大蒜胞外囊泡活性成分解析及其缓解小鼠结肠炎作用

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

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/6J小鼠口服给予GENs(20、100、500 mg/(kg m_(b)·d)),评估GENs的毒性,同时考察GENs对质量分数3%葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导结肠炎小鼠的疾病活动指数(disease activity index,DAI)评分和结肠长度的影响;采用苏木精和伊红对结肠组织切片进行染色;采用ELISA和试剂盒法测定小鼠血清中IL-6、IFN-γ、TNF-α和NO的水平。结果:GENs为纳米级杯状囊泡,含有26种脂质、62种蛋白质和317种miRNA,脂质中磷脂酰胆碱(phosphatidylcholine,PC)含量最高,预测出5种miRNAs可靶向编码人炎性细胞因子的基因。GENs能显著减轻LPS引起Caco-2单层细胞的损伤,抑制NO的产生,调节细胞因子IL-6、IFN-γ、TNF-α和IL-1β分泌水平。体内实验表明,各剂量范围内GENs无毒性;当GENs剂量为100 mg/(kg m_(b)·d)时能显著降低小鼠DAI评分(P<0.05),改善DSS引起的结肠缩短和病理损伤,下调DSS引起的血清中IL-6、IFN-γ及NO水平升高。结论:GENs可能通过磷脂酰胆碱和特定miRNA共同作用,保护肠道屏障损伤,调节炎性介质分泌水平,抑制细胞内炎症反应,缓解DSS诱导的小鼠结肠炎。

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-γand TNF-αas 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-γand 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-γand 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.