Study On Effects Of Lactobacillus Plantarum G83 On Treatment Of Intestinal Inflammation In Giant Panda Mice Derived From Giant Panda Intestinal Microbiota-associated Mice

Captive giant pandas are prone to intestinal microbiota dysbiosis and various intestinal infections due to long-term artificial feeding and their special diet and intestinal physiology can easily cause intestinal damage.Probiotic treatment is a very promising biological method for animal intestinal diseases.Lactobacillus plantarum BSG201683(L.plantarum G83)is a giant panda-derived probiotic with excellent beneficial functions.It is a promising candidate probiotic strain for the prevention and treatment of intestinal inflammatory diseases in giant pandas.In this study,we conducted a preliminary study on its anti-inflammation functions in vitro and generated a giant panda intestinal microbiota-associated(PMA)mouse model by using antibiotics and 5%DSS,and carried out a study on anti-inflammatory effects and mechanisms in vivo.1.Effects of L.plantarum G83 on LPS-induced inflammation on Raw 264.7.The anti-inflammatory effects and mechanisms of L.plantarum G83 were assessed through pre-treatment,co-culture,and post-treatment with different doses in LPS-induced inflammatory Raw 264.7 cell.The phagocytic function of macrophage was significantly improved after different L.plantarum G83 treatments.Post-treatment and co-culture group have a positive anti-inflammatory effect on LPS-induced inflammation by significantly reducing the secretion of TNF-α,IL-6,IL-10,and NO in the cell supernatant,and significantly reducing the expression of inflammation-related genes and the phosphorylation of NF-κB/p65 protein.2.Effects of L.plantarum G83 on tight junctions of Caco-2 cell.The protect effect and mechanism of different doses L.plantarum G83 on tight junctions were assessed through pre-treatment,co-culture,and post-treatment in LPS-impaired Caco-2 cell monolayer.Permeability of Caco-2 cell monolayer to FITC-D4 was significantly reduced,and concentration of TNF-αwas suppressed,while that of IL-10 was promoted in the supernatant after co-culture and post-treatment with L.plantarum G83.Tight junction proteins were significantly increased at protein and transcription level.The results showed above demonstrated that L.plantarum G83 exhibited a good property to reduce inflammation and improve the tight junction of intestinal epithelial cells in vitro.3.Generation of PMA mouse.Two different methods are used to generate the PMA mice.1)After mice drinking water with antibiotics cocktail for 7 days,they were gavaged with giant panda intestinal microbiota to generate antibiotic induced panda microbiota associated(APMA)mouse.The result of viable bacterial count showed that the abundance of intestinal microbe was significantly reduced after the antibiotic drinking water treatment.There was no difference in alpha diversity of intestinal microbiota among conventional mice,APMA mice and giant panda.The significant differences in beta diversity indicated that the different groups showed unique microbiota.However,the abundance of E.coli in the intestinal microbiota of APMA mice and giant panda was significantly higher than that of conventional mice.There was significantly different of beta diversity among all groups.2)5%DSS drinking water was introduced to generate another panda microbiota associated(DPMA)mouse model after the antibiotic drinking water treatment and microbiota gavage.The analysis found that alpha diversity of intestinal microbiota had no significant difference among APMA mice,DPMA mice and giant panda.However,higher relative abundances of Proteobacteria,Enterobacteriaceae,and Enterococcus in the giant panda microbiota were also observed in the DPMA mice;beta diversity of intestinal microbiota showed significant differences among the two type of PMA mice and giant panda.LEf Se analysis was used to determine the different bacterial genera associated with DSS intestinal inflammation.Together,the intestinal microbiota of DPMA mice had a more similar microbiota to that of giant panda.4.Effects of L.plantarum G83 on the colon inflammation of DPMA mice.After DPMA mice were treated with different doses of L.plantarum G83,we analyzed the effect of L.plantarum G83 on DPMA mice disease activity index barrier function,immune reaction,and intestinal microbiota.The results are as below:(1)Effects of L.plantarum G83 on the barrier function of DPMA mice.H&E staining results showed that the intestinal epithelial integrity was improved and fewer ulcers and inflammatory cells were observed in the medium-and high-dose treatment groups;D-lactic acid content in the peripheral blood was significantly lower in each treatment group than in the blank group;Low-dose treatment group had the highest concentration of s Ig A in the intestinal tract content;was used to determine the expression of ZO-1,Occludin and Claudin-1 protein in colon tissue of DPMA mice,the ZO-1 and Occludin in the intestinal tissue of the medium-and high-dose treatment groups were significantly increased based on immunohistochemistry result,and there was no significant difference in the expression of Claudin-1 among groups.q PCR results of ZO-1,Occludin,and Claudin-1 gene in the intestinal tissue were consistent with immunohistochemistry results.(2)Effects of L.plantarum G83 on intestinal inflammation of DPMA mice.ELISA results showed that the L.plantarum G83 treatment could decrease the concentration of TNF-αand IFN-γin peripheral blood,but that was higher than these in the mesalazine treatment group.Also,higher IL-10 concentration was observed in the medium-and high-dose treatment group;TNF-αand MPO in the intestinal tissue were lower in the medium-and high-dose group.Gene expression level determined by q PCR were consistent with these results;The ratio of CD3~+CD4~+/CD3~+CD8~+T lymphocyte in the Payer’s patch,mesenteric lymph node,and spleen tissue were increased after treatment with L.plantarum G83,while the ratio of CD3~+CD4~+/CD3~+CD8~+T lymphocyte in peripheral blood was negatively correlated with the treatment doses.The ratio of Th1/Th2 and Th1/Th17 in the spleen increased significantly in all treatment groups.The NF-κB and i KKβgene and NF-κB/p65 phosphorylation level were significantly reduced after treatment with L.plantarum G83,while My D88 gene expression was improved.Further analysis showed that IL1RL1,TLR negative regulatory gene,was significantly increased in medium L.plantarum G83 treatment group.(3)Effects of L.plantarum G83 on intestinal microbiota of DPMA mice.Alpha diversity of intestinal microbiota in DPMA mice showed a dose-dependent on L.plantarum G83treatment.Beta diversity result of L.plantarum G83 treatments were significantly different from Blank D group,while there was no significant difference observed between mesalazine treatment group and Blnk D group.Relative abundance of Lactobacillus and Bifidobacterium in intestinal tract were significantly increased in L.plantarum G83 treatment groups,however,relative abundance of Proteobacteria and Enterobacteriaceae significantly decreased when compared with Blank D and Treat Mz group.Also,concentration of SCFAs was increased after L.plantarum G83 treatments in DPMA mice.FAPROTAX gene prediction showed that abundance of disease-related bacteria after treatment with L.plantarum G83 was significantly lower than that of Blank D group.In summary,the result indicates that L.plantarum G83 exhibits ability to reduce the inflammatory response induced by LPS and alleviate tight junction proteins impaired in vitro.L.plantarum G83 can effectively prevent intestinal antigens from entering the colon tissue by promoting the intestinal epithelial integrity and improving expression of tight junction proteins in DPMA mice.Reduction of inflammatory factors in peripheral blood and colon tissue and increased ratio of Th1/Th2 and Th1/Th17 cells in spleen further demonstrated L.plantarum G83 can reduce the symptom of colon inflammation in DPMA mice.The increase of NF-κB/p65 protein dephosphorylation by L.plantarum G83 may be the molecular basis of its anti-inflammatory function.Moreover,L.plantarum G83 can improve microbiota balance to reduce intestinal inflammatory response by suppressing abundance of opportunistic pathogenic bacteria and increasing the abundance of beneficial bacteria and the concentration of SCFAs in the colon.