茶皂素对志贺菌感染的保护作用及其机制研究

Protective effect and related mechanism of tea saponin on Shigella infection

目的:探究茶皂素(TS)对志贺菌感染性肠炎的保护作用及其机制研究。方法:实验分体外实验和体内实验。体外实验:通过微量肉汤稀释法检测TS对志贺菌的抗菌活性;使用酶标仪检测不同浓度TS作用下菌液的600 nm下的吸光度(A_(600)),绘制生长曲线;通过平板菌落计数检测细菌数。体内实验:将15只小鼠分为对照组、福氏2a志贺菌301株(Sf301)组和TS组,每组5只。Sf301组和TS组连续8 d分别灌胃无菌水或TS,于第3天制备志贺菌感染小鼠模型。用疾病活动指数(DAI)评价小鼠的一般情况。于第8天处死小鼠,测量各组小鼠结肠长度,取结肠组织行HE染色,观察病理学变化;取小鼠盲肠内容物与粪便进行平板计数,检测志贺菌载量;用实时荧光定量PCR(RT-qPCR)检测结肠炎症因子的mRNA表达水平。结果:(1)体外实验:TS的最小抑菌浓度(MIC)为1024μg/ml,不同浓度的TS(256、512、1024μg/ml)的平板菌落计数、A_(600)依次减小,与对照组比较,差异均有统计学意义(均P<0.05)。(2)体内实验:对照组、TS组小鼠结肠分别为(7.70±0.24)、(7.35±0.41)cm,与Sf301组[(6.13±0.05)cm]比较,差异有统计学意义(P<0.05)。组织病理学结果显示Sf301组结肠上皮细胞脱落,杯状细胞减少,炎性细胞浸润。而TS组结肠黏膜较完整,无炎性细胞浸润。与Sf301组比较,TS组小鼠盲肠内容物细菌载量更低(P<0.05)。TS组的肿瘤坏死因子-α水平低于Sf301组,白细胞介素-10水平高于Sf301组(均P<0.05)。结论:TS可有效抑制志贺菌,通过减少志贺菌载量和抑制炎症缓解志贺菌感染性肠炎。

Objective:To explore the protective effects and related mechanism of tea saponin(TS)on intestinal inflammation due to Shigella infection.Methods:In vitro,the antibacterial activity of TS was detected by standard broth microdilution method.The absorbance at 600 nm of the bacterial liquid was detected by Microplate Reader under different concentrations of TS,and the growth curve was drawn.Bacterial count was obtained by plate colony counting.In vivo,15 mice were divided into 3 groups(n=5 each):control group,Sf301 group and TS group.Sterile water or TS was applied to mice in the Sf301 group and the TS group per gavage once a day for 8 days,and the mouse model of Shigella infection was established on day 3.The disease activity index(DAI)was used to evaluate the general condition of mice.The mice were sacrificed on day 8.Colon length was measured and colon tissues were stained with HE to analyze the pathological changes.The cecal contents and feces of mice were taken for plate counting and Shigella load was obtained.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the mRNA expression levels of inflammatory factors.Results:(1)In vitro,the MIC of TS was 1024μg/ml.The plate counting and A_(600) of TS decreased in proportion to increasing concentrations(256,512,1024μg/ml)in comparison with the control group(all P<0.05).(2)In vivo,colon length was(7.70±0.24)cm in the control group and(7.35±0.41)cm in the TS group,which was significantly longer than that of the Sf301 group([6.13±0.05]cm,P<0.05).Histopathological examination evidenced colonic epithelial cells shedding,decreased goblet cells,and inflammatory cell infiltration in the colon tissue of the Sf301 group.In the TS group,the colonic mucosa was intact without significant inflammatory cell infiltration.Bacterial load in cecal contents was significantly lower in the TS group than in the Sf301 group(P<0.05).The level of tumor necrosis factor-αwas significantly lower,and the level of interleukin-10 was significantly higher in the TS group than in the Sf301 group(all P<0.05).Conclusion:TS can effectively inhibit Shigella and alleviate Shigella infective enteritis by reducing Shigella load and inhibiting inflammation.