of NLRP3 inhibitor on IL-1? expression in PAM cells infected with lipopolysaccharide of Haemophilus parasuisAfter HPS(LZ strain)LPS treatment,the NLRP3 inflammatory complex was significantly activated,while the activation of NLRP3 inflammatory complex was significantly inhibited in the cells treated with MCC950 inhibitor,which also inhibited the expression of IL-1?.3.Effect of LPS activation of TLR4 on NF-?B signaling pathway activationAfter LPS stimulation and TLR4 siRNA treatment,compared with uninfected control PAM cells,the concentration of I?B and p65 increased while the concentration of p-I?B and p-p65 decreased in the cytoplasm,and the concentration of p-p65 in the nucleus also increased.The results showed that after TLR4 was induced,the LPS of Haemophilus parasuis stimulated PAM cells to activate TLR4-induced NF-?B pathway and phosphorylated I?B and part of NF-?B,and partially dissociated NF-?B rapidly transferred I?B to the nucleus,inducting the expression of related genes.In summary,Haemophilus parasuis lipopolysaccharides stimulate secretion of IL-1? and activation of NLRP3 inflammatory complex in porcine alveolar macrophages,and activate TLR4 protein to cause activation of the I?B kinase complex,leading to phosphorylation of I?B and NF-p65.This study revealed the role of LIpopolysaccharides in the infection of haemophilus porcine,and provided research data for the prevention and treatment of haemophilus porcine,which is of great significance.