| Objective:The importance of intestinal flora to the giant pandas is increasingly being valued.Studies have found that there is an inevitable connection between intestinal microbes and intestinal diseases.Probiotics are active microorganisms that can maintain the ecological balance of intestinal flora and are beneficial to physiological functions and health.The giant panda source Bacillus subtilis HH2(B.subtilis HH2)can secrete a variety of antibacterial substances and balance the microecology in the intestine,and can play a probiotic function in the intestine of the giant panda.Intestinal flora the panda-derived animal simulates the characteristics of the giant panda’s intestinal flora and can be used to study the effects of probiotics on the intestinal diseases of the giant panda.In order to explore the relationship between probiotics,intestinal diseases and intestinal flora,and to provide an important theoretical basis for the field of giant panda microecological preparations,the present study used 5%TNBS induces colitis in panda-derived rabbits with intestinal flora,to explore the role and possible mechanism of B.subtilis HH2 in colitis.Mehtods:1.In order to construct a panda-derived rabbit model for intestinal flora,the experiment collected fresh stool samples of 8 healthy adult giant pandas.21 New Zealand white rabbits were randomly divided into a control group and a test group.The rabbits in the control group were fed with sterile normal saline every day,and the rabbits in the test group were fed with a syringe every day with the panda feces suspension after treatment.On the 15th and 25th days of continuous feeding,collected the fecal samples of two groups of rabbits,extract fecal flora DNA,used 16S rRNA sequencing technology to detect the changes and colonization status of the panda intestinal flora in the experimental rabbits.2.After establishing the panda-derived model of intestinal flora,the rabbits that have completed the transplantation of panda intestinal flora were randomly divided into four groups.5%TNBS was used to induce acute colitis in rabbits.Observed rabbits condition and the disease activity index score.3.After induced TNBS colitis model,except for the normal group and the model group,mesalazine and B.subtilis HH2 were used for intervention.Collecting colon tissues for HE staining and histopathological score;Western blot analyzed claudin-1,occludin,and ZO-1 expression levels;ELISA to detect the levels of TNF-α,IL-10 and MPO in rabbit serum;RT-qPCR determination of TNF-α,IL-1β,IL-10 and claudin-1,occludin,and ZO-1 mRNA expression in colon tissue;16S rRNA high-throughput sequencing analysis to study the structure of probiotics on panda-derived rabbit intestinal flora and its diversity.Results:(1)Successful construction of rabbit model of intestinal flora sourced from giant panda.16S rRNA sequencing analysis found that the similarity of the fecal flora of rabbits with continuous fecal transplantation for 25 days was significantly higher than transplantation for 15 days.The intestinal flora of rabbits were closely related to the intestinal flora of giant pandas.At the phylum level,the similarity of the average abundance of the flora is more than 50%.The clustering results showed that at the phylum level,Firmicutes,Proteobacteria,Bacteroides,and Actinomycetes in the intestine of giant pandas could be colonized in the intestines of rabbits in the stool transplantation group.(2)The effect of B.subtilis HH2 on the intestinal flora of panda-derived rabbit colitis.① After the first day of modeling,the weight of the rabbits in the TNBS model group decreased very significantly(P<0.001),diarrhea with blood in the stool,and serious perianal pollution.From the first day after B.subtilis HH2 was administered,the rabbit had loose or unshaped feces and weight loss.After 2 days of intervention with B.subtilis HH2,the diarrhea and blood in the stool decreased and the weight gradually increased.② At the end of modeling,the DAI score of the TNBS group reached 5.0±4.43,which was significantly higher than that of the control group(P<0.01).The DAI score of HH2 group was lower than that of the TNBS model group;Compared with the TNBS group,HH2 group histopathological score of the rabbit colon was significantly reduced(P<0.05);③ ELIS A of the rabbit serum:compared with the control group,the rabbits in the TNBS model group,the concentration of TNF-α and MPO increased significantly(P<0.01),and the concentration of IL-10 decreased significantly(P<0.05).TNF-α and MPO concentrations in HH2 group decreased significantly(P<0.05);④Rabbit colonic tissue mRNA expression level:Compared with the TNBS group,HH2 group were significantly down-regulated IL-1β.IL-10 and occludin were significantly up-regulated(P<0.05);⑤Rabbit colonic intestinal epithelial cells tight junction protein expression level:Compared with the control group,the Western blot results of the TNBS group showed that the protein expression of ZO-1 and occludin was significantly reduced(P<0.01),and the expression of claudin-1 was significantly reduced(P<0.05).The expression of occludin protein in HH2 group increased significantly(P<0.05)⑥16S rRNA analysis of intestinal flora changes:Compared with TNBS model group,the observed species of the HH2 group increased significantly(P<0.05).The relative abundance of rumen cocci and bifidobacteria in rabbits fed with B.subtilis HH2 also increased.Conclusion:(1)The average abundance of the intestinal flora of the rabbits transplanted with panda feces is similar to more than 50%at the level of the flora.The hierarchical cluster analysis showed that the intestinal flora of the FMT rabbits and giant pandas were closely related and the similarity.The FMT rabbits can better simulate the microbial flora environment of the giant panda’s intestine.(2)B.subtilis HH2 had a therapeutic and protective effect on panda-derived rabbit colitis induced by TNBS.B.subtilis HH2 can reduced the level of intestinal barrier damage by regulating the expression of tight junction proteins in epithelial cells,protected the intestinal mucosa and deep tissues from the invasion of harmful substances and inflammation,and increased the expression of the anti-inflammatory IL-10 in colon tissues. |
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