Effect Of Lactobacillus On The Production Of Autophagy And Inflammasome In Piglets Intestine Infected With Salmonella

Background and Aims:Salmonella is the main pathogen causing bacterial diarrhea in weaned piglets.Certain probiotics as a potential antibiotic substitute can be against Salmonella-induced diarrhea in weaned piglets,but the specific regulatory mechanisms are not yet clear.Autophagy is an evolutionarily conserved degradative process that is required to maintain host health and facilitates the capture and clearance of invading pathogens by the immune system.This study aimed to explore the role of probiotics and potential regulatory mechanisms in the prevention and control of diarrhea in weaned piglets with SalmonellaMethods:Twenty-four 21-day-old weaned piglets were randomly divided into three groups:1)control group,oral administration of sterile physiologic saline;(2)Salmonella group,oral administration of sterile physiologic saline at first week and oral challenge with Salmonella infantis(S.infantis)(1012 CFU/piglet)on day 8;(3)Lactobacillus and Salmonella group,oral administration of Lactobaillus johnsonii(L.johnsonii)(1010 CFU/piglet),and oral challenge with S.infantis(1012 CFU/piglet)on day 8.At 9 AM.Feed intake,body weight were recorded daily.On the 18th day,the pigs in the trial were all anaesthetised and slaughtered.Transmission electron microscopy to observe intestinal damage.The pig intestinal epithelial cells IPEC-J2 were pre-incubated with 107 CFU/mL L.johnsonii and then infected with S.infantis 108 CFU/mL.Cell viability was detected,electron damage was observed by electron microscopy,mitochondrial membrane potential was detected by JC-1,intracellular oxide content was detected by DHE,and expression of inflammatory factors and receptors related to IPEC-J2 cells was detected by Real-time PCR.The expression of autophagy-related proteins were detected by Western blotting.Immunofluorescence was used to locate intracellular Salmonella and autophagosomes.Twenty-four 21-day-old weaned piglets were randomly divided into three groups as described above(1010 CFU/mL monophasic variant Salmonella,109 CFU/mL Lactobacillus rhamnosus GG strain(LGG)).On the 15th day,the experimental pigs were anaesthetised and slaughtered to evaluate diarrhea and intestinal inflammation.Peripheral blood lymphocyte was counted,Real-time PCR method was used to detect the expression of intestinal ion channels,inflammatory factors and receptors.Western blotting method was used to detect intestinal inflammatory correlative protein expression.Cell flow assay was used to analyze dynamic changes of innate lymphoid cells in peripheral blood and intestinal tract related lymphoid tissues.Results:Oral L.johnsonii significantly ameliorated fever,intestinal inflammation,and other symptoms of piglets caused by S.infantis.S.infantis infection of IPEC-J2 cells decreased the mitochondrial membrane potential,induced the release of ROS,and promoted the expression of NLRP3 and ASC in cells and ileum.S.infantis induced the mRNA expression of TLR9 and Cytochrome C in cells.Pre-incubation of L.johnsonii inhibits mitochondrial damage and promotes the gene expression of Parkin and LC3 protein in the late phase of infection but does not cause the expression of PINK1.In weaned pigs,monophasic variant Salmonella induced the expression of TLR5 and TLR4 in the jejunum,while increasing IL-8 and IL-6 mRNA expression.Monophasic variant Salmonella stimulated the expression of NOD1 and NOD2 mRNA in the ileum,promoted the phosphorylation of IκB-α,activated the NF-κB pathway and promoted the release of inflammatory cytokines.Oral administration of LGG reduced the increase in NOD1 mRNA expression induced by Salmonella in the jejunum and ileum,and promoted the secretion of sIgA in different intestinal segments.LGG prevented activation of the NLRP6/ASC/Caspase-1 inflammasome during Salmonella infection and reduced IL-18 production in the ileum.However,there was no significant change in NLRP3 inflammasome in the experiment.Conclusions:S.infantis can cause mitochondrial damage and cellular oxidative stress in IPEC-J2 cells.L.johnsonii can activate the selective mitochondrial autophagy and inhibit the activation of NLRP3 inflammasome by promoting the activity of Parkin ubiquitin ligase in host cells.The infection of piglets with monophasic variant Salmonella does not affect their growth performance,fecal grade,etc.,but it will lead to the occurrence of enteritis.Orally fed LGG accelerated Salmonella monophasic variant shedding,and also inhibited the NLRP6 inflammatory response by blocking the activation of the canonical NF-κB pathway to overcome the expression of proinflammatory cytokines.