五味止痢合剂对大肠杆菌致小鼠腹泻的治疗作用

Therapeutic Effect of Wuwei Zhili Mixture on Diarrhea Induced by Escherichia coli in Mice

【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。

【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-αin 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-αand 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-αin 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-αexpression 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-αamong 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-αand IL-4 secreted by intestinal mucosa,effectively inhibiting intestinal inflammatory response,stimulating the expression of intestinal mucosal repair factors PCNA and TGF-α.