中药联合益生菌对大肠杆菌致小鼠腹泻保护机制研究

【目的】前期研究表明中药联合益生菌对致病性大肠杆菌所致小鼠腹泻具有明显的保护作用,本研究旨在进一步探讨中药联合益生菌对致病性大肠杆菌致小鼠腹泻的保护机制。【方法】选取小鼠60只,随机分为5组,空白组、模型组、中药组、益生菌组及中药联合益生菌组,每组12只。除空白组外,其余各组小鼠通过腹腔注射1×10⁷CFU/kg大肠杆菌分离株菌悬液建立小鼠腹泻模型,空白组腹腔注射等量生理盐水。中药组灌胃给予中药液(2.5 g/kg蒲公英提取物、0.5 g/kg黄芪总黄酮及0.5 g/kg黄芪多糖),益生菌组灌胃给予枯草芽孢杆菌悬液(2×10⁸CFU/kg),中药联合益生菌组灌胃给予中药液(2.5 g/kg蒲公英提取物、0.5 g/kg黄芪总黄酮及0.5 g/kg黄芪多糖)与枯草芽孢杆菌悬液(5×10⁷CFU/kg)的混合液,1 次/d,连续给药7 d。HE染色观察小肠组织病理学变化,扫描电镜观察小肠组织超微结构变化,ELISA法检测小鼠小肠匀浆和血清中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量,RT-PCR法检测小肠组织及血液中TNF-α和IL-6 mRNA表达,免疫组织化学染色检测小肠组织中核转录因子-κB(NF-κB)p65蛋白表达。【结果】中药联合益生菌能改善小肠病理组织学病变、修复小肠绒毛高度脱水萎缩和微绒毛脱落明显的损伤。同时,中药联合益生菌可明显降低小肠组织及血清中TNF-α和IL-6的含量(P＜0.05;P＜0.01),显著抑制小肠组织及血液中TNF-α和IL-6 mRNA的表达(P＜0.05;P＜0.01)。中药联合益生菌可明显降低小肠组织NF-κB p65蛋白的表达。【结论】中药联合益生菌可通过调控NF-κB信号通路,抑制小肠组织及血液TNF-α和IL-6 mRNA的过度表达,降低小肠组织及血清TNF-α和IL-6含量,改善肠腺变性和黏膜炎性细胞浸润以及小肠绒毛和微绒毛损伤,从而对大肠杆菌所致小鼠腹泻起到保护作用。

Study on the Protective Mechanism of Traditional Chinese Medicine Combined with Probiotics on Diarrhea Caused by E.coli in Mice

【Objective】 Our previous study had shown that traditional Chinese medicine (TCM) combined with probiotics had obvious protective effect on diarrhea caused by pathogenic E.coli in mice.This study was aimed to further explore the protective mechanism of TCM combined with probiotics on diarrhea caused by pathogenic E.coli in mice.【Method】 Sixty mice were randomly divided into five groups: Blank group,model group,TCM group,probiotics group and TCM combined probiotics group.The diarrhea model of mice was established by intraperitoneal injection with 1×10⁷CFU/kg E.coli isolate suspension,TCM group was gavaged with TCM (2.5 g/kg dandelion extract,0.5 g/kg Astragalus total flavone and 0.5 g/kg Astragalus polysaccharide),and probiotics group was gavaged with Bacillus subtilis suspension (2×10⁸CFU/kg),TCM combined with probiotics group was gavaged with the corresponding dose of TCM and Bacillus subtilis (5×10⁷CFU/kg) mixture,once a day for 7 days.The pathological changes of small intestine tissue were observed by HE staining,the ultrastructural changes were observed by scanning electron microscope.The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in intestine homogenate and serum of mice were determined by ELISA,the expression of TNF-α and IL-6 mRNA in intestine tissue and blood were determined by RT-PCR.The expression of nuclear factor-κB (NF-κB) p65 protein in small intestine was determined by immunohistochemical staining.【Result】 TCM combined with probiotics could improve the pathological and histological changes of small intestine,repair the phenomenon of high dehydration and atrophy of small intestinal villi and obvious shedding of microvilli.At the same time,TCM combined with probiotics could significantly reduce the contents of TNF-α and IL-6 in small intestine homogenate and serum (P＜0.05 or P＜0.01),significantly inhibit the expression of TNF-α and IL-6 mRNA in small intestine tissue and blood (P＜0.05 or P＜0.01).Furthermore,TCM combined with probiotics could significantly decrease the high expression of NF-κB p65 protein in small intestine.【Conclusion】 TCM combined with probiotics regulatesd NF-κB signaling pathway,inhibited the excessive expression of TNF-α and IL-6 mRNA,reduced the contents of TNF-α and IL-6 in intestine homogenate and blood,improved intestinal gland degeneration and mucosal inflammatory cell infiltration,as well as small intestinal villi and microvilli damage,thus playing a protective role against diarrhea caused by E.coli in mice.