Study On The Mechanism Of Alleviating DSS-induced Ulcerative Colitis By The Combination Of Probiotics With Different Functions

Ulcerative colitis(UC)is a chronic inflammatory disease that affects the colon.UC can induce colorectal cancer and a variety of extra-intestinal inflammatory manifestations and complications,which requires high-cost treatments and results in great damage to human health and the economy.The incidence of UC is increasing worldwide and has become a global public health threat.The first-line drugs in UC treatment are not effective in a large number of UC patients,and some may even cause serious adverse effects.Therefore,the development of new therapeutic strategies is urgent.Studies have shown that administration of certain probiotic strains can reduce intestinal inflammation and enhance intestinal homeostasis.However,the effects of different pribiotic strains are quite different,and more studies are needed to illuminate the advantages and mechanism of muli-strain probiotic strains.In addition,the understanding and usage patterns of probiotics are changing as the accumulation of knowledge related to intestinal microorganisms.The limited range of traditional probiotics has clearly failed to meet the demand for treatment of more diseases.Therefore,the exploration of symbiotic microbial resources in the gastrointestinal tract helps to find new and valuable microorganisms and drug molecules.In traditional Chinese medicine,Massa Medicata Fermentata(MMF)and Euphorbia humifusa Willd.(E.humifusa)are used to treat gastrointestinal diseases.However,more studies are needed to illuminate the therapeutic effects of MMF and E.humifusa on UC,as well as the effect on intestinal microorganisms.In addition,the mechanism and side effects of MMF and E.humifusa are still unclear.Evaluating the therapeutic effects and mechanism of MMF and E.humifusa on intestinal inflammation and damage is of great significance for the development of new agents in UC treatment.This study aims to develop intestinal symbiotic microorganisms in mammals,and explore the potential and mechanism of probiotics and traditional Chinese medicine in UC treatment.This study mainly includes the following three parts:(1)Isolation and safety analysis of intestinal microorganisms in Songliao black piglets.Songliao black pig is a local pig breed in northeast China.Its excellent growth performance benefits from its intestinal microorganisms.Therefore,the intestinal microorganisms of Songliao black pigs have great value.In order to screen safe and antibiotic-sensitive probiotics,weaning piglets fed with probiotics fermented Massa Medicata Fermentata were selected.Microorganisms from different digestive tract parts of piglets were isolated and cultured with five media(MRS medium,LB medium,Modified M17 medium,M2 GSC medium and PTYG medium).The isolates were classified according to bacterial colony morphology,gram staining and restriction fragment length polymorphism(RFLP).Then,hemolytic analysis was performed to screen the non-hemolytic strains.The isolates were identified by 16 S rRNA sequencing,and results showed that they belonged to four different phyla,including Deinococcus – Thermus.The similarity of three of these strains to the closest microorganisms in the NCBI database was 95.15%,95.81% and 97.25%,respectively,indicating that they may be new microorganisms.Then,the sensitivity of these strains to eight antibiotics(ampicillin,vancomycin,gentamicin,kanamycin,streptomycin,clindamycin,tetracycline and chloramphenicol)was analyzed by Disk diffusion test.Lactobacillus reuteri RAM0101(number: BMMA-MRS-2),Bacillus coagulans RAM1202(number: BMMA-MRS-3)and Clostridium butyricum RAM0216(number: BMJA-M2GSC-31)were sensitive to all of these antibiotics.(2)Study on the therapeutical effects of probiotics and traditional Chinese medicine on intestinal inflammation and injury in colitis mice.Dextran sulfate sodium(DSS)-induced colitis mouse model was used to evaluate the potential of multi-strain probiotic mixture(L.reuteri RAM0101,B.coagulans RAM1202,Bifidobacterium longum CICC6197 and C.butyricum RAM0216),MMF and E.humifusa in UC treatment.Sulfasalazine(SASP)was used as a positive control.The effects of different drugs on weight loss,diarrhea,hematochezia,histopathology,level of inflammatory factors and barrier function of colonic mucosa were analyzed.The results showed that the SASP reduced inflammatory cell infiltration and the level of pro-inflammatory cytokines,but SASP could not repair the damage of colonic mucosa and microbial ecology.MMF had no effects of DSS-induced colitis.E.humifusa alleviated disease activity index(DAI)and reduced mortality in colitis mice,but E.humifusa increased the proportion of pro-inflammatory bacteria in the colonic mucosa.The combination of the four probiotic strains significantly alleviated the symptoms of colitis in mice,decreased colonic injury and levels of pro-inflammatory cytokines and increased the intestinal barrier function.In addition,the probiotic mixture restored the proportion of core microbiota in colonic mucosa,reduced the proportion of pro-inflammatory bacteria and increased the proportion of beneficial bacteria,such as Bifidobacterium and Blautia.These results suggested that the multi-strain probiotic mixture was more beneficial to the repair of intestinal microecology than clinical anti-inflammatory drug and traditional Chinese medicines,and thus alleviated intestinal inflammation and damage more effectively.(3)The mechanism of probiotic mixture to improve intestinal inflammation and injury.On the basis of the above study,we further compared the therapeutic effect of single probiotics and multi-strain probiotic mixture on DSS-induced colitis,aiming to explore the mechanism of probiotic mixture in alleviating colitis.Results showed that the functions of the four probiotic strains were different in regulating intestinal immunity and barrier function.The four-species probiotic cocktail was more effective than the species individually and anti-inflammatory drugs in repairing the dysbiosis of mucosal microbial ecology and reducing intestinal inflammation.In addition,probiotic mixture significantly enhanced the expression of IL-10 and intestinal barrier function.Probiotic mixture reduced the expression of IL-22 and Reg3?,suggesting that probiotic mixture decreased the injury of intestinal barrier and the invasion of antigens.The above results indicated that the effect of probiotic mixture was due to the synergy of probiotics with different functions,which restored the balance of interaction between intestinal microorganisms and immunological niches.In conclusion,intestinal symbiotic microorganisms were isolated from digestive tract of Songliao black pigs,which had never been fed with antibiotics.Animal experiments showed that different probiotic strains regulated different intestinal immune response pathways and barrier functions.The four-species probiotic cocktail was more effective than the species individually and anti-inflammatory drugs in repairing the dysbiosis of mucosal microbial ecology and reducing intestinal inflammation.The combination of probiotics with different functions repaired the microbial ecology of intestinal mucosa,restored the balance of interaction between microbes and immunological niches and increased the expression of IL-10 and barrier function,thus alleviating intestinal inflammation and injury.