大黄与小鼠小肠抗生素肽表达变化的相关性研究

Correlation Study of Rhubarb and Expression Changes of Antimicrobial Peptides in the Small Intestine of Mice

目的通过观察大黄对正常小鼠小肠抗生素肽(antimicrobial peptide,AP)的作用,研究大黄与小肠AP之间的相关性。方法将30只健康ICR小鼠随机分为实验组和对照组,每组15只,分别给予10%大黄煎剂及等量的生理盐水灌胃。24h后处死小鼠,剖腹切取从屈氏韧带到回盲部的小肠。以10%乙酸溶液冲洗肠腔收集灌洗液,并以30%乙酸溶液制备小肠组织匀浆。称取等量的乙酸溶解物分别进行12·5%酸性尿素-聚丙烯酰胺凝胶电泳(AU-PAGE)、Tricine-十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(Tricine-16·5% SDS-PAGE),考马斯亮蓝R-250或氨银染色显示多肽条带,然后进行凝胶成像分析和电泳凝胶琼脂糖弥散法杀菌实验(gel overlay assay)。比较两组标本的杀菌活性、小肠AP表达的变化,测定小肠AP的分子量。结果大黄灌胃后,小鼠小肠匀浆的AP含量变化不明显,但是AP分子量不同;而小肠灌洗液中AP含量增加明显,分子量介于14·3～18·4kDa;但是其杀菌能力与对照组比较差异无统计学意义(P>0·05)。结论大黄可增加小肠部分AP表达,这可能是大黄保护、增强肠道屏障的作用机制之一。

Objective To study the correlation between rhubarb and expressions of antimicrobial peptides（AP） in the small intestine of mice by observing the effect of rhubarb on it.Methods Thirty healthy ICR mice were randomly divided into the test group and the control group,fifteen in each.Ten percent rhubarb decoction and equal volume of normal saline solution was respectively given to mice of the two groups by gastrogavage.Mice were sacrificed 24 h later.The small intestines from the ligament of Treitz to the ileocecal junction were removed by ventrotomy.The lumen of each resected intestine was rinsed with 10% acetic acid solution and the perfusate was collected.The small intestine homogenate was prepared using 30% acetic acid solution.The equivalent samples were eletrophoresed on 12.5% AU-PAGE and Tricine-16.5% SDS-PAGE,and peptide bands were visualized with Coomassie brilliant blue R-250 or ammoniacal silver-stained respectively.And then analysis of gel imaging and gel overlay assay were performed.The bactericidal activity and expressions of AP in the small intestine of samples in the two groups were compared.The molecular weight of AP in the small intestine was measured.Results After gastrogavaged with rhubarb decoction,changes of AP contents in the small intestine homogenate of mice were not obvious,but the molecular weights of AP were different.But the AP contents in the small intestine perfusate obviously increased,with the molecular weights being 14.3 to 18.4 kDa.But its bactericidal capacity showed no statistical difference when compared with that of the control group（P0.05）.ConclusionRhubarb enhanced the expressions of partial APs in the small intestine,which might possibly be one of its mechanisms of protecting and strengthening the intestinal barrier.