基于转录组学研究毛冬青三萜皂苷对脂多糖诱导小鼠肠损伤的保护作用及其机制

Effect and mechanism of Ilex pubescens triterpenoid saponins on lipopolysaccharide-induced intestinal barrier injury in mice based on transcriptome

目的探讨毛冬青三萜皂苷(Ilex pubescens triterpenoid saponin,IPTS)对脂多糖(lipopolysaccharide,LPS)诱导的小鼠肠损伤的作用及潜在机制。方法C57BL/6小鼠随机分为对照组、模型组和IPTS低、中、高剂量(88、176、264mg/kg)组,给予药物干预7 d,ip LPS诱导小鼠小肠损伤,造模7 h后,采用苏木素-伊红(HE)染色法观察小鼠回肠组织病理变化;检测小鼠血清中丙二醛(malondialdehyde,MDA)水平和超氧化物歧化酶(superoxide dismutase,SOD)活性;采用qRT-PCR和Western blotting法检测回肠组织炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)m RNA和肠屏障紧密连接蛋白ZO-1、Occludin mRNA及蛋白表达。通过转录组学测序挖掘IPTS作用于回肠的潜在机制。结果与模型组比较,IPTS组小鼠回肠组织完整,氧化应激损伤改善(P<0.001),回肠炎症因子水平降低(P<0.01),肠屏障恢复(P<0.05、0.01、0.001)。RNA-seq测序结果显示,与对照组比较,模型组共筛选出2584个差异表达基因;与模型组比较,IPTS高剂量组共筛选出259个差异表达基因;这些差异表达基因主要富集在免疫球蛋白A(immunoglobulin A,IgA)相关的肠道免疫、花生四烯酸代谢、B细胞受体、瞬时受体电位(transient receptor potential,TRP)通道炎症调控、亚油酸代谢等信号通路。结论IPTS可以改善LPS诱导的小鼠小肠损伤,可能与花生四烯酸代谢、IgA相关的肠道免疫代谢以及TRP通道炎症调控通路密切相关。

Objective To investigate the effect and potential mechanism of Ilex pubescens triterpenoid saponins(IPTS)on lipopolysaccharide(LPS)-induced small intestinal injury in mice.Methods C57BL/6 mice were randomly divided into control group,model group and IPTS low-,medium-and high-dose(88,176,264 mg/kg)groups.After 7 d of drug intervention,mice were ip LPS to induce small intestine injury.After 7 h of modeling,hematoxylin eosin(HE)staining was used to observe pathology of ileum in mice;Level of malondialdehyde(MDA)and activity of superoxide dismutase(SOD)in serum of mice were detected;qRT-PCR and Western blotting were used to detect inflammatory factor interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)mRNA and intestinal barrier tight junction protein ZO-1,Occludin m RNA and protein expressions in ileum.The potential mechanism of IPTS on ileum was explored by transcriptome sequencing.Results Compared with model group,ileum in IPTS group was intact,oxidative stress injury was improved(P<0.001),level of ileitis factor was decreased(P<0.01),and intestinal barrier was restored(P<0.05,0.01,0.001).RNAseq sequencing results showed that 2584 differentially expressed genes were screened from model group compared with control group;Compared with model group,259 differentially expressed genes were screened out in IPTS high-dose group;These differentially expressed genes were mainly enriched in signaling pathway such as immunoglobulin A(IgA)related intestinal immunity,arachidonic acid metabolism,B cell receptor,transient receptor potential(TRP)channel inflammation regulation,linoleic acid metabolism,etc.Conclusion IPTS can improve LPS induced intestinal injury in mice,which may be closely related to arachidonic acid metabolism,IgA related intestinal immune metabolism and TRP channel inflammatory regulation pathway.