中药联合益生菌对大肠杆菌性小鼠腹泻的保护作用

doi:10.16431/j.cnki.1671-7236.2022.08.038

摘要/Abstract

摘要： 【目的】研究中药联合益生菌对大肠杆菌性小鼠腹泻的保护作用。【方法】通过正交试验筛选中药联合益生菌最佳因素配比。取60只小鼠随机分为空白组、模型组、中药组、益生菌组和中药联合益生菌组,每组12只。空白组小鼠腹腔注射生理盐水(0.2 mL/只),模型组、中药组、益生菌组和中药联合益生菌组均先腹腔注射分离菌悬液1×10⁷ CFU/kg,然后中药组按10 mL/kg灌胃中药液(其中蒲公英提取物浓度为0.25 g/mL,黄芪总黄酮和黄芪多糖浓度均为0.05 g/mL),益生菌组按4 mL/kg灌胃枯草杆菌悬液(菌含量5×10⁷ CFU/mL),中药联合益生菌组按上述量灌胃中药液和枯草杆菌混合液,每天定时灌胃给药1次,连续3 d。每日记录小鼠腹泻、体重、采食量等情况。末次给药12 h后分离心脏、肝脏、肾脏、脾脏、肺脏,称重并计算脏器系数;血液分析法检测血液学指标;取粪便及小肠内容物检测粪便隐血情况并进行细菌计数;ELISA法检测小鼠血清和小肠组织中髓过氧化物酶(MPO)活性。【结果】中药联合益生菌最佳因素配比为A₁B₂C₂D₂。与空白组相比,模型组小鼠腹泻率100%且无自愈现象,症状和剖检病变明显,腹泻指数极显著上升(P＜0.01),体重、采食量均极显著下降(P＜0.01),肝脏、脾脏器官指数均显著升高(P＜0.05),淋巴细胞比率(LYM)、中间细胞比率(MID)均显著下降(P＜0.05),粒细胞比率(GRAN)极显著上升(P＜0.01);与模型组相比,中药组和中药联合益生菌组腹泻指数极显著下降(P＜0.01),体重、采食量显著上升(P＜0.05),中药组肝脏、脾脏器官指数显著下降(P＜0.05),中药联合益生菌组脏器系数极显著下降(P＜0.01),中药组、益生菌组及中药联合益生菌组LYM显著上升(P＜0.05)、GRAN显著下降(P＜0.05)。粪便隐血检测结果显示,模型组检测呈强阳性;中药组、益生菌组呈弱阳性,中药联合益生菌组呈阴性。与空白组相比,模型组小鼠肠内大肠杆菌数、血清及小肠组织中MPO活性均极显著上升(P＜0.01);与模型组相比,中药组和中药联合益生菌组小鼠肠内大肠杆菌数、血清及小肠组织中MPO活性均显著下降(P＜0.05)。【结论】中药联合益生菌可降低腹泻指数、肝脏和脾脏器官指数,增加体重、饮食量,使粪便隐血转阴并降低肠内大肠杆菌数、血清及小肠组织中MPO活性,从而对大肠杆菌性小鼠腹泻产生协同保护作用。

关键词: 中药联合益生菌; 细菌性腹泻; 小鼠; 大肠杆菌

Abstract: 【Objective】 The purpose of this experiment was to study the protective effect of traditional Chinese medicine (TCM) combined with probiotics on E.coli diarrhea in mice.【Method】 The best factor ratio of TCM combined with probiotics was selected by orthogonal test. 60 mice were randomly divided into blank group, model group, TCM group, probiotics group and TCM combined probiotics group, with 12 mice in each group. Mice in blank group were intraperitoneally injected with normal saline (0.2 mL/mouse), model group, TCM group, probiotics group and TCM combined probiotics group were intraperitoneally injected with E.coli isolate 1×10⁷ CFU/kg. The TCM group was gavaged with mixed TCM solution for 10 mL/kg(the concentration of dandelion extract was 0.25 g/mL, and the concentration of total flavonoids of Astragalus membranaceus and Astragalus polysaccharides were 0.05 g/mL), the probiotic group was gavaged with Bacillus subtilis suspension for 4 mL/kg (Bacillus subtilis contents 5×10⁷ CFU/mL), the TCM combined with probiotics group was gavaged with the mixture of TCM and Bacillus subtilis, they were given by gavage once a day for 3 days. The diarrhea, body weight and dietary quantity of the mice were recorded every day. 12 hours after the last administration, the heart, liver, kidney, spleen and lung were separated, weighed and the viscera coefficient were calculated. Hematological indicators were analyzed by blood test. Fecal and small intestine contents were taken to observe fecal occult blood and count bacteria. The activity of MPO in serum and small intestine were detected by ELISA.【Result】 The best factor ratio of TCM combined with probiotics was A₁B₂C₂D₂. The results showed that compared with blank group, the diarrhea rate of the model group was 100% without self-healing and there were obvious symptoms and pathological changes. The diarrhea index was extremely significantly increased (P<0.01), body weight and dietary amount were extremely significantly decreased (P<0.01), liver and spleen indexes were increased (P<0.05), the lymphocyte ratio and intermediate cell ratio were decreased (P<0.05) and the granulocyte ratio were extremely significantly increased (P<0.01) in model group. Compared with model group, diarrhea index in TCM group and TCM combined with probiotics group was extremely significantly decreased (P<0.01), body weight and dietary quantity were increased (P<0.05), liver and spleen indexes in TCM group were decreased (P<0.05), viscera indexes in TCM combined with probiotics group were extremely significantly decreased (P<0.01). LYM was increased significantly (P<0.05) and GRAN was decreased significantly (P<0.05) in TCM group, probiotics group and TCM combined probiotics group. Hematological indexes were greatly improved (P<0.05). Fecal occult blood test results showed that model group was strong positive, TCM group and probiotics group were weak positive, while TCM combined with probiotics group was negative. Compared with blank group, the number of E.coli in intestine in model group was extremely significantly increased (P<0.01), and the activity of MPO was extremely significantly increased (P<0.01). Compared with model group, the number of E.coli in intestine and the activity of MPO in serum and small intestine of mice in TCM group and TCM combined probiotics group were significantly decreased (P<0.05).【Conclusion】 TCM combined with probiotics can reduce diarrhea index, liver and spleen indexes, increase body weight and diet, turn fecal occult blood into negative, and reduce the number of E.coli in intestine and the activity of MPO in serum and small intestine, so as to provide a synergistic protective effect on E.coli induced diarrhea in mice.

Key words: traditional Chinese medicine (TCM) combined with probiotics; bacterial diarrhea; mice; E.coli

中图分类号:

S859.7

李春亭, 王沙沙, 赵欣, 王巍, 葛冰洁, 张雪梅. 中药联合益生菌对大肠杆菌性小鼠腹泻的保护作用[J]. 中国畜牧兽医, 2022, 49(8): 3226-3234.

LI Chunting, WANG Shasha, ZHAO Xin, WANG Wei, GE Bingjie, ZHANG Xuemei. Protective Effect of Traditional Chinese Medicine Combined with Probiotics on E.coli Diarrheain Mice[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3226-3234.

参考文献

[1] LAWLER J V, WALLACE M R. Diagnosis and treatment of bacterial diarrhea[J].Current Gastroenterology Reports, 2003, 5(4):287-294.
[2] 张龙舟,高青山,崔莲花,等.基于肠道菌群调控治疗动物腹泻的相关研究进展[J].饲料研究, 2021, 4(12):108-112. ZHANG L Z, GAO Q S, CUI L H, et al. Research progress in the treatment of animal diarrhea bases on the regulation of intestinal flora[J]. Feed Research, 2021, 44(12):108-112.(in Chinese)
[3] 屈平平.应用噬菌体治疗畜禽细菌性腹泻研究进展[J].畜牧兽医科学(电子版), 2021,7:33-34. QU P P. Research progress of bacteriophage in the treatment of livestock bacterial diarrhea[J]. Graziery Veterinary Sciences (Electronic Version), 2021,7:33-34.(in Chinese)
[4] 吴陈俊.禽大肠杆菌病的分析、诊断和防治[J].现代畜牧科技, 2021,9:153-154. WU C J. Analysis, diagnosis and prevention of avian Escherichia coli disease[J]. Modern Animal Husbandry Science&Technology, 2021,9:153-154.(in Chinese)
[5] 王哲,潘帮婷,迟长安,等.燥湿止泻颗粒对大肠杆菌引起腹泻小鼠血清和小肠组织中MPO的影响[J].养殖与饲料, 2020, 19(8):22-24. WANG Z, PAN B T, CHI C A, et al. Effect of dryness dampness Zhixie granule on MPO in serum and small intestine of diarrhea mice caused by E.coli[J]. Animals Breeding and Feed, 2020, 19(8):22-24.(in Chinese)
[6] DENG Y, HAN X, TANG S, et al. Magnolol and honokiol attenuate apoptosis of enterotoxigenic Escherichia coli-induced intestinal epithelium by maintaining secretion and absorption homeostasis and protecting mucosal integrity[J]. Medical Science Monitor, 2018, 24:3348-3356.
[7] MOSQUITO S, RUIZ J, BAUER J C L, et al.Molecular mechanisms of antibiotic resistance in Escherichia coli-associated diarrhea[J]. Revista Peruana De Medicina Experimental Y Salud Publica, 2011, 28(4):648-656.
[8] ROUSSEL C, SIVIGNON A, DEWIELE T V, et al. Foodborne enterotoxigenic Escherichia coli:From gut pathogenesis to new preventive strategies involving probiotics[J]. Future Microbiology, 2017, 12:73-93.
[9] LI L, CUI H, LI T, et al. Synergistic effect of berberine-based Chinese medicine assembled nanostructures on diarrhea-predominant irritable bowel syndrome in vivo[J]. Frontiers Pharmacology, 2020, 11:1210.
[10] XU P, XU X B, KHAN A, et al. Antibiofilm activity against Staphylococcus aureus and content analysis of Taraxacum Officinale phenolic extract[J]. Polish Journal Veterinary Sciences, 2021, 24(2):243-251.
[11] LABED A, FERHAT M, LABED Z I, et al. Compounds from the pods of astragalus armatus with antioxidant, anticholinesterase, antibacterial and phagocytic activities[J]. Pharmaceutical Biology, 2016, 54(12):3026-3032.
[12] 陈奥运,吴明谦,孟伯龙,等.枯草芽孢杆菌与乳酸杆菌联合应用对断奶仔猪肠道菌群的影响[J].畜牧与兽医, 2017, 49(4):61-64. CHEN A Y, WU M Q, MENG B L, et al. Effects of joint application of Bacillus subtilis and Lactobacilli on intestinal flora in weaned piglets[J]. Animal Husbandry&Veterinary Medicine, 2017, 49(4):61-64.(in Chinese)
[13] 柳成东,肖明霞,李琦华,等.饲用益生菌凝结芽孢杆菌抑菌性能的研究[J].中国畜牧杂志,2021, 57(10):236-241. LIU C D, XIAO M X, LI Q H, et al. Study on bacteriostatic performance of feeding probiotics Bacillus coagulans[J]. Chinese Journal of Animal Science, 2021, 57(10):236-241.(in Chinese)
[14] 潘兴亮,王玉田,郑瑞峰,等.抑菌型枯草芽孢杆菌在蛋鸡生产上的应用效果评价[J].当代畜禽养殖业, 2020,12:5-6. PAN X L, WANG Y T, ZHENG R F,et al. Evaluation of application effect of bacteriostatic Bacillus subtilis in laying hens[J]. Modern Animal Husbandry, 2020,12:5-6.(in Chinese)
[15] 秦枫,刘云,吴植,等.中药复方对大肠杆菌感染雏鸭的防治效果研究[J].中国畜牧兽医, 2020, 47(10):3371-3378. QIN F, LIU Y, WU Z, et al. Study on the prevention and control effect of Chinese herbal compound on E.coli infected ducklings[J]. China Animal Husbandry&Veterinary Medicine, 2020, 47(10):3371-3378.(in Chinese)
[16] HARRIES J T. The problem of bacterial diarrhoea[J]. Ciba Foundation Symposium, 1976,(42):3-25.
[17] RAHIMZADEH G. Encapsulation of bacteriophage cocktail into chitosan for the treatment of bacterial diarrhea[J]. Scientific Reports, 2021, 11(1):15603.
[18] 邹素华.葎草提取物对羔羊血液生化指标及体液免疫的影响[D].郑州:河南农业大学, 2010. ZOU S H. Effects of humulus scandens extract on blood biochemical indices and humoral immunity of lambs[D]. Zhengzhou:Henan Agricultural University, 2010.(in Chinese)
[19] GOMES T A, ELIAS W P, SCALETSKY I C, et al. Diarrheagenic Escherichia coli[J]. Brazilian Journal of Microbiology, 2016, 47:3-30.
[20] 谷巍,孙明杰,王丽荣,等.嗜酸乳杆菌发酵复方中药成分变化对腹泻小鼠的影响[J].中国预防兽医学报, 2019, 41(5):520-525. GU W, SUN M J, WANG L R, et al. Effect of changes in traditional Chinese medicine components of Lactobacillus acidophilus fermented compound on diarrhea mice[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(5):520-525.(in Chinese)
[21] MA Y, ZHANG Q, LIU W, et al. Preventive effect of depolymerized sulfated galactans from eucheuma serra on enterotoxigenic E.coli caused diarrhea via modulating intestinal flora in mice[J]. Marine Drugs, 2021, 19(2):80.
[22] 曹玲芝.四君子汤对小鼠大肠杆菌性腹泻防治机制的研究[D].保定:河北农业大学, 2009. CAO L Z. The Study on prevention mechanism of Sijunzi tang on E.coli induced diarrhea of mice[D]. Baoding:Hebei Agricultural University, 2009.(in Chinese)
[23] 张晓利.白头翁复方对腹泻小鼠肠道粘膜双糖酶的影响[D].保定:河北农业大学, 2010. ZHANG X L. The effects of compound radix pulsatillae on the activities of disaccharidase in intestinal mucous in diarrhea mice[D]. Baoding:Hebei Agricultural University, 2010.(in Chinese)
[24] VANJARI H, MOHAN M, SAUDAGAR P, et al. Protective effect of SKB_gutbiotic against castor oil and E.coli induced diarrhea in laboratory animals[J]. Microbial Pathogenesis, 2020, 143:104078.
[25] COOPER R E, HUTCHINSON E K, IZZI J M. Evaluation of the guaiac fecal occult blood test for detection of gastrointestinal bleeding in the Rhesus macaque(Macaca mulatta)[J]. Journal of Medical Primatology, 2020, 49(1):16-25.
[26] 王凌云.糖尿病肾病患者髓过氧化物酶水平与代谢指标的相关性[J].中国医学创新, 2021, 18(25):175-178. WANG L Y. Correlation between the level of myeloperoxidase and metabolic indexes in patients with diabetic nephropathy[J]. Medical Innovation of China, 2021, 18(25):175-178.(in Chinese)
[27] SIRAKI A G. The many roles of myeloperoxidase:From inflammation and immunity to biomarkers, drug metabolism and drug discovery[J]. Redox Biology, 2021, 46:102109.
[28] LEDWABA S E, COSTA D V S, BOLICK D T, et al. Enteropathogenic Escherichia coli infection induces diarrhea, intestinal damage, metabolic alterations, and increased intestinal permeability in a murine model[J]. Front Cell Infect Microbiology, 2020, 10:595266.
[29] 赵宝凯,耿健,钟灵,等.金香颗粒预防小鼠大肠杆菌性腹泻的效果观察[J].中国兽医杂志, 2020, 56(12):108-112. ZHAO B K, GENG J, ZHONG L, et al. Prophylaxis effect of Jinxiang granule against mouse diarrhea caused by E.coli[J]. Chinese Journal of Veterinary Medicine, 2020, 56(12):108-112.(in Chinese)
[30] 张亚辉.鹳榆止泻散对小鼠大肠杆菌性腹泻的治疗效果研究[D].兰州:甘肃农业大学, 2019. ZHANG Y H. Study on therapeutic effect of Guyuzhixiesan on E.coli induced diarrhea in mice[D]. Lanzhou:Gansu Agricultural University, 2019.(in Chinese)
[31] XU B, YAN Y, HUANG J, et al. Cortex phellodendri extract's anti-diarrhea effect in mice related to its modification of gut microbiota[J]. Biomedicine and Pharmacotherapy, 2020, 123:109720.