| Inflammatory bowel disease(IBD)is a kind of disease affected by environmental,microbial,immune response and genetic factors,including ulcerative colitis(UC)and Crohn’s disease(CD).The incidence rate of UC is high,which seriously affects people’s normal life.It’s conventional therapeutic drugs are limited and may have side effects,so it is urgent to develop new therapeutic drugs or methods.Some probiotics can help decompose toxic substances or drugs,prevent bacteria from invading the body,and have the potential to alleviate inflammation.The therapeutic advantages of some traditional Chinese medicine on IBD have also been verified in animal experiments or clinical experiments.Objective: Probiotics with broad-spectrum antibacterial activity were screened from traditional fermented foods in Sichuan,and the safety,colonization ability and function of probiotics were tested.It was used to intervene in the treatment of acute UC model constructed by dextran sulfate sodium(DSS)in advance,in order to explore whether probiotics can alleviate or prevent the recurrence of acute UC.Probiotics and traditional Chinese medicine were combined to explore the differences in the therapeutic effects between combined and single drugs,so as to provide a new reference for the prevention and treatment of acute UC.Methods: using bromocresol purple medium and Gram staining microscopy,we screened lactic acid bacteria from different fermented foods in Sichuan.Then we used Staphylococcus aureus,Escherichia coli,Acinetobacter baumannii,Pseudomonas aeruginosa and Streptococcus pneumoniae as indicator strains to screen probiotics with wide antibacterial activity and sequenced.The simultaneous interpreting of Bauman’s bacteria was carried out.The safety of screened strains was evaluated by using biological amino factors,virulence factor genes and MIC test results.The colonization ability of the screened strains was evaluated by hydrophobicity test,automatic aggregation ability test,acid and alkali resistance test and bile salt resistance test.The functions of the screened strains,such as cholesterol lowering ability and hydroxyl radical scavenging ability,were tested.Finally,a probiotic strain with strong comprehensive ability was selected for subsequent animal experiments.Using traditional Chinese medicine or L9 alone,and using traditional Chinese medicine combined with L9 to intervene 7 days in advance,the UC mouse model was constructed with 3% DSS and divided into 9 groups: blank group(N),modeling group(DSS),and L9 preventive treatment Group(Z),Astragalus preventive treatment group(H),Atractylodes prophylaxis group(B),Astragalus combined with L9 preventive therapy group(H+Z),Atractylodes combined with L9 preventive therapy group(B+Z),Astragalus and Atractylodes preventive therapy Group(HH).Observe the status of mice every day,record the weight,diarrhea and bloody stool of mice,and score the disease activity index.The colon length of mice was measured.The activity of colonic myeloperoxidase and the expression of colonic IL-1β,IL-6,IFN-γ,Occludin,Reg3 g and Reg3 b were measured.Histological score was performed by hematoxylin staining.Comprehensive evaluation of the degree of inflammation in mice to judge the efficacy of the experimental intervention.Results: 138 strains of lactic acid bacteria were screened from traditional fermented foods in Sichuan,and 7 strains of probiotics with broad antibacterial activity were screened: L9,6-2,4-2,XD-2,YL1-5,XD1-5,D1-4,gene identification results are all Lactobacillus plantarum.All screened strains do not contain biological amino genes hdc,tdc and odc,and do not contain virulence factor genes Ace,Gel E and Cyl A.All strains were sensitive to gentamicin,levofloxacin,chloramphenicol and meropenem.The resistance of L9 to polymyxin B and tetracycline was mediated,and the rest were resistant.Strain 6-2 and 4-2 were intermediate to ampicillin,others were sensitive.L9 was sensitive to kanamycin,6-2,4-2,YL1-5 and standard strains were intermediate,and the rest were resistant.Compared with CGMCC 1.2469,6-2,4-2,XD-2,YL1-5 and L9 have higher hydrophobicity.The auto-aggregation ability of 4-2 and L9 is similar to that of CGMCC 1.2469.Compared with the standard strains,the 7 strains showed the advantage of acid resistance,especially under the condition of p H=2.5.All strains were highly tolerant to simulated intestinal fluid and 0.3% or 0.5% bile salts.The cholesterol lowering ability of XD1-5,XD-2 and L9,and the hydroxyl radical scavenging ability of XD-2,6-2 and XD1-5 were better than CGMCC 1.2469.L9 was selected as a potential strain for the treatment of acute UC mice.Experimental therapeutic interventions attenuated weight loss and prolonged colon length in mice to varying degrees.On the 5th day of modeling,the DAI score of the H+Z group was higher than that of the DSS group,and the DAI values of the other groups were all lower than those of the DSS group,with the DAI value of the B group being the smallest.The results of MPO showed that the MPO activity in the colon of mice in group B,B+Z and Z group was significantly lower than that in DSS group.Group Z,group B,group B+Z,group H and group H+Z can effectively reduce the expression of IL-1β and IL-6,and the IFN-γ of DSS group is statistically compared with the Z group and HH group in the experimental group Although the B+Z group,the H+Z group and the HH+Z group decreased the content of IFN-γ in the colon of mice,there was no statistical significance.The content of Occludin in DSS group was significantly decreased,but there was no significant difference between Z group,B group and HH+Z group.Compared with the N group,the experimental intervention significantly promoted the transcription levels of Reg3 g and Reg3 b.Compared with DSS group,B group,B+Z group,H group,H+Z group significantly increased Reg3 b content,B+Z group significantly increased Reg3 g content.The transcription levels of Reg3 g and Reg3 b were relatively weak in groups Z and B.The results of colon HE staining showed that the HH group and HH+Z group did not significantly reduce inflammation,which was similar to the DSS group.Group Z,group B,group B+Z,group H,group H+Z reduced the pathological damage of colonic mucosa to different degrees,among which group Z,group B,group B+Z and group H had significant difference compared with DSS.The B+Z group had the best anti-inflammatory effect and preserved some intestinal crypts.Conclusions: This experiment successfully screened out a potential probiotic L9 with broad-spectrum antibacterial activity,safety,good colonization ability and functionality,and used it for the treatment of acute UC mice.In the experiment,mice in group Z,group B and group B+Z showed better anti-inflammatory advantage.Among them,the B+Z group has the best effect,which can reduce the expression of IL-1β,IL-6and IFN-γ in the colon,reduce the activity of MPO in the colon,enhance the expression of Occludin,reduce the infiltration of inflammatory cells,and can promote the transcription level of Reg3 g and Reg3 b,resist the invasion of bacteria.And can effectively relieve colon shortening and reduce DAI score.The treatment effect of HH group and HH+Z group was not good.The H group and H+Z group showed the effect of reducing the infiltration of intestinal inflammatory cells in mice in the detection of inflammatory factors and colon HE staining,but their DAI score and MPO detection effect were not good,and the experiments need to be repeated for comparison.The results show that compared with the use of traditional Chinese medicine or probiotics alone,the therapeutic effect of probiotics combined with traditional Chinese medicines is not obvious.In the follow-up,the amount of specimens should be increased,and the concentration gradient of drugs and probiotics should be further set for combined experiments. |
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