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香草乙酮改善葡聚糖硫酸钠诱发溃疡性结肠炎小鼠的炎症反应与NOXs-ROS-p38MAPK信号通路的关系

魏丹丹, 林旭红, 王慧超, 王斌, 白春洋, 王亚强, 李国恩, 任学群

河南大学淮河医院检验科；第一附属医院肾内科；淮河医院消化内科；淮河医院普通外科，开封 475000

摘要

本文旨在探讨香草乙酮(apocynin)治疗葡聚糖硫酸钠(dextran sulfate sodium, DSS)诱导的溃疡性结肠炎(ulcerative colitis, UC)小鼠的分子机制。以饮用水配制5% DSS诱发UC动物模型，2%香草乙酮治疗UC小鼠，采样并应用HE染色进行结肠组织病理学评估。采用鲁米诺化学发光方法检测结肠组织活性氧自由基(reactive oxygen species, ROS)生成及DPI抑制后的NADPH消耗率分析NADPH氧化酶(NADPH oxidases, NOXs)活性，Western blot法检测p38MAPK磷酸化水平，Griess方法分析NO，酶联免疫法检测前列腺素E2 (prostaglandin E2, PGE2)，real time PCR及Western blot法检测iNOS、COX2的表达，酶联免疫吸附实验测定细胞因子TNF-α、IL-6、IFN-γ、IL-1β的水平。体外实验采用分离的结肠组织中性粒细胞，检测其ROS生成、NOXs活性、NO、PGE2的变化，并应用Western blot检测其NOX1、p-p38MAPK的表达。结果显示，香草乙酮治疗后UC小鼠结肠组织NOXs活性及ROS生成被抑制(P < 0.01)，p38MAPK磷酸化水平降低，NO、PGE2及相关细胞因子生成减少(P < 0.01)，UC炎症反应得到缓解。在炎症部位的中性粒细胞中，香草乙酮抑制ROS生成及NOX活性(P < 0.01)，并降低NOX1的表达、p38MAPK的磷酸化及NO、PGE2的生成(P < 0.01)。因此，香草乙酮可能通过NOXs-ROS-p38MAPK信号通路缓解DSS诱发UC小鼠的炎症反应，中性粒细胞是参与其保护机制的主要炎症细胞。

关键词： 香草乙酮; NADPH氧化酶; 氧自由基

分类号：R333.3

[Apocynin relieves inflammation in dextran sulfate sodium-induced ulcerative colitis mice: the role of NOXs-ROS-p38MAPK pathway.] [Article in Chinese]

WEI Dan-Dan, LIN Xu-Hong, WANG Hui-Chao, WANG Bin, BAI Chun-Yang, WANG Ya-Qiang, LI Guo-En, REN Xue-Qun

Department of Clinical Laboratory, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China； Department of Nephrology, the First Hospital Affiliated to Henan University, Kaifeng 475000, China； Department of Digestive Medicine, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China； Department of General Surgery, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China

Abstract

The study is aimed to explore the molecular mechanism of the treatment of apocynin in dextran sulfate sodium (DSS)-
induced ulcerative colitis (UC) mice. 5% DSS was used to mimic the UC model, and 2% apocynin was applied to treat the UC mice. HE staining was used for histopathological evaluation. Chemiluminescence technique was used to measure reactive oxygen species (ROS) production, and the rate of consumption of NADPH inhibited by DPI was detected to determine the NADPH oxidases (NOXs) activity. Western blot was applied to identify the level of p38MAPK phosphorylation, Griess reaction assay to analyze NO production, immunoenzymatic method to determine prostaglandin E2 (PGE2) production, real time RT-PCR and Western blot to identify the expression of iNOS and COX2, and enzyme linked immunosorbent assay to detect inflammatory cytokines TNF-α, IL-6, IFN-γ, IL-1β. Rat neutrophils were separated, and then ROS production, NOXs activity, NO and PGE2 production, NOX1 and p-p38MAPK expression were detected. Compared with the UC group, apocynin decreased ROS over-production and NOXs activity (P < 0.01), reduced p38MAPK phosphorylation, inhibited NO, PGE2 and cytokines production (P < 0.01). Apocynin also decreased NOXs activity and ROS over-production (P < 0.01), inhibited p38MAPK phosphorylation and NOX1 expression, and reduced NO and PGE2 production (P < 0.01) in separated neutrophils from UC mice. Therefore, apocynin could relieve inflammation in DSS-induced UC mice through inhibiting NOXs-ROS-p38MAPK signal pathway, and neutrophils play an important role.

Key words: apocynin; NADPH oxidase; reactive oxygen species

收稿日期：2014-07-10　　录用日期：2014-08-21

通讯作者：任学群　　E-mail: lxh80726@126.com

引用本文：

魏丹丹, 林旭红, 王慧超, 王斌, 白春洋, 王亚强, 李国恩, 任学群. 香草乙酮改善葡聚糖硫酸钠诱发溃疡性结肠炎小鼠的炎症反应与NOXs-ROS-p38MAPK信号通路的关系[J]. 生理学报 2015; 67 (1): 74-82.

WEI Dan-Dan, LIN Xu-Hong, WANG Hui-Chao, WANG Bin, BAI Chun-Yang, WANG Ya-Qiang, LI Guo-En, REN Xue-Qun. [Apocynin relieves inflammation in dextran sulfate sodium-induced ulcerative colitis mice: the role of NOXs-ROS-p38MAPK pathway.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (1): 74-82 (in Chinese with English abstract).