Study On Probiotic CMS-H002 Strain And Probiotic CMS-H003 Strain For The Therapy Of Mice With Acute Ulcerative Colitis

Chapter one Signature analysis of probiotic CMS-H002 strain and CMS-H003strainObjective:To investigate genetic stability and the drug'value of probioticCMS-H002 strain and CMS-H003 strain and their sensitiveness toantibiotic.Methods:1. Study of the survival activity of probiotic CMS-H002 strain andCMS-H003 strain in the imitating gastrointestinal environmentAntagonisic properties of probiotic CMS-H002 strain and CMS-H003strain from the seed bank of Hunan Yitai company to acid and bile saltwere detected in the imitating gastrointestinal environment, in whichhydrochloric acid, pepsin and bilis bovina were used.2. Analysis of genetic stability of probiotic CMS-H002 strain andCMS-H003 strain.If probiotic CMS-H002 strain and CMS-H003 strain had goodAntagonisic properties to acid and bile salt, their G+C mol% contentwould be detected to evaluate their genetic stability.3. Investigate the sensitiveness of probiotic CMS-H002 strain and CMS-H003 strain to antibiotic.Their sensitiveness to antibiotic were detected by K-B methodResults:1. After probiotic CMS-H002 strain and CMS-H003 strain werecultured in a PH value 3.0 or bile concentration 0.5% environment and ina PH value and bile gradient medium for four hours, their bacterialnumbers did not decrease significantly. Furthermore, their bacterialnumbers in the imitating gastrointestinal environment for four hourswhich were 1×10~8 cfu/ml and 1×10~7cfu/ml individiually did not decreasemarkedly too.2. The G+C mol% content of probiotic CMS-H002 strain andCMS-H003 strain who had experienced imitating gastrointestinalenvironment was 46.6% and 47.2% respectively, which was accord tooriginal identity result.3. Probiotie CMS-H002 strain was sensitive to thirty-three kinds ofantibiotic including carbenicillin and streptomycin and was not sensitiveto nine kinds of antibiotic including Cefuroxime. CMS-H003 strain wassensitive to eighteen kinds of antibiotic including alficetin andstreptomycin and was not sensitive to twenty-four kinds of antibioticincluding cidomycin.Conclusions:Probiotic CMS-H002 strain and CMS-H003 strain have good antagonisic properties to acid and bile salt. Probiotic CMS-H002 strainwhich G+C mol% content is 46.6% is sensitive to thirty-three kinds ofantibiotic including carbenicillin and streptomycin and is not sensitive tonine kinds of antibiotic including Cefuroxime in vitro. ProbioticCMS-H003 strain which G+C mol% content is 47.2% is sensitive toeighteen kinds of antibiotic including alficetin and streptomycin and isnot sensitive to twenty-four kinds of antibiotic including cidomycin invitro. Chapter two Pharmacodynamic study of probioticCMS-H002 strain and CMS-H003 strainObjective:To analysis the relationship between doses of probiotic and theirtreatment effect on UC, furthermore, to investigate the effect of probioticcombination in treating UC and their partial mechanisms.Methods:1. Establish mice acute UC modelColitis was induced by exposing BALB/c mice to 5% DSS indrinking water for 13 days.2 Dose-effect analysis of probioticsThe BALB/c mice were randomly divided into ten groups(controlgroup, model group, NS group, positive group(boalsalazide),higher doseprobiotic CMS-H002 strain group, middle dose probiotic CMS-H002strain and lower dose probiotic CMS-H002 strain group, higher doseprobiotic CMS-H003 strain group, middle dose probiotic CMS-H003strain group and lower dose probiotic CMS-H003 strain group). Colitiswas induced by exposing BALB/c mice to 5% DSS in drinking water for13 days. The control group and the model group did not accept anytreatment.NS group was infused with 0.4ml 0.9% NaCl per day. Thepositive group was infused with balsalazide (8mg/10g)per day. ThreeCMS-H002 groups in doses of 4.0×10~9cfu/10g, 4.0×10~7cfu/10g, 4.0×10~5 cfu/10g, and three CMS-H003 groups in doses of 4.0×10~7cfu/10g,4.0×10~5cfu/10g, 4.0×10~3 cfu/10g were administered orally once a dayindividually, beginning concurrently with exposure to DSS. The bodyweight and the character of feces as well as occult blood were observed toknow disease activity index of every mouse per day.The colon length wasmeasured and the colony tissue was handled with HE stained after 13days of treatment with DSS.3. Investigate the effect of probiotic CMS-H002 strain combinedwith probiotic CMS-H003 strainThe BALB/c mice were randomly divided into seven groups(controlgroup,model group, balsalazide group, probiotic CMS-H002 strain group,probiotic CMS-H003 strain group and combined treatment group). Thecontrol group and the model group did not accept any treatment. NS groupwas fed with 0.4ml 0.9% NaCl per day and probiotic CMS-H002 straingroup and probiotic CMS-H003 strain group as well as balsalazide groupwere fed with probiotic CMS-H002 strain (4.0×10~7cfu/10g), probioticCMS-H003 strain (4.0×10~7cfu/10g) and balsalazide (8mg/10g) per dayrespectively. The combined treatment group was fed with probioticCMS-H002 strain (4.0×10~9cfu/10g) and probiotic CMS-H003 strain(4.0×10~7cfu/10g)per day. The body weight and the character of feces aswell as occult blood were observed to know disease activity index ofevery mouse per day.At the first and last day, the mice feces were handled with analysising flora. Thirteen days later, all mice weresacrificed, the colon length and weight were measured, and histologicaldamage and myeloperoxidase (MPO) activity were assessed in colonictissue,furthermore, the colony tissue was handled with HE stained.Results:1 The result of the dose-effect of probioticsAfter BALB/c mice drank 5% DSS water freely for 13 days,thelength of the colon was shortened from 10.85cm to 8.65cm (P＜0.05), anddisease activity index (DAI)rose from 0 to 8.4.Oral administration ofdifferent doses of probiotic CMS-H002 strain from 4.0×10~9cfu/10g to4.0×10~5cfu/10g and different doses of probiotic CMS-H003 strain a from4.0×10~7cfu/10g to 4.0×10~3cfu/10g significantly suppressed the shorteningof colon length(P＜0.05) and the level of DAI and markedly improvedcolonic histological damage. Our study showed that the higher doses ofprobiotics were used for UC mice, the better therapeutic effects wereproduced,for example the length of the colon and the level of DAI fromthe higher dose probiofic groups were similar to those of normal group.The lower dose probiotics had less influence on colon length(P＜0.05) andthe level of DAI, but their therapeutic effects were similar to those ofbalsalazide, and better than those of NS group (P＜0.05). In a word, thehigher dose probiotics had most influence on above indexes in all dosesof probiotic CMS-H002 and CMS-H003 strain. 2 The effects of combined treatmentThere were the lightest histological damage and the best generalstate of health in probiotic CMS-H002 strain combined with probioticCMS-H003 strain,in which group the length of colon was 9.98cm and theweight of colon was 0.142g/g and the level of DAI changed from 1.0 to2.0 and histological score was 1.9.3. The result of faecal flora analysisAfter BALB/c mice drank 5% DSS solution freely for 13 days,faecalbacterium numbers of lactobacilli were decreased significantly, comparedto those of normal group,however the bacterium numbers ofenterococcus faecalis and escherichia were increased markedly, comparedto their first day bacterium numbers and the bacterium numbers ofbacteroid and staphylococcus aureaus and bifidobacteria did not changesignificantly during modeling. When BALB/c mice drinking 5% DSSsolution were given probiotic CMS-H002 and CMS-H003 strain at thesame time, we discovered that the faecal flora from combinationtreatment group was the closest to the normal faecal flora in all treatmentgroups,in which group the bactserium numbers of lactobacilli andbifidobacteria and clostridium butyricum were increased significantly andthe bacterium numbers of escherichia and enterococcus faecalis weredecreased markedly, compared to their first day bacterium numbersindividually, furthermore the bacterium numbers of bacteroidstaphylococcus aureaus did not change significantly during treatment.Balsalazide had been used to cure UC patients, but we discovered balsalazide had little influence on mice faecal flora in our experiment,sodid NS.4. The result of colon tissue MPO activityAfter BALB/c mice drank 5% DSS water freely for 13 days,theactivity of MPO of colon was increased markedly, other treatment drugsexcept for NS could decrease the activity of MPO of colonictissue, furthermore we discovered that the combination, treatment hadmore influence on the activity of MPO than probiotic CMS-H002 strainand probiotic CMS-H003 strain alone.Conclusions:1. The improved method of establishing UC model is not onlysimple and economic but also have the lower mortality rate than originalmethod and have the symptoms and colonic tissue pathology charactersimilar to humanbeing UC patients, moreover that is suitable formechanism study of the severe and moderate UC and the drug's functionfurther.2. Probiotic CMS-H002 and CMS-H003 strain by oral administrationcan alleviate DSS-induced ulcerative colitis and combination treatmentshows the coeffects on DSS-induced mice acute ulcerative colitis andanti-inflammation and regulating abnormal flora are their possiblemechanism of treating UC. Chapter three Mechanism of combination of probioticCMS-H002 and CMS-H003 strain in treating ulcerative colitisObjective:To elucidate the mechanism of the UC and the drug's functionfurther.Methods:Thirteen days later, all mice were sacrificed, colonic tissue were reservedto detect the expression of cytokines in the colonic mucosa byimmunohistochemical staining, EUlSA, western-blotting and semi-quantatitivereserve transcriptional (RT)PCR respectively.Results:1. After BALB/c mice drank 5% DSS water freely for 13 days, theexpression of EMAP-Ⅱ,TNF-alpha,IL-1beta,IL-6,MCP-1,KC,P-selection, TF and Caspase-1 in the colonic mucosa were significantlyincreased and the expression of PCNA were markedly decreased.2. Balsalazide, probiotic CMS-H002 strain and probiotie CMS-H003strain by oral administration significantly suppressed the expression ofEMAP-Ⅱ,TNF-alpha,IL-1beta,IL-6,MCP-1,KC,P-selection,TF and Caspase-1 and promoted the expression of PCNA in the colonicmucosa.3. The expression of EMAP-Ⅱ,TNF- alpha,IL-1beta,IL-6, P-selection,TF and Caspase-1 was lower and the expression PCNA washigher in the colonic mucosa of combination treatment group than thosein the balsalazide and probiotic groups respectively(P＜0.05).Conclusions:1. Probiotic CMS-H002 and CMS-H003 strain may cure UC byalleviating inflammation, inhibiting thrombogenesis,stopping apoptosisand promoting proliferation.2. EMAP-Ⅱperhaps plays some important roles in the course ofonset of UC, because EMAP-Ⅱhas the property of pro-inflammationand chemotaxis, by which the inflammation of colon is launched and itcan also regulates up the expression of other pro-inflammation cytokines,adherence factors and tissue factor, by which the inflammation of colon isenlarged many times in the short time. Probiotic CMS-H002 andCMS-H003 strain can treat UC partly by down-regulating it's expression3. There are lower expression of EMAP-Ⅱ,TNF-alpha,IL-1beta,IL-6,P-selection,TF and Caspase-1 in the combination group than thatin CMS-H002 and CMS-H003 strain group alone,which may be themolecule mechanism of the coeffect on mice UC.