Screen Of Probiotics To Prevention And Cure To Ulcerative Colitis And Part Of Research Of The Mechanism

Chapter oneFlora analysis of ulcerative colitis patients and health controlsObjective:To investigate the changes of fecal flora in ulcerative colitis patients.Methods:The fecal flora is analyzed by gradient dilution method. Results:Enterobactefia(8.55±0.34,P＜0.05),staphylococcus aureus(8.31±0.52,P＜0.05)as well as the total count of aerobian (8.76±0.68,P＜0.05)is increased significantly in acute UC patients compared with controls,while Bifidobacterium(8.06±0.42,P＜0.05)and Lactobacillus(7.95±0.62,P＜0.05)as well as the total count of anaerobian(8.39±0.38,P＜0.05)is reduced sharply,but Bacteroides (8.34±0.41,P＜0.05)is increased.Conclusions:Dysbacteria is founded by the analysis of fecal flora in acute ulcerative colitis patients,Enterobacteria and staphylococcus aureus increase while Bifidobacterium and Lactobacillus decrease. Chapter two Screen assay of probiotics to prevent and cure to ulcerative colitisObjective:Screen probiotics to effectively treat experimental colitis,and evaluate the curative effect,approach their partial mechanisms.Methods:1.Establish acute mice UC modelColitis was induced by exposing BALB/c mice to 5%DSS in drinking water for 13 days.2.Investigate the effect of probiotics80 BALB/c mice were randomly divided into eight groups(normal control group,model control group,normal saline control group, balsalazide group,probiotic CMS-H002 strain group,probiotic CMS-H003 strain group,probiotic CMS-H005 strain group and probiotic CMS-H006 strain group).The normal control group and the model group did not received any treatment.NS group was fed with 0.4ml 0.9%NaCl per day and probiotic CMS-H002 strain group,probiotic CMS-H003 strain group,probiotic CMS-H005 strain group and probiotic CMS-H006 strain group as well as balsalazide group were fed with probiotic CMS-H002 strain(4.0×10¹⁰cfu/10g),probiotic CMS-H003 strain (4.0×10¹⁰cfu/10g),probiotic CMS-H005 strain(4.0×10¹⁰cfu/10g), probiotic CMS-H006 strain(4.0×10¹⁰cfu/10g)and balsalazide (8mg/10g)per day respectively.The body weight and the character of feces as well as occult blood were observed.At the first and last day,the mice feces were handled with analysising flora.Thirteen days later,all mice were sacrificed,the colon length and weight were measured, histological damage and myeloperoxidase(MPO)activity were assessed in colonic tissue,furthermore,the colony tissue was handled with HE stained and electron microscope.Results:1.Occult blood occurred on the fourth day,bloody stools occurred on the seventh day after drinking DSS in model group,these symptoms are similar to typical model;the colon length is shortened,colon wet weight is increased,large haematocele can be seen in enteric cavity at the experiment termination on the thirteenth day.Our model is successful with the envidence of histopathology and electron microscope.2.Each index is insignificant in NS group compared with model group.Each index such as body weight,colon length,colon wet weight,DAI index,histopathology,ultrastructure in balsalazide group and probiotics groups is better than model group.General therapeutic effect of CMS-H002group,CMS-H003group,CMS-H005group is equilent to balsalazide group,but each index of CMS-H005group is better than balsalazide group.3.The mRNA and protein expression of IL-1βin normal control is very little,yet model control is highest,NS control is insignificant to model group,all of balsalaside group and each probiotics group are less than model group;The mRNA and protein expression of IL-4 in normal control is highest,yet model control is least,all of balsalaside group and each probiotics group are higher than model group,and probiotics group is higher than balsalazide group.3.The result of faecal flora analysisAfter BALB/c mice drank 5%DSS solution freely for 13 days,faecal bacterium numbers of lactobacilli were decreased significantly,compared to those of normal group,however the bacterium numbers of enterococcus faecalis and escherichia were increased markedly,compared to their first day bacterium numbers and the bacterium numbers of bacteroid and staphylococcus aureaus and bifidobacteria did not change significantly during modeling.When BALB/c mice drinking 5%DSS solution were given probiotic CMS-H002,CMS-H003,CMS-H005,CMS-H006 strains,we discovered that the faecal flora from CMS-H006 group was the closest to the normal faecal flora in all treatment groups,in which group the bactserium numbers of lactobacilli and bifidobacteria and clostridium butyricum were increased significantly and the bacterium numbers of escherichia and enterococcus faecalis were decreased markedly,compared to their first day bacterium numbers individually, furthermore the bacterium numbers of bacteroid staphylococcus 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,so did NS.4.The result of colon tissue MPO activityAfter BALB/c mice drank 5%DSS water freely for 13 days,the activity of MPO of colon was increased markedly,other treatment drugs except for NS could decrease the activity of MPO of colonic tissue,and the activity of probiotic CMS-H006 strain group is the lowest.5.The mRNA and protein expression of IL-1βin normal control is very little,yet model control is highest,NS control is insignificant to model group,all of balsalaside group and each probiotics group are less than model group;The mRNA and protein expression of 1L-4 in normal control is highest,yet model control is least,all of balsalaside group and each probiotics group are higher than model group,and probiotics group is higher than balsalazide group.Conclusions:1.The improved model of DSS induced colitis is successful,the improved method of establishing UC model is not only simple and economic but also have the lower mortality rate than original method and have the symptoms and colonic tissue pathology character similar to human being UC patients.2.Probiotics CMS-H002,CMS-H003,CMS-H005,CMS-H006 strains by oral administration can alleviate DSS-induced ulcerative colitis,the curative effect is equivalent or surpass to balsalazide,and CMS-H006 strain is the best.Regulating abnormal flora in intestinal and anti-inflammation are their possible mechanisms of treating UC. Chapter three Effect of CMS-H006 Strain on Expression of TLR2,TLR4 in Colonic Mucosa of UC MiceObjective:To investigate the effect of CMS-H006 strain on expression of Toll like recep tor(TLR)2,4 in colonic mucosa of ulcerative colitis(UC)mice.Methods:60 mice were divided into six groups as follows:normal controlgroup(NC),model control group(MC),normal salinegroup(NS), balsalazide group(B),probiotics CMS-H006 group(P)and antibiotics group(A),TLR2,TLR4 expression in colonic mucosa was measured by reverse transcription polymerase chain reaction(RT-PCR)and Western blot,at the same time,IL-lbeta and IL-4 expression was measured by ELISA and RT-PCR.Results:1.Symtoms of antibiotics group is earlier and severer than model group,3 mice were killed every time at the fourth day,the seventh day,one mouse died at the sixth day,the other 3 mice all died before the end of the experiment.2.At the fourth day,the seventh day,the thirteenth day after drinking 5%DSS,the expression of IL-lbeta in colon mucosa ascend in each group,while the expression of IL-4 in colon mucosa descend;At every time,the expression of IL-lbeta in probiotics group is the lowest,while the expression of IL-4 is the highest;At the fourth day and the seventh day,the expression of IL-lbeta of antibiotics group is the highest,while the expression of IL-4 is the lowest,and decends obviously.3.At the fourth day,the seventh day,the thirteenth day after drinking 5%DSS,the expressions of TLR2 and TLR4 ascend in all groups including model group,probiotics group and antibiotics group,while the expression of probiotics group is the lowest,the antibiotics group is the highest.Conclusions:1.Refrain the expression of IL-lbeta of colon mucusa,prevent the descending of IL-4,adjust the disequilibrium between the proinflammatory factor and anti-inflammatory factor maybe one of the mechanisms of the treatment to UC with probiotic CMS-H006 strain.2.Down regulate the over expression of TLR2 and TLR4 maybe one of the mechanisms of the treatment to UC with probiotic CMS-H006 strain.3.Oral administration with broadspeetrum antibiotics aggravate the symptoms of UC mice induced by DSS,the ascendment of the proinflammatory factor IL-lbeta and descendment of anti-inflammatory factor IL-4 accompanied by the over expression of TLR2 and TLR4 maybe involved in the mechanism,but advanced research should be done.