Study On Antagonistic Pathogens Of Lactobacillus Rhamnosus By Reducing ERBIN Protein Expression

Innate immune response refers to the innate immune response of the body to various pathogenic microorganisms and foreign body invasion.Nod protein family is a large class of pattern recognition molecules.Bacterial cell wall can stimulate Nod protein binding oligomyelization,activate NF-κB and MAPK signaling pathways,and eventually induce the production of IL-1,INF-γ and other cytokines in the body to resist the invasion of exogenous pathogens,and play an important role in the innate immune response.However,ERBIN can reverse regulate Nod2 proteins in the Nod family and inhibit the NF-κB pathway.At present,there is no report on the effect of ERBIN on probiotics against intestinal pathogens.In this study,we investigated whether Lactobacillus rhamnosus FLRH93 could relieve the inhibition of Nod2 by interfering the expression of ERBIN protein,activate the NF-κB pathway,enhance the immunity of the body and resist the invasion of foreign pathogens.Using Listeria monocytogenes CMCC54001,Salmonella typhimurium ATCC13311,Escherichia coli O157:H7,lentivirus and Coxackie virus as the representative pathogens,by quantitative PCR and western blot,The effect of L.rhamnosus FLRH93 on the transcription and expression of ERBIN protein in intestinal epithelial cells was detected,and the changes of related signaling pathways controlled by Nod2 downstream in normal cells were compared,and the antagonistic effect against pathogens was observed through the adhesion and invasion rate of pathogens to intestinal epithelial cells.The results showed that L.rhamnosus FLRH93 significantly reduced the transcription and expression of ERBIN in intestinal epithelial cells,uninhibited the inhibition of Nod2,activated the NF-κB signaling pathway,and significantly reduced the adhesion and invasion rate of food-borne pathogens to intestinal epithelial cells.Fluorescence microscopy showed that the adhesion and invasion rates of lentivirus and Coxsackie virus decreased by 76.9% and 80%,respectively.It is speculated that L.rhamnosus FLRH93 can enhance the immunity of the body by regulating ERBIN.By gavage of L.rhamnosus FLRH93,the effects of intestinal ERBIN protein on pathogen resistance were evaluated.Combined with fluorescence quantitative PCR and immunohistochemistry,the effect of intestinal ERBIN protein on pathogen resistance was proved.The results showed that compared with the model group,the survival rate of BALB/c-nu in nude mice was increased by about 50% after L.rhamnosus FLRH93 intervention.After infecting BALB/c with Coxsackievirus,the concentrations of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum of mice were significantly increased,and pancreatitis was found in the slices stained by HE.However,intragastric administration of L.rhamnosus FLRH93 could significantly improve the liver injury and pancreatitis caused by Coxsackievirus.It is indicated that L.rhamnosus FLRH93 can play a beneficial role both in vivo and in vitro.In order to verify whether ERBIN is a key gene in the antagonistic pathogen of L.rhamnosus FLRH93,ERBIN overexpressed cell lines were constructed,and it was found that the antiviral infection ability of L.rhamnosus FLRH93 in this cell line was significantly decreased,thus proving the expected derivation.The receptor protein binding to intestinal cell surface of L.rhamnosus FLRH93 was further screened and identified as Annexin A2(ANXA2)by sequencing.It is speculated that L.rhamnosus FLRH93 may regulate the transcription and expression of ERBIN through specific binding to Annexin A2,so as to enhance the immunity of the body.To further verify whether other probiotics have similar effects,Lactobacillus plantarum SFUL25,Lactobacillus delbrueckii YPAL28 and Lactobacillus campylobacter KSWL33 were selected,and it was also found that they could reduce the expression of ERBIN.Therefore,the mechanism of probiotics enhancing immunity by reducing ERBIN expression has certain universality.In this paper,through in vitro cell experiments and in vivo animal experiments,we have explored the mechanism of L.rhamnosus FLRH93 to improve the body’s resistance to foreign pathogens by regulating the expression of ERBIN protein,thus enriching the molecular mechanism of probiotic antagonism against pathogens,and providing a new molecular marker for the subsequent screening of probiotic strains that enhance the body’s immunity.