五味止痢合剂对大肠杆菌致小鼠腹泻的治疗作用

doi:10.16431/j.cnki.1671-7236.2022.05.042

摘要/Abstract

摘要： 【目的】研究五味止痢合剂对大肠杆菌致小鼠腹泻的治疗效果。【方法】选择4周龄清洁级ICR小鼠，分为空白对照组(BC)、模型对照组(MC)、五味止痢合剂高(W-H)、中(W-M)、低剂量组(W-L)和盐酸环丙沙星药物对照组(CPFX)。每组20只，除BC组外均腹腔注射大肠杆菌，构建小鼠腹泻模型，建模5 h后，W-H、W-M、W-L组小鼠分别灌胃25、20、15 mL/kg BW五味止痢合剂(相当于生药2.5、2.0、1.5 g/kg BW)，CPFX组小鼠灌胃盐酸环丙沙星20 mL/kg BW，MC组和BC组灌胃等量生理盐水，2次/d，连用5 d。停药后14 d计算腹泻率和保护率。另取100只小鼠按照上述方法分组和给药，分别于给药后第2天，停药后第1、7和14天采集十二指肠和空肠。HE染色观察各组十二指肠黏膜层形态，采用ELISA试剂盒测定空肠分泌型免疫球蛋白A(sIgA)和十二指肠α-干扰素(IFN-α)和白介素-4(IL-4)细胞因子的含量，采用免疫组织化学法检测十二指肠黏膜修复因子增殖细胞核抗原(PCNA)和转化生长因子-α(TGF-α)表达情况。【结果】在停药后第14天，W-H和W-M组腹泻保护率分别高达95%和90%，死亡率均为0，均与CPFX组相当。在治疗过程中，W-H、W-M组小鼠小肠绒毛逐步趋于完整，淋巴细胞浸润逐渐减少，黏膜形态逐步趋于正常。W-H、W-M和CPFX组的空肠sIgA含量均显著高于MC组(P<0.05)。与MC组比较，在停药后第1、7和14天各给药组均能显著抑制空肠黏膜IFN-α的分泌(P<0.05)，显著提升IL-4的分泌(P<0.05);在停药后第14天，W-H组IFN-α和IL-4含量与BC组接近。在停药后第1和7天，W-H和CPFX组肠道修复因子PCNA表达量显著高于MC组(P<0.05);在停药后第1天，W-H和CPFX组肠道修复因子TGF-α表达量显著高于MC组(P<0.05)，在停药后第7天W-H和CPFX组TGF-α表达与MC组相比无显著差异(P>0.05);在停药后第14天各组间PCNA和TGF-α的表达均无显著差异(P>0.05)。【结论】 2.5 g/kg BW五味止痢合剂灌胃给药能显著控制大肠杆菌引起的小鼠腹泻，保护率达95%。其主要通过显著提升空肠灌洗液中sIgA的含量，充分激活免疫细胞，调节肠道黏膜分泌的细胞因子IFN-α和IL-4，有效抑制肠道炎症反应，刺激肠道黏膜修复因子PCNA和TGF-α的表达发挥治疗作用。

关键词: 五味止痢合剂; 大肠杆菌; 腹泻; 细胞因子; 修复因子

Abstract: 【Objective】The purpose of this experiment was to study the therapeutic effect of Wuwei Zhili mixture on diarrhea induced by Escherichia coli in mice.【Method】 Four-week-old clean grade ICR mice were divided into blank control (BC),model control (MC),Wuwei Zhili mixture high (W-H),medium (W-M),low (W-L) doses and ciprofloxacin hydrochloride control (CPFX) groups.20 mice in each group were intraperitoneally administered with Escherichia coli except BC group to construct the diarrhea model.When diarrhea symptoms appeared,the treatment group was intragastrically administered with high,medium and low doses of Wuwei Zhili mixture (25,20,15 mL/kg BW,equivalent to crude drug 2.5,2.0,1.5 g/kg BW),CPFX group was intragastrically administered with ciprofloxacin hydrochloride (20 mL/kg BW),and MC group and BC group were intragastrically administered with the same amount of normal saline,twice a day for 5 successive days.Diarrhea rate and protection rate were determined 14 days after drug withdrawal.Another 100 mice were divided and administrated according to the above methods,the duodenum and jejunum were collected on the days 2 after administration and the day 1,7 and 14 after drug withdrawal.The expressions of duodenal mucosa repair factor proliferating cell nuclear antigen (PCNA) and transforming growth factor-α (TGF- α) were detected by immunohistochemistry.【Result】 On the day 14 after drug withdrawal,the protection rates of diarrhea in W-H and W-M groups were 95% and 90%,and the mortality rates were both 0,which was similar to CPFX group.In the course of treatment,the small intestinal villi of mice in W-H and W-M groups gradually tended to be intact,the infiltration of lymphocytes gradually decreased,and the mucosal morphology gradually tended to be normal.The sIgA contents in W-H,W-M and CPFX groups were significantly higher than that in MC group (P<0.05).The secretion of IFN-α in intestinal mucosa of W-H,W-M,W-L and CPFX groups were significantly inhibited (P<0.05),and the secretion of IL-4 were significantly increased (P<0.05) when compared to that of MC group at day 1,7 and 14 after drug withdrawal.On the day 14 after drug withdrawal,the contents of IFN-α and IL-4 in W-H group were similar to that in BC group.On day 1 and 7 after drug withdrawal,the expression level of intestinal repair factor PCNA in W-H and CPFX groups was significantly higher than that in MC group (P<0.05).On day 1 after drug withdrawal,the expression level of intestinal repair factor TGF-α in W-H and CPFX groups was significantly higher than that in MC group (P<0.05),and compared with MC group,there was no significant difference in TGF-α expression in W-H and CPFX groups on day 7 after drug withdrawal (P>0.05).There were no significant differences in the expression of PCNA and TGF-α among all groups on day 14 after drug withdrawal (P>0.05).【Conclusion】 2.5 g/kg BW Wuwei Zhili mixture could significantly control diarrhea caused by Escherichia coli in mice,and the protection rate was 95%.It played a prominent therapeutic role by increasing the content of sIgA in jejunal lavage fluid,fully activating immune cells,regulating the cytokines IFN-α and IL-4 secreted by intestinal mucosa,effectively inhibiting intestinal inflammatory response,stimulating the expression of intestinal mucosal repair factors PCNA and TGF-α.

Key words: Wuwei Zhili mixture; Escherichia coli; diarrhea; cytokines; repair factor

中图分类号:

S854

陈毓, 陈晓兰, 杨海峰, 罗军, 张婷, 左海萍, 郑义. 五味止痢合剂对大肠杆菌致小鼠腹泻的治疗作用[J]. 中国畜牧兽医, 2022, 49(5): 2001-2009.

CHEN Yu, CHEN Xiaolan, YANG Haifeng, LUO Jun, ZHANG Ting, ZUO Haiping, ZHENG Yi. Therapeutic Effect of Wuwei Zhili Mixture on Diarrhea Induced by Escherichia coli in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 2001-2009.

参考文献

[1] 龚争,唐响响,刘硕.中草药对畜禽致病性大肠杆菌体外抑菌效果试验[J].安徽科技学院学报,2017,31(3):1-5. GONG Z,TANG X X,LIU S.In vitro bacteriostasis test of Chinese herbal medicine on pathogenic E.coli from livestock and poultry[J].Journal of Anhui Science and Technology University,2017,31(3):1-5.(in Chinese)
[2] LI S S,QI Y L,CHEN L X,et al.Effects of Panax ginseng polysaccharides on the gut microbiota in mice with antibiotic-associated diarrhea[J].International Journal of Biological Macromolecules,2019,124:931-937.
[3] 杨洪早,王东升,董书伟,等.仔猪腹泻的病因及中药防治研究进展[J].动物医学进展,2016,37(10):89-93. YANG H Z,WANG D S,DONG S W,et al.Progress on causes of diarrhea in piglets and the comprehensive prevention and treatment the use of TCM[J].Progress in Veterinary Medicine,2016,37(10):89-93.(in Chinese)
[4] LV C H,WANG T,REGMI N,et al.Effects of dietary supplementation of selenium-enriched probiotics on production performance and intestinal microbiota of weanling piglets raised under high ambient temperature[J].Journal of Animal Physiology and Animal Nutrition,2015,99(6):1161-1171.
[5] 张世昌,王志祥,孙永刚,等.复方中草药饲料添加剂对断奶仔猪生长性能、养分消化率及肠道菌群的影响[J].江西农业学报,2009,21(12):166-169. ZHANG S C,WANG Z X,SUNG Y G,et al.Effects of compound Chinese medicine herb additives on growth performance digestibility of main nutrients and intestional bacteria of weaned piglets[J].Acta Agriculturae Jiangxi,2009,21(12):166-169.(in Chinese)
[6] 王天宁,刘玉婷,肖凤琴,等.马齿苋化学成分及药理活性的现代研究整理[J].中国实验方剂学杂志,2018,24(6):224-234. WANG T N,LIU Y T,XIAO F Q,et al.Chemical composition and pharmacological activity of Portulaca oleracea[J].Chinese Journal of Experimental Tradiontional Medical Formulae,2018,24(6):224-234.(in Chinese)
[7] SEEBALUCK R,GURIB-FAKIM A,MAHOMOODALLY F.Medicinal plants from the genus Acalypha (Euphorbiaceae)-A review of their ethnopharmacology and phytochemistry[J].Journal of Ethnopharmacology,2015,159:137-157.
[8] 杨海峰,邱树磊,陈晓兰,等.五味止痢合剂组方依据及其防治仔猪腹泻的机制探析[J].中兽医医药杂志,2019,38(2):85-88. YANG H F,QIU S L,CHEN X L,et al.Prescription and pharmacological mechanisms analysis of Wuwei Zhili mixture on prevention and treatment of diarrhea in piglets[J].Journal of Traditional Chinese Veterinary Medicine,2019,38(2):85-88.(in Chinese)
[9] 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[J].兰州:甘肃农业大学,2019. ZHANG Y H.Study on therapeutic effect of Guyuzhixiesan on Escherichia coli-induced diarrhea in mice[J].Lanzhou:Gansu Agricultural University,2019.(in Chinese)
[10] BARNARD J.Epidermal growth factor receptor blockade:An emerging therapeutic modality in gastroenterology[J].Gastroenterology,2001,120(7):1872-1874.
[11] 颜爱,李波,李润成,等.香菇多糖和黄芪多糖对免疫抑制小鼠免疫功能调节的研究[J].中国免疫学杂志,2012,28(11):999-1005. YAN A,LI B,LI R C,et al.Effect of lentinan and Astragalus polysaccharides on immunological function inimmunosuppressed mice[J].Chinese Journal of Immunology.2012,28(11):999-1005.(in Chinese)
[12] 陶贵斌,何慧楠,李雪惠,等.马齿苋多糖体外免疫调节活性研究[J].食品研究与开发,2017,38(17):176-179. TAO G B,HE H N,LI X H,et al.The study of immunoregulatory activity in vitro of purslane polysaccarides[J].Food Research and Development,2017,38(17):176-179.(in Chinese)
[13] 吴娟,姚美村,戴聪,等.地榆对大鼠免疫功能的影响[J].沈阳药科大学学报,2018,35(8):663-668. WU J,YAO M C,DAI C,et al.Study on the effect of Sanguisorba officinalis in healthy and immunosuppressed rats[J].Journal of Shengyang Pharmaceutical University,2018,35(8):663-668.(in Chinese)
[14] 陈秋三,韦建缘,尹雪冬,等.不同浓度铁苋菜对胃肠蠕动加速性腹泻的治疗效果分析[J].亚太传统医药,2018,14(9):6-8. CHEN Q S,WEI J Y,YIN X D,et al.Effect of different concentration of Acalypha copperleaf in the treatment of gastrointestinal peristaltic diarrhea[J].Asia-Pacific Traditional Medicine,2018,14(9):6-8.(in Chinese)
[15] 张松莲,赵龙岩,袁清霞,等.仙人掌多糖主要组分对糖尿病小鼠的免疫调节作用[J].中国生化药物杂志,2012,33(5):532-536. ZHANG S L,ZHAO L Y,YUAN Q X,et al.Immunomodulatory effect of main component of Opuntia dillenii Haw.polysaccharides on mice with streptozotocin and cyclophosphamidle-induced diabetes[J].Chinese Journal of Biochenmical Pharmaceutics,2012,33(5):532-536.(in Chinese)
[16] 徐芳琴,杜小波,姚敏娜,等.马齿苋水提物对溃疡性结肠炎保护作用的实验研究[J].现代生物医学进展,2016,32:6239-6253. XU F Q,DU X B,YAO M N,et al.Protective effects of aqueous extract from Portulacaoleracea L.on ulcerative colitis[J].Progress in Modern Biomedicine,2016,32:6239-6253.(in Chinese)
[17] MANTIS N J,ROL N,CORTHÉSY B.Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut[J].Mucosal Immunology,2011,4(6):603-611.
[18] 赵春萍.姜黄素对大肠杆菌攻毒仔猪生长性能及肠道黏膜屏障功能的影响[D].海口:海南大学,2015. ZHAO C P.Effects of curcumin ongrowth performance andintestinal mucosal barrierfunction of weaned piglets challenged with Escherichia coli[D].Haikou:Hainan University,2015.(in Chinese)
[19] 高学军.大鼠小肠缺血再灌注损伤中细胞凋亡与增殖动态变化及葛根素干预研究[D].天津:天津医科大学,2006. GAO X J.Research on dynamic alteration of epithelial cells apoptosis and proliferation with the protection of puerarin on the model of intestinal ischemia-reperfusion injury in rats[D].Tianjin:Tianjin Medical University,2006.(in Chinese)
[20] 张桃.牛源致病性大肠杆菌O157:H7的分离鉴定及小鼠疾病模型的中药治疗效果[J].乌鲁木齐:新疆农业大学,2016. ZHANG T.Isolation identification of E.coli O157:H7 strainfrom cattle and chinese medicine treatment effect of thedisease model of mouse[J].Urumqi:Xingjiang Agricultural University,2016.(in Chinese)
[21] 徐倩倩,陈立功,刘颖,等.白头翁素对腹泻小鼠肠黏膜修复因子PCNA和TGFα表达的影响[J].河北农业大学学报,2013,36(2):99-102. XU Q Q,CHEN L G,LIU Y,et al.Effect of anemonin on intestinal mucosa repair factors PCNA and TGFα expression of diarrheal mice[J].Journal of Hebei Agricultural University,2013,36(2):99-102.(in Chinese)