大蒜胞外囊泡活性成分解析及其缓解小鼠结肠炎作用

目的：解析大蒜胞外囊泡(garlic-derived extracellular nanovesicles,GENs)的活性成分并探究其对小鼠结肠炎的作用。方法：采用超速离心与蔗糖密度梯度离心法从蒜汁中分离纯化GENs，并对其形貌和结构进行表征。采用液相色谱串联质谱联用仪对GENs进行脂质分析和蛋白质组学分析；采用RNA转录组测序技术进行小RNA(microRNA,miRNA)序列鉴定和靶基因预测分析。构建Caco-2单层肠上皮细胞模型，脂多糖(lipopolysaccharide,LPS)刺激后，通过荧光光谱测定细胞渗透表观系数；采用试剂盒法测定上清液中一氧化氮(nitric oxide,NO)含量；采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)测定细胞上清液中白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、干扰素-γ(interferon-γ,IFN-γ)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)质量浓度。C57BL/...

Garlic-Derived Extracellular Nanovesicles: Bioactive Components and Their Roles in Alleviating Colitis in Mice

Objective: This study aimed to analyze the bioactive components of garlic-derived extracellular nanovesicles (GENs) and explore their anti-inflammatory activity against colitis in mice. Methods: GENs were isolated from garlic juice by ultracentrifugation and sucrose density gradient centrifugation, and the morphology and structure of GENs were characterized. Lipids and proteins were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzed by online databases. Meanwhile, RNA sequencing was carried out for sequence identification and target gene prediction of microRNAs (miRNAs). Caco-2 cells were cultured as a model of monolayer intestinal epithelial cells and were determined for apparent permeability coefficient by fluorescence spectroscopy after lipopolysaccharide (LPS) stimulation. The level of nitric oxide (NO) in the supernatant was measured by a commercial assay kit, and interleukin-6 (IL-6), interleukin-1β (IL-1β), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay (ELISA). C57BL/6J mice were orally administrated with GENs (20, 100 and 500 mg/(kg mb·d)) to evaluate the toxicity of GENs. The effect of GENs on the disease activity index (DAI) score and colon length of mice with 3% dextran sulphate sodium salt (DSS)-induced colitis was investigated. Colonic tissue sections were stained with hematoxylin and eosin (H&E) and observed. The levels of IL-6, IFN-γ and TNF-α as well as NO in the serum were measured by ELISA and a commercial assay kit, respectively. Results: GENs were goblet nanovesicles containing 26 lipids, 62 proteins and 317 microRNAs. Phosphatidylcholine (PC) was the most abundant lipid component. Five miRNAs targeting human genes encoding inflammatory factors were predicted. GENs significantly reduced LPS-induced damage to the permeability of Caco-2 cell monolayers, inhibited the production of NO, and regulated the secretion of IL-6, IL-1β, IFN-γ and TNF-α. GENs were non-toxic towards mice at all doses tested. GENs at 100 mg/(kg mb·d) significantly decreased the DAI score of mice (P < 0.05), improved shortening of colon length and pathological damage induced by DSS and repressed the increase in serum IL-6, IFN-γ and NO levels. Conclusion: GENs exert an anti-inflammatory activity probably through their bioactive molecules such as phosphatidylcholine and specific miRNAs. GENs have great potential for alleviating intestinal inflammation and preventing DSS-induced colitis in mice.