夏枯草蜂蜜提取物对硫酸葡聚糖诱导肠上皮细胞损伤的保护作用及其机制

Protective Effect and Mechanism of Prunella vulgaris Honey Extract on Dextran Sulfate Sodium-Induced Injury in Caco-2 Cells

目的:在细胞分子层面揭示夏枯草蜂蜜提取物对硫酸葡聚糖(dextran sulfate sodium,DSS)诱导的人结肠上皮细胞(Caco-2)损伤的保护作用及其作用机制。方法:以洋槐蜂蜜为对照,分别采用福林-酚法和硝酸铝法测定两种蜂蜜中总酚酸含量和总黄酮含量;通过测定1,1-二苯基-2-三硝基苯肼自由基、2,2’-联氮-双(3-乙基苯并噻唑啉-6-磺酸)自由基清除能力、半抑制浓度和铁离子抗氧化能力评估两种蜂蜜的体外抗氧化能力;通过建立DSS诱导大鼠溃疡性结肠炎模型,结合实时荧光定量聚合酶链式反应、免疫印迹实验及细胞迁移实验研究夏枯草蜂蜜提取物对溃疡性结肠炎的保护作用。结果:夏枯草蜂蜜的总酚酸含量为(52.4±0.4)μg/g,总黄酮含量为(7.0±0.3)μg/g;通过建立体积分数2.5%DSS诱导Caco-2细胞损伤模型,发现夏枯草蜂蜜提取物在质量浓度50~100μg/mL时能显著缓解DSS处理所导致的细胞活力下降;实时荧光定量聚合酶链式反应发现夏枯草蜂蜜提取物预处理可显著上调抗氧化蛋白基因(NQO-1、Txnrd1和Nrf2)和紧密连接蛋白基因(ZO-1)的表达;免疫荧光实验表明夏枯草蜂蜜提取物预处理可显著拮抗DSS对紧密连接蛋白(ZO-1)的损伤;免疫印迹实验分析表明夏枯草蜂蜜提取物预处理可显著促进抗氧化蛋白(NQO-1、Txnrd1和Nrf2)的表达。结论:夏枯草蜂蜜可缓解DSS对肠上皮细胞屏障功能的破坏,具有良好的开发利用价值。

Purpose:The aim of this study was to reveal the protective effect and underlying mechanism of Prunella vulgaris honey(PVH)extract on dextran sulfate sodium(DSS)-induced human colon epithelial cell(Caco-2)injury at cellular and molecular levels.Methods:The contents of total polyphenols and flavonoids in PVH and acacia honey(AH)as a control were determined by Folin-phenol reagent method and aluminum nitrate method,respectively.2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radical cation and 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical scavenging capacity,50%maximum inhibitory concentration(IC50),and ferric ion reducing antioxidant power(FRAP)were used to evaluate in vitro antioxidant activity of the two honeys.Then,the protective effect of PVH on ulcerative colitis in rats was studied using a rat model of DSS-induced ulcerative colitis by real-time quantitative polymerase chain reaction(RT-qPCR),Western blotting and Transwell migration analysis.Results:The contents of total phenolic acid and total flavonoids in PVH were respectively(52.4±0.4)and(7.0±0.3)μg/g.PVH extract at concentrations of 50–100μg/mL could significantly alleviate the decrease of cell viability caused by 2.5%(V/V)DSS treatment.Based on RT-qPCR analysis,PVH significantly up-regulated the mRNA expression of antioxidant cytokine genes such as NQO-1,Txnrd1 and Nrf2 and tight junction gene such as ZO-1.Based on immunofluorescence analysis,PVH extract could protect the key tight junction protein ZO-1 in Caco-2 cell monolayers against DSS-induced damage.In addition,PVH extract could significantly up-regulate the expression of antioxidant proteins such as NQO-1,Txnrd1 and Nrf2 as evaluated by Western blot assay.Conclusion:PVH has great potential for development and utilization in alleviating DSS-induced damage to the barrier function of intestinal epithelial cells.