| Lactobacillus rhamnosus has been widely used due to its function of improving intestinal health, enhancing growth and reducing the rate of diarrhea in the host. Pre-sently, much attention is being focused on the immunomodulatory properties of Lac-tobacillus rhamnosus. However, little is known about the immunomodulatory proper-ties of Lactobacillus rhamnosus and its mechanisms of effects as an intestinal barrier in the piglets. Therefore, the present study was designed to investigate the effects of Lactobacillus rhamnosus6001(LR6001) as an immunomodulatory properties and intestinal barrier in vivo and in vitro. The main results are listed as follows:Trial1:The effects of orally administered Lactobacillus rhamnosus6001(LR6001) on the intestinal barrier of sucking piglets. Six litters (n=24) newborn piglets (Large White×Landrace×Duroc) were randomly divided into two groups,3litters per group. After birth and before the first sucking, piglets of control group orally received10%sterilized skim milk2mL piglet-1day-1. The LR6001group re-ceived10%sterilized skim milk2mL piglet-1day-1in addition to viable LR6001(5-6×108CFU·mL-1) for the first time, and another two of oral gavages were admi-nisted on the alternative odd days (3rd and5th day) of post partum. The feeding trial was conducted for25days and during experimental no antibiotic was added in the diet of sows and piglets throughout the trial. Results showed that oral administration of LR6001increased daily body weight gain by14.68%, while decreased diarrhea rate30.73%(P<0.05). Significantly Modulatory changes were appeared in the diversity of intestinal flora and the balance of microflora significantly, which led the beneficial effects by exerting prominent decrease in the growth of gram-negative bacteria (e.g., Bacteroidetes spp., Proteobacteria spp., Fusobacteria spp., and Enterobacteria), and an increased number of gram-positive bacteria noted (e.g., Firmicutes spp.). The ratio of villus height and crypt depth increased by20.13%(P<0.05) in jejunum of treat-ment group piglets, while the crypt depth decreased by10.01%(P<0.05). The gene expression of OCLN decreased by48.12%(P<0.05) in jejunum, but the expression of ZO-1remained unchanged. The gene expression of pBD-1and PMAP-37increased in jejunum of LR6001treated group, but LYS mRNA level noted lower significanlty. It was observed that the gene expression of MUC2and pH values numerically altered in the jejunum. The activities of DAO and ITF in ileul mucosa increased by375.15%(P<0.01) and100.44%(P<0.01) in the treatment group, respectively, while the activities of DAO and ITF in jejunal mucosa decreased by60.51%(P<0.01) and33.98%(P<0.01). The activity of maltase increased by41.29%(P<0.05) in jejunal mucosa of LR6001group, but the activities of sucrose and lactase were unaltered. Moreover, the concentrations of sIgA and C3in ileal mucosa increased by25.70%(P<0.05) and37.54%(P<0.05) in treatment group respectively, while the con-centrations of sIgA, C3and IgG in jejunal mucosa and IgG in ileal mucosa numari-cally improved. Additionally, LR6001treatment up-regulated the anti-inflammatory cytokines of TGF-β1and1L-10in jejuna mucosa, while the production of pro-inflammatory cytokines of IL-1, IL-6, IL-12and TNF-a, and chemokine of IL-8down-regulated (P<0.01) in both jejunal and ileal mucosa. It was also observed that pro-inflammatory cytokines of TNF-α, IFN-γ and chemokine of MCP-1up-regulated (P<0.01). Therefore, it provides the information that LR6001adminis-tration modulates the immune response by regulating the secretion of an-ti-inflammatory cytokines, pro-inflammatory cytokines, and chemokine in both jejun-al and ileal mucosa. Moreover, the gene expressions of MD-2, TLR2, TLR9, and TRAF6up-regulated21.29%(P<0.05),230.77%(P<0.01),107.34%(P<0.01), and81.92%(P<0.05), respectively, which suggests that MD-2, TLR2, TLR9, and TRAF6activated by immunomodulatory function of LR6001. All together, oral administra-tion of LR6001after birth promotes growth performance and improves the intestine health, regulates microbial ecological balance, functions of chemical barrier and physical barrier beside the TLR-meditated signaling pathway, as well as innate im-mune responses to reduce diarrhea in sucking piglets.Trial2:The effect of LR6001on intestinal mucosa barrier of weanling piglets was evaluated in the2nd trail. The same experimental protocol was used as described in Trial1. The results as compared to control group showed that average daily weight gain slightly increased in LR6001-treated group, and diarrhea incidence decreased by 15.32%(P>0.05). Oral administration of LR6001increased the diversity of intestinal flora, and also improved the composition of microflora. The amount of Firmicutes bacteria increased remarkably, while the amount of Bacteroid and Proteus reduced markedly. The structure of jejunal mucosa was more integrated in LR6001group than that in control group. The ratio of villus length and villus height to crypt depth in-creased by13.92%(P<0.01) and34.93%(P<0.01) in LR6001group respectively, and the crypt depth reduced by14.53%(P<0.05). Additionally, pH values in stomach re-duced in LR6001-treated piglets group. There was a decrease in the jejunal mucosal DAO activity by21.77%(P<0.01), whereas the serum concentrations of ITF and en-dotoxin were remained unchanged. Lactase activity in jejunal mucosa reduced13.60%(P<0.05) in LR6001group, but no significant difference was observed for the activities of sucrase and maltase. The mucosal concentration of sIgA increased by50%(P<0.01) in jejunum of LR6001-treated piglets, but the concentrations of IgG and C3slightly changed (P>0.05). Furthermore, LR6001treatment increased (P<0.01) anti-inflammatory cytokine TGF-β1and pro-inflammatory cytokine TNF-α, but re-duced (P<0.05) the level of inflammatory cytokine of IL-1β,IL-6,IL-12, IFN-γ and chemokine IL-8. Conclusively, we found that LR6001administered orally after birth improves the intestine health, prevents harmful effects of weaning stress in weaning piglets. It induces the increased capacity of absorption and balance the ecological en-vironment of intestine. Further, enhances the functions of chemical and physical bar-riers as well as innate immune responses in the piglets during the first week of wean-ing.Trial3:The effects of LR6001on expression level of TLR in piglets epithelial cells (IPEC-J2). The cultured IPEC-J2cells were randomly assigned into four treat-ment groups. Control (CK), treatmentⅠ (T6), treatmentⅡ (T7) and treatmentⅢ(T8), and were stimulated with0,106,107,108CFU·mL-1of Lactobacillus rhamnosus6001(LR6001). After stimulation period of3h, the IPEC-2cells were collected to investi-gate the mRNA expression of TLR2, TLR4, TLR6, TLR9and TLR10using real-time PCR. The results showed that the T8group increased the mRNA levels of TLR2(P<0.01) and TLR9(P<0.01), however TLR4mRNA levels reduced significantly as compared to CK, while T7showed increased level of TLR6mRNA expression. Among the experimental groups, the expression levels of TLR2and TLR9were lower in T6and T7as compared to T8(P<0.01), however, the TLR4gene expression level was higher in T6and T7in comparision of T8(P<0.01), and mRNA expression of TLR10in T7group was significanlty lower than T8group. The results indicated that the different concentrations of LR6001promote the proinfalammatory and antiinfa-lamatory response through activation of TLR.In summary, the mechanism of LR6001for the promotion of the intestinal health in piglets is due to the enhancement functions of biological, physical, chemical and immune barriers. Moreover, LR6001can also regulate the non-specificity immune response via TLR-meditated signaling pathways. |
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