从肠道微生态探讨清肠化湿汤治疗溃疡性结肠炎黏膜损伤的机制研究

【作者】 李萍；

【导师】 汪红兵； 张会存；

【作者基本信息】 北京中医药大学 ， 中医内科临床（专业学位）， 2018， 硕士

【摘要】 目的和意义:通过体内外实验两种途径,证明清肠化湿汤可通过促进益生菌的生长及抑制致病菌的生长,恢复溃疡性结肠炎大鼠肠道菌群微生态平衡,抑制肠黏膜损伤,为清肠化湿法治疗溃疡性结肠炎提供依据和支撑。研究方法:1、体外实验:(1)将配置成0.5麦氏浓度的双歧杆菌及鼠李糖乳杆菌菌液,分别接种于双歧杆菌BS培养基(不含琼脂)、MRS培养基及6.25%,12.5%,25%,50%浓度的清肠化湿汤培养基中,37℃厌氧培养双歧杆菌14h,16h,18h以及鼠李糖乳杆菌12h,14h,16h后,稀释10倍,测其吸光度OD57。,比较OD对照与OD实验值评估其对双歧杆菌、鼠李糖乳杆菌体外生长的影响。(2)将已灭菌的浓度为3.8g/L(该浓度定义为100%)的清肠化湿汤提取液1mml与等体积营养肉汤混匀后加入24孔板第1孔中,以连续倍比稀释法稀释至第11孔,第12孔为不含清肠化湿汤的阳性对照组,以未接种大肠杆菌的相对应的中药培养基为空白组,加入0.5麦氏浓度的大肠杆菌、金黄色葡萄球菌、沙门氏菌,37℃恒温培养22h后,以目视比浊法观测抑菌效果,并于每孔中取适量混合液,以划线法37℃恒温箱中培养18h后观察杀菌效果。2、体内实验:将48只、雄性SD大鼠随机分成正常对照组,模型组,清肠化湿汤高、中、低剂量组,柳氮磺吡啶组,共6组。应用TNBS灌肠法制备溃疡性结肠炎大鼠模型,造模成功后,每天予清肠化湿汤高、中、低浓度及柳氮磺吡啶定时灌胃,持续12天后处死。通过对大鼠体质量、大便性状和隐血或肉眼血便情况进行评估疾病活动指数(DAI),肉眼观测肠道黏膜进行结肠黏膜损伤指数评分,并在显微镜下观察肠黏膜的病理改变。研究成果:1、6.25%、12.5%、25%、50%浓度的清肠化湿汤对双歧杆菌均具有促生长的作用(P<0.01),其中,25%浓度的清肠化湿汤促生长作用最明显。2、25%、50%浓度的清肠化湿汤在12h、14h时,对鼠李糖乳杆菌具有明显的促生长作用(P<0.01),但在16h出现抑制作用;6.25%、12.5%浓度的清肠化湿汤在12h时,对鼠李糖乳杆菌具有明显的促生长作用(P<0.05),在14h时促生长作用不明显(P>0.05),16h时出现抑制作用。3、清肠化湿汤对大肠杆菌、金黄色葡萄球菌有抑菌及杀菌作用,两者最低抑菌浓度依次为0.030g/L、0.007g/L,最低杀菌浓度依次为0.119g/L、0.238g/L;对沙门氏菌有抑菌作用,最低抑菌浓度为0.030g/L。4、清肠化湿汤可显著降低溃疡性结肠炎大鼠的DAI评分及结肠黏膜损伤指数评分,与模型组相比差异显著(P<0.05)。结论及意义:1、清肠化湿汤可促进双歧杆菌体外生长,对鼠李糖乳杆菌体外生长有双向调节作用。2、清肠化湿汤对大肠杆菌、金黄色葡萄球菌有抑菌及杀菌作用;对沙门氏菌有抑菌作用。3、清肠化湿汤可减轻溃疡性结肠炎大鼠的疾病活动情况及结肠黏膜损伤情况。更多还原

【Abstract】 Objective and significance:Through in vitro and in vivo experiments,it was proved that Qingchanghuashi Decoction can promote the growth of probiotics,inhibit the growth of pathogenic bacteria,restore the micro-ecological balance of intestinal microflora in rats with ulcerative colitis,and inhibit intestinal mucosal damage.To provide basis and support for the treatment of ulcerative colitis with clean up the intestinal tract and remove moisture method.Research methods:1.The in vitro experiments:(1)Bifidobacterium and Lactobacillus rhamnosus bacillus liquids configured to have a concentration of 0.5 McFarland were inoculated into Bifidobacterium BS medium(without agar)and concentrations of 6.25%,12.5%,25%,50%Qingchanghuashi Decoction culture medium,respectively.After anaerobic incubation at 37℃ for 14h,16h,18h of Bifidobacteria and Lactobacillus rhamnosus 12h,14h,16h of Lactobacillus rhamnosus,then diluted 10 times and absorbance OD570 was measured to compare OD control and OD the Chinese medicine value was evaluated for its influence on the growth of Bifidobacterium and Lactobacillus rhamnosus in vitro.(2)Add 1ml of Qingchanghuashi Decoction extract with sterilized concentration of 3.8g/L(defined as 100%)to an equal volume of nutrient broth and add it to the first well of 24-well plate.Diluted to the 11th well by serial multiple dilution method.The 12th well was a positive control group without Qingchanghuashi Decoction,and the corresponding Chinese herbal medicine culture medium not inoculated with Escherichia coli was used as a blank group.Each well was added with Escherichia coli,Staphylococcus aureus,and Salmonella at a concentration of 0.5 McFarland.After incubation at 37℃ for 22 hours,the bacteriostatic effect was observed with a visual turbidimetric method,and an appropriate amount of the mixture was taken in each well to draw a line.The bactericidal effect was observed after incubation in a 37℃ incubator for 18 hours.2.Forty-eight male Sprague-Dawley rats were randomly divided into normal control group,model group,high-,middle-,and low-dose Qingjiehuashi Decoction group,and sulfasalazine group,a total of 6 groups.A rat model of ulcerative colitis was prepared by TNBS enema.After the model was established,high-,middle-,and low-concentrations of Qingchanghuashi Decoction were regularly administered orally and the rats were sacrificed after 12 days.The disease activity index(DAI)was assessed by assessing the body mass,stool characteristics,occult blood or gross bloody stools,visually observing the intestinal mucosa for colonic mucosal injury index scores,and observing the pathological changes of the intestinal mucosa under a microscope.Research findings:1、The 6.25%,12.5%,25%,and 50%concentrations of Qingchanghuashi Decoction all had a growth-promoting effect on bifidobacteria(P<0.01).Among them,25%concentration of Qingchanghuashi Decoction had the most obvious effect on promoting growth.2、The 25%and 50%concentrations of Qingchanghuashi Decoction had obvious growth-promoting effect on Lactobacillus rhamnosus at 12h and 14h(P<0.01),but it showed inhibitory effect at 16h.The 6.25%,12.5%concentration Qingchanghuashi Decoction had significant growth promoting effect on Lactobacillus rhamnosus at 12h(P<0.05).It had no obvious growth promoting effect at 14h(P>0.05),it inhibited at 16h.3.Qingchanghuashi Decoction has bacteriostasis and bactericidal effect on Escherichia coli and Chrysosporium.The MIC of the two drugs was 0.030g/L and 0.007g/L in turn,MBC were 0.119g/L and 0.238g/L in turn.And has bacteriostasis effects on Salmonella,the MIC was 0.030g/L.4.Qingchanghuashi Decoction can significantly reduce the DAI score and colonic mucosal injury index score in rats with ulcerative colitis,and there is a significant difference compared with the model group(P<0.05).Conclusion and significance:1.Qingchanghuashi Decoction can promote the growth of Bifidobacterium in vitro and has a two-way effect on the growth of Lactobacillus rhamnosus in vitro.2.The Qingchanghuashi Decoction has antibacterial and bactericidal effects on Escherichia coli and Staphylococcus aureus,and it has antibacterial effect on Salmonella.3.Qingchanghuashi Decoction can reduce the disease activity and colon mucosal injury in rats with ulcerative colitis.更多还原