The Therapeutic Effects And The Influence Of Bacteria Flora Of Different Concentration Of Lactobacillus Acidophilus On Experimental Colitis In Balb/c Mice And The Research Of The Mechanism

Chapter One The therapeutic effects of different concentration of Lactobacillus acidophilus on experimental colitis in Balb/c miceObject:In order to find the appropriate concentration of Lactobacillus acidophilus for experimental colitis,we have investigated the therapeutic effects under different concentrations of Lactobacillus acidophilus on the DSS-induced mouse model of experimental colitis in Balb/c mice.Methods:A total of 72 Balb/c mice were enrolled in this experiment, 6-8-week-old females weighing 20.0±2.0g.These mice were purchased from the Dongchuang Scientific Service Department of Experimental Animals in Hunan Agricultural University and raised in animal rooms of clean grade.Mice were randomly divided into 9 groups,n=8 in each group,reared under standard conditions(humidity 50%±10%;12h daytime/12h night rotation) and fed with ordinary mouse forage.Groups were as follows:(1) Model group:drinking 5%DSS for modeling;(2) NS group:5%DSS for modeling + NS(1mL/10g) by gavage;(3)(C4 group): 5%DSS for modeling +1×10~4CFU/10g Lactobacillus acidophilus by gavage;(4)(C5 group):5%DSS for modeling +1×10~5CFU/10g Lactobacillus acidophilus by gavage;(5)(C6 group):5%DSS for modeling +1×10~6 CFU/10g Lactobacillus acidophilus by gavage;(6)(C7 group):5%DSS for modeling +1×10~7CFU/10g Lactobacillus acidophilus by gavage;(7)(C8 Group):5%DSS for modeling +1×10~8CFU/10g Lactobacillus acidophilus by gavage;(8) Prednisone acetate group:5%DSS for modeling + prednisone acetate(45ug/10g) by gavage;(9) Normal group:normal diet without special treatment.We observed the general conditions of mice,like food intake,drinking and activities,and weighed the mice daily;we also observed their fecal properties,fecal occult blood and hemafecia,calculated their DAI scores and assessed the severity of colitis everyday.At day 7,mice were executed under ether anesthesia and their abdomens were anatomized,for observation of the overall changes of the colon and measurement of lengths.About 0.Scm colonic segments were respectively cut 1cm to the ileocecum and anus for bacterial analyses;about 0.5cm colonic structure adjacent to the anus was cut and fixed in neutral formalin and sliced into paraffin-embedded sections for H&E staining;the colonic remnant was preserved with liquid nitrogen for other use.Results:Moderate concentration of Lactobacillus acidophilus(C6 group,10~6CFU/ml concentration level) obvious relieved the symptom of experimental colitis,and had the lowest DAI scores and histological scores. Conclusion:The therapeutic effects of Lactobacillus acidophilus on experimental colitis might correlate with the high concentration of Lactobacilli in colon.The phenomenon that C6(10~6CFU/ml concentration level) group had the best therapeutic effect suggested that there should be a correlation with Lactobacilli.Our further study will focus on the relationship between Lactobacillus acidophilus of gavage and the Lactobacilli in colon. Chapter Two The influence of bacteria flora in proximal and distal colon on experimental colitis in Balb/c mice with different concentration of Lactobacillus acidophilusObject:Aim to investigate the influence of bacteria flora in proximal and distal colon on experimental colitis in Balb/c mice with different concentration of Lactobacillus acidophilus.Method:The design principle of animal was the same as chapter one.On day 7,mice were executed under ether anesthesia and their abdomens were anatomized.Long colonic segments of a respective 0.5cm were cut at 1cm respectively to the ileocecum and anus,and diluted with NS by the serial dilution method after weighing and homogenate with a high-pressure and temperature sterilized homogenizer. Three types of representative anaerobic bacteria(Lactobacilli, Bifidobacteria and Bacteroides),three types of aerobic bacteria (Staphylococcus aureus,Escherichia coli and Enterococci) and total aerobic bacteria were chosen from the intestinal flora.Anaerobic bacteria (Lactobacilli and Bifidobacteria) and Enterococci were tested by the API test bar.And the digital maps were applied to determine the outcome of identification.Result:The amount of bacteria in the proximal colon of all groups had no significant difference in 7 culture media.Proximal colonic bacteria of various experimental groups were insignificantly different from each other in concentrations and the number of Lactobacilli and Bifidobacteria in mice of the distal colon was the highest in the C6 (10~6CFU/ml-concentration level) group(p<0.05),Staphylococcus aureus the lowest(p<0.05)and other bacteria insignificantly different..Conclusion:1.Lactobacillus acidophilus can't permanent plant on the proximal colon,and it has no effect on the microflora on the proximal colon.2.Lactobacillus acidophilus can permanent plant on the distal colon and it will increase the amount of lactobacilli and Bifidobacteria, decrease the amount of Staphylococcus aureus.The 10~6CFU/mL concentration level Lactobacillus acidophilus exerts the best effect on experimental colitis and the impact on other bacteria. Chapter Three The effect of Lactobacillus acidophilus on experimental colitis in the Th17/IL23 axisObject:Aim to investigate immune mechanism of different concentrations of lactobacillus acidophilus on experimental colitis.We focus on the effect on main cytokines of Th17/IL23 axis:IL17,IL23, TGFβ,TNFa,STAT3 and P-STAT3 to provide further therapy for the treatment of experimental colitis.Method:The design principle was the same as chapter one.On the 7 day,mice were executed under ether anesthesia and their abdomens were anatomized,the colon which was preserved with liquid nitrogen was tested by RT-PCR and western blotting for cytokine IL17,IL23,TGFβ, TNFa,STAT3,P-STAT3.The colon which was cut and fixed in neutral formalin and sliced into paraffin-embedded sections was tested by immunohistochemistry for cytokine STAT3,P-STAT3.Result:The expression of all the tested factors in mRNA and protein level as follows:the model groups had the highest expression level of all the factors,followed by the NS group.The prednisone group had significant decrease of the expression of all the factors.The result of different concentrations of lactobacillus acidophilus as follows:The 10~5CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression level of IL23 and TGFβ,the factor which was related by the differentiation of Th17 cell,which was significant different from the prednisone group(p<0.05) The 10~5CFU/mL-concentration level and 10~6CFU/mL concentration level of Lactobacillus acidophilus had the lowest expression level of STAT3 and P-STAT3,the signal factor which was related by the differentiation of Th17 cell,which was significant different from the prednisone group(p<0.05).In the RT-PCR and immunohistochemistry test,the 10~6CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression level of STAT3, while in the WB test,the 10~5CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression amount of STAT3 in protein level.Contrary with the protein level of P-STAT3,the 10~6CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression level tested by the immunohistochemistry method which was significant different from the prednisone group(p<0.05) and the 10~5CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression level tested by the WB method which was significant different from the prednisone group(p<0.05)The 10~5CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression level of IL17,the pro-inflammatory factor which was related by the incidence of experimental colitis,which was significant different from the prednisone group(p<0.05).While the 10~6CFU/mL-concentration level of Lactobacillus acidophilus had the lowest expression level of TNFa,the pro-inflammatory factor which was also related by the incidence of experimental colitis,which was significant different from the prednisone group(p<0.05)Conclusion:1.Lactobacillus acidophilus could reduce the expression of the cytokine which was related by the differentiation of Th17 cell(IL23,TGFβ),the signal factor which was related by the differentiation of Th17 cell(STAT3,P-STAT3) and the pro-inflammatory factor which was also related by the incidence of experimental colitis(IL17,TNFa).The 10~6CFU/mL-concentration level of Lactobaciltus acidophilus had obvious effects of the above mentioned factors.