| Yak is a special cattle breed living at high altitude,which is resistant to low temperature and suitable for low oxygen compared with plain animals,studies have shown that it has a unique gut microbial composition.Lactic acid bacteria can keep the gut homeostasis,and have the effects of anti-inflammatory,anti-oxidation,promoting the content of lipids and improving immunity,which often used to improve intestinal disorders.Intestinal mucositis(IM)refers to inflammatory lesions and ulcers of the intestinal mucosa,mainly caused by chemotherapy or radiotherapy.5-Fluorouracil,a pyrimidine antimetabolite,is widely used in cancer therapy,but it also affects nucleic acid and protein synthesis in normal cells,resulting in cell and tissue injury,such as basal cells and mucosal cells of the gastrointestinal tract.At present,there are few therapeutic drugs for IM,most of whom have obvious side-effects or are too costly.Therefore,in this study,yak intestine and feces were selected for the isolation of lactic acid bacteria,hoping to select lactic acid bacteria with excellent performance and explore their intervention effects on 5-FU-induced intestinal mucositis in mice.The results are as follows:(1)Isolation and identification and in vitro test of lactic acid bacteria of yak originFour lactic acid bacteria with good probiotic performance were isolated from 10samples of yak rumen contents,which were Lactobacillus fermentum A4,Pediococcus pentosaceus A3.4 and A1.2,and Pediococcus lactis B1.9.Compared with the remaining three strains,L.fermentum A4 treated for 3 h in broth with 0.3%bile salt had the highest survival rate(p<0.001),while in broth with p H=3.0,P.pentosaceus A3.4 had the highest survival rate(p<0.001).The hydrophobicity and autoagglutination ability of P.pentosaceus A3.4 were better than those of the other three strains.The four strains of Lactobacillus showed good growth performance,all reaching the plateau stage at around10 hours of incubation and had good bacterial inhibition properties.Safety tests showed that the four strains were not haemolytic and showed sensitivity to most common antibiotics.In conclusion,P.pentosaceus A3.4,which performed the best in vitro test,was used as the animal test strain for subsequent tests.(2)Effect of lactic acid bacteria on intestinal mucositis in miceThe animal test was breaked into 4 groups,control group(Control group),probiotic group(Probiotic group),model group(5-FU group),and protection group(Protection group).On days 1–14,the Control and 5-FU groups were gavaged with normal saline,and the Probiotic and Protection groups were gavaged with P.pentosaceus A3.4(10~9CFU/m L).On days 15–19,the 5-FU and Protection groups were injected with5-FU(i.p)(50 mg/kg),and other groups were injected with saline.It could be observed that,the body weight of model group decreased(p<0.01).According to the spleen index and liver index,it could be found that the both index were obviously decreased in relation to the control group(p<0.001),while the index in the Protection group was raised in relation to the 5-FU group(p<0.01);at the same time,the index of the Probiotic group was raised than that of the Control group,and there was a variance in the spleen index(p<0.05),but there was no notable variance in the liver index.Pathological changes were heeded in HE stained sections of jejunal tissue,which were unremarkable in the Control group and Probiotic group,and the intestinal tissue was intact without pathological changes.However,the sections in the 5-FU model group could observe the severe jejunal tissue injury,which involved the damage and shortening of mucosa,and submucosa and muscle layer,including the damage and shortening of intestinal villi,loss of crypt structure,infiltration of inflammatory cells and thinning of intestinal wall.However,in the protected group,the length and thickness of intestinal were recovered and the infiltration of inflammatory cells decreased,indicating the integrity of gut was better.The detection of antioxidant indicators and MPO kit in the serum of mice in each group revealed that the antioxidant indicators in the model group were decreased in relation to the control group(p<0.01),and the antioxidant indicators of the Protection group were significantly higher than 5-FU group(p<0.01);the MPO activity in serum in the model group was absolutely higher than control group(p<0.01),and the protection group could reduce the MPO content,in relation to control group there was a little difference.The results of fluorescence quantification of jejunal tissue showed that mice in the 5-FU group could up-regulate the inflammatory response mediated through NLR receptors,resulting in the up-regulation of downstream TNF-αand IL-6 cytokines.At the same time,the expression of chemokines CXCL-9 and CXCL-5 was up-regulated.Moreover,the expression of IL-10 was down-regulated.After treatment with P.pentosaceus A3.4,the expression of NLRP3 in the jejunal enteritis bodies of mice decreased(p<0.05),while CXCL-9,CXCL-5,IL-6,and TNF-αcytokines related to inflammation showed a decreasing trend(p<0.05),while the expression of IL-10 increased(p<0.05).(3)Effect of lactic acid bacteria on intestinal bacteria in mice with intestinal mucositisThe dominant bacterial phyla in the mouse cecum are Bacteroidetes,Firmicutes,Epsilonbacteraeota,Proteobacteria,and Verrucomicrobia.The dominant genera were Bacteroides,uncultured＿bacterium＿f＿Muribaculaceae,uncultured＿bacterium＿f＿Lachnospiraceae,Helicobacter,and Lachnospiraceae＿NK4A136＿group.Abundance of Epsilonbacteraeota increased significantly in the model group compared with Control group(p<0.05);Acinetobacter,Acetobacter,and Xanthomonas increased significantly(p<0.05).In relation to the 5-FU group,the Protection group had a significant rise in Tenericutes and Bacteroidetes(p<0.05);and a significant rise in Pediococcus spp.,Ruminococcus spp.,and Akkermansia spp.(p<0.05).The treatment with 5-FU resulted in a disruption of the intestinal flora of the mice,increased abundance of harmful bacteria such as Actinobacillus,Acetobacter,and Xanthomonas.P.pentosaceus A3.4 treatment in the Protection group resulted in an increase in the relative levels of Lactococcus and Akkermansia spp.in the intestine,which can regulate the inflammatory response of the body and is negatively associated with some metabolic diseases.Thus,the advance treatment of Lactobacillus improves the symptoms of intestinal mucositis induced by5-FU. |
