Effects Of Lactobacillus Delbrueckii On Intestinal Immune Regulation In Suckling Piglets

The major function of the Lactobacillus on the animals is to enhance the immune response in the gastrointestinal tract of the animals,thus the investigation of the underlying mechanisms on the effects of Lactobacillus on the muscoal immune function may shed light on the development of Lactobacillus-based probiotic and its good application.In this study,the effects and mechanism of intestinal immune regulation of Lactobacillus delbrueckii were studied at the cellular and molecular levels by combining animal experiments and in vitro cell culture experiments,providing a theoretical basis for the development and application of Lactobacillus delbrueckii in piglet production.Six litters piglets with similar calving date,parity and litter size about 7-12,were adjusted litter number to eight.Every litter was divided between control group and experimental group at random.The control groups were administrated with normal saline,the experimental groups were change to Lactobacillus delbrueckii.Each piglet was orally administrated with 1,2,3 and 4ml probiotics or normal saline at the birth age of 1,3,7 and 14d.At the end of the21-day weaning experiment,six piglets in each group were randomly selected for blood collection and slaughter to take intestinal tissue samples.Serum biochemical indexes,intestinal barrier function,antioxidant indexes,intestinal mucosa cytokines,chemokines and receptors,COX2 and i NOS protein expression,TLR and its downstream signaling molecule expression were analyzed.Dendritic cells in good state in logarithmic growth phase were prepared into 10~6/ml cell suspension in RPMI-1640 complete medium and placed in a 6cm culture dish,divided into blank group,LPS group(LPS concentration 1μg/m),LAB1 group(heat inactivated Lactobacillus delbrueckii 10~6cfu/ml),LAB2 group(heat inactivated Lactobacillus delbrueckii 10~7cfu/ml),LAB3 group(living Lactobacillus delbrueckii 10~6cfu/ml),LAB4group(living Lactobacillus delbrueckii 10~7cfu/ml).After the cells are attached to the wall,LPS or Lactobacillus delbrueckii were added to cell culture medium,5%CO2,37℃cultivating for 24 h.The culture supernatant and cells were collected and the expressions of cell phenotype molecules,cytokines,chemokines and receptors,TLR,intracellular downstream signaling molecules and transcription factors were analyzed.The main test results are as follows:Lactobacillus delbrueckii was administered during lactation can significantly increase weaning weight and average daily gain of piglets(P<0.05),significantly reduce diarrhea rate(P<0.01),significantly increase serum Ig G and Ig A levels(P<0.05),decrease serum urea nitrogen levels(P<0.05),,and effectively improve the body antioxidant capacity of piglets.The results showed that Lactobacillus delbrueckii could promote growth by enhancing nutrition metabolism and antioxidant capacity of piglets.Lactobacillus delbrueckii was administered during lactation can significantly increase jejunum and ileum villi height,tight junction protein ZO-1 and Occludin m RNA abundance(P<0.05),promote the expression levels of cytokine proteins in jejunum and ileum mucosa,such as s Ig A,INF-γ,IL-4,IL-6,IL-10,IL-12,and increase CD80,CD86,MHC-II,CX3CL1,MIP-3α,CX3CR1,CCR6 m RNA abundance,and significantly increase the expression of COX2 and i NOS.The results showed that Lactobacillus delbrueckii improved the intestinal immune function of suckling piglets.The m RNA abundance and protein expression levels of TLR2,TLR4,My D88,TRIF,NF-κB,AP-1 and IRF-3 were significantly increased(P<0.01).It indicates that Lactobacillus delbrueckii can activate intestinal TLR2 and TLR4,activate intracellular junction molecules My D88 and TRIF,activate transcription factors NF-κB,AP-1 and IRF-3,promote the expression and secretion of IL-4,IL-6,IL-10 and other cytokines,mediate the Th2 immune response,promote the synthesis and secretion of s Ig A,and thus play the role of immune regulation.Lactobacillus delbrueckii and heat inactivated Lactobacillus delbrueckii act on dendritic cells in vitro,the expression levels of mature surface markers CD80,CD86 and MHC-Ⅱwere all improved in varying degrees.The expression levels of Th1 and Th2 cytokines IL-2、IL-4、IL-6、IL-10、IL-12、GM-CSF、TNF-αand IFN-γwere significantly increased in the Lactobacillus delbrueckii group(p<0.05)and the expression level of inflammatory cytokines were lower than that of LPS group.These results suggest that Lactobacillus delbrueckii is not an inflammatory reaction but a moderate immune activation.The expression levels of Th2 cytokines IL-4 and IL-10 were significantly up-regulated(P<0.05)after the heat inactivated Lactobacillus delbrueckii treatment,showing a dose-effect relationship.In addition,Lactobacillus delbrueckii could promote the expression of chemokines MIP-3α,CX3CL1 and CCR6 in dendritic cells,while the heat-inactivated bacteria had no significant effect.The research results show that Lactobacillus delbrueckii act on dendritic cells in vitro,it can promote the maturation of DC,through the high expression of cytokines,chemokines and receptors,etc.,so as to play the immune regulatory function of DC.Lactobacillus delbrueckii mainly exerts Th1/Th2 immunomodulatory effect through moderate immune activation,while heat-inactivated bacteria mainly exerts Th2 immunomodulatory effect.Heat inactivated Lactobacillus delbrueckii can up-regulate the expression of DC TLR2and TLR4,while living Lactobacillus delbrueckii can only up-regulate the expression of DC TLR2.Heat inactivated Lactobacillus delbrueckii can up-regulate the expression of DC My D88 and TRIF,while living Lactobacillus delbrueckii can only up-regulate the expression of DC My D88.Both inactivated and viable bacteria could induce the phosphorylation of ERK,JNK,p38,IκB-αand p65 proteins,and increase the protein expression of AP-1,these results suggest that Lactobacillus delbrueckii can induce the phosphorylation cascade of NF-B and MAPK pathways in dendritic cells,activation of NF-κB and MAPK pathways.However,it had no effect on the expression of IRF-3.In this study,it was suggested that living Lactobacillus delbrueckii can activate the DC TLR2pathway,while the inactivated lactobacillus can activate TLR2 and TLR4 pathway,and both can induce the phosphorylation cascade reaction of the NF-κB and MAPK pathways,promote the activation of the TLR pathway,and further promote the expression of relevant immune molecules and genes,so as to play an immunomodulatory role.In summary,Lactobacillus delbrueckii can can improve the performance of suckling piglets by improving intestinal barrier function,antioxidant capacity and intestinal mucosal immune function.Lactobacillus delbrueckii can regulate the immune function of dendritic cells through TLR-mediated signaling pathways such as NF-κB and MAPK.There were some differences in immune function and signal transduction pathway between living and heat-inactivated bacteria.