藜芦酸通过激活Nrf2/HO-1信号通路减轻氧化应激缓解葡聚糖硫酸钠诱导的小鼠实验性结肠炎

doi:10.12122/j.issn.1673-4254.2026.02.18

摘要/Abstract

摘要：

目的探讨藜芦酸（VA）通过调控Nrf2/HO-1信号通路改善葡聚糖硫酸钠（DSS）诱导的结肠炎氧化应激损伤及肠屏障功能障碍的分子机制。方法将30只雄性C57BL/6小鼠随机分为对照组（WT组）、DSS模型组及VA治疗组，10只/组。通过评估体质量变化、疾病活动指数（DAI）、结肠长度及组织病理学损伤。ELISA检测结肠组织中炎症因子（TNF-α、IL-6、IL-10）水平，以及分析氧化应激标志物（SOD、GSH、MDA、COX-2）的表达。免疫荧光及Western blotting检测紧密连接蛋白（ZO-1、Claudin-1）及Nrf2/HO-1通路相关蛋白的表达。体外构建H₂O₂（150 μmol/L）诱导的Caco-2细胞氧化应激模型，流式细胞术及DCFH-DA荧光探针检测活性氧（ROS）蓄积，并联合Nrf2抑制剂ML385验证VA的调控机制。结果 VA干预缓解DSS小鼠体质量下降（P<0.05）、结肠缩短（P<0.05）及DAI评分升高（P<0.05），以及修复肠黏膜隐窝结构、紧密连接蛋白（ZO-1和Claudin-1）表达（P<0.05）和增加杯状细胞数量（P<0.05）。VA降低结肠组织中促炎因子TNF-α、IL-6水平（P<0.05），同时上调抗炎因子IL-10表达水平（P<0.05）。VA逆转DSS诱导的SOD和GSH活性降低，以及MDA和COX-2水平升高（P<0.05）。在H₂O₂诱导的Caco-2细胞模型中，流式细胞术和免疫荧光染色结果提示：VA降低ROS阳性细胞率和细胞内ROS蓄积；免疫荧光染色和Western blotting数据分析显示：VA增加了Claudin-1和ZO-1蛋白的表达。机制上，VA促进Nrf2及下游HO-1蛋白表达（P<0.05），以及Nrf2抑制剂ML385部分拮抗了VA对H₂O₂诱导的ROS阳性细胞比例的下降。结论 VA通过激活Nrf2/HO-1通路增强抗氧化防御、抑制炎症反应并修复肠屏障功能，为IBD治疗提供新策略。

关键词: 炎症性肠病, 藜芦酸, Nrf2/HO-1信号通路, 氧化应激, 肠屏障修复

Abstract:

Objective To investigate the molecular mechanism by which veratric acid (VA) ameliorates oxidative stress injury and intestinal barrier dysfunction in mice with dextran sulfate sodium (DSS)-induced colitis. Methods Thirty male C57BL/6 mice were randomized equally into control group, DSS model group, and VA treatment group. The mice were assessed for changes in body weight, disease activity index (DAI), colon length, and colonic histopathology. Colonic expressions of TNF-α, IL-6, and IL-10 and oxidative stress markers (SOD, GSH, MDA, and COX-2) were determined using ELISA, and the expressions of tight junction proteins (ZO-1 and claudin-1) and Nrf2/HO-1 pathway proteins were detected using immunofluorescence staining and Western blotting. In Caco-2 cells with H₂O₂-induced oxidative stress, ROS accumulation was examined using flow cytometry and a DCFH-DA probe, and Nrf2 inhibitor (ML385) was used to validate the mechanism of VA for ameliorating oxidative stress. Results VA treatment significantly alleviated DSS-induced body weight loss, colon shortening and the increase of DAI score of the mice, resulting also in improved crypt structure and increased expressions of ZO-1 and claudin-1 and the number of goblet cells. VA obviously reduced colonic levels of TNF‑α and IL-6, increased the level of IL-10, and reversed DSS-induced decreases in SOD and GSH activity and increases in MDA and COX-2 levels. In H₂O₂-treated Caco-2 cells, VA decreased ROS-positive cell rate and intracellular ROS accumulation, and increased cellular expressions of claudin-1 and ZO-1. Mechanistically, VA promoted the expressions of Nrf2 and the downstream HO-1 protein, and ML385 partially reversed ROS-reducing effect of VA. Conclusion VA enhances antioxidant defense, inhibits inflammation, and repairs intestinal barrier function in mice with DSS-induced colitis by activating the Nrf2/HO-1 pathway, suggesting a novel strategy for treatment of inflammatory bowel disease.

Key words: inflammatory bowel disease, veratric acid, Nrf2/HO-1 signaling pathway, oxidative stress, intestinal barrier repair

尹林, 张可妮, 乔通, 牛民主, 殷丽霞, 刘馨悦, 耿志军, 李静, 胡建国. 藜芦酸通过激活Nrf2/HO-1信号通路减轻氧化应激缓解葡聚糖硫酸钠诱导的小鼠实验性结肠炎[J]. 南方医科大学学报, 2026, 46(2): 403-411.

Lin YIN, Keni ZHANG, Tong QIAO, Minzhu NIU, Lixia YIN, Xinyue LIU, Zhijun GENG, Jing LI, Jianguo HU. Veratric acid relieves oxidative stress and DSS-induced colitis in mice by activating the Nrf2/HO-1 signaling pathway[J]. Journal of Southern Medical University, 2026, 46(2): 403-411.

图/表 8

图1 VA对DSS诱导的小鼠肠炎症状的影响

Fig.1 Effect of VA on DSS-induced colitis symptoms in mice (n=10). A: Changes of body weight. B: Changes of DAI scores. C, D: Comparison of colon length among different groups. *P<0.05 vs WT group; #P<0.05 vs DSS group.

图2 VA对DSS小鼠肠组织病理学的影响

Fig.2 Effect of VA on intestinal histopathology in DSS-treated mice (n=10). A: HE staining and inflammatory scores of the intestinal tissues of mice in each group. B: Histopathological scores of mice in each group. C: AB-PAS staining of the intestinal tissues in each group. D: Comparison of goblet cell counts in each group. *P<0.05 vs WT group; #P<0.05 vs DSS group.

图3 VA对DSS小鼠结肠组织中炎症因子表达的影响

Fig.3 Effect of VA on expressions of inflammatory cytokines in the colonic tissues of DSS-treated mice (n=10). A: Levels of interleukin-6 (IL-6) in the colonic mucosa of the mice in each group detected by enzyme-linked immunosorbent assay (ELISA). B: Levels of interleukin-10 (IL-10) in the colonic mucosa of the mice in each group detected by ELISA. C: Levels of tumor necrosis factor-α (TNF-α) in the colonic mucosa of the mice in each group detected by ELISA. *P<0.05 vs WT group; #P<0.05 vs DSS group.

图4 VA对DSS小鼠结肠组织中紧密连接蛋白的影响

Fig.4 Effect of VA on tight junction proteins in the colonic tissues of DSS-induced mice (n=10). A: Immunofluorescence staining for claudin-1 and ZO-1 in the colons of the mice in each group. B: Western blotting for detecting expression levels of claudin-1 and ZO-1 in the colons of the mice in each group. *P<0.05 vs WT group; #P<0.05 vs DSS group.

图5 VA对DSS小鼠结肠组织中氧化应激的调控作用

Fig.5 Regulatory effect of VA on oxidative stress in the colonic tissues of DSS-induced mice (n=10). A: Detection results of glutathione (GSH) in each group. B: levels of superoxide dismutase (SOD) in each group. C: Malondialdehyde (MDA) levels in each group. D: Cyclooxygenase-2 (COX-2) levels in each group. *P<0.05 vs WT group; #P<0.05 vs DSS group.

图6 VA对H₂O₂诱导的Caco-2细胞活性氧的影响

Fig.6 Effect of VA on reactive oxygen species (ROS) production induced by H₂O₂ in Caco-2 cells (n=10). A: ROS staining with the fluorescent probe (DCFH-DA) in each group. B: ROS detection by flow cytometry in each group. *P<0.05 vs Control group. #P<0.05 vs H₂O₂ group.

图7 VA 对H₂O₂诱导的Caco-2细胞肠屏障的影响

Fig.7 Effect of VA on intestinal barrier function of H₂O₂-induced Caco-2 cells (n=10). A: Immunofluorescence staining for claudin-1 and ZO-1 in each group. B: Western blotting for detecting expression levels of claudin-1 and ZO-1 in each group. *P<0.05 vs Control group; #P<0.05 vs H₂O₂ group.

图8 　VA 通过激活Nrf2/HO-1通路改善结肠炎小鼠肠道氧化应激损伤

Fig.8 VA alleviates intestinal oxidativestress in colitis mice by activating the Nrf2/HO-1 pathway. A: Immunofluorescencestaining of Nrf2/HO-1 in the colons of themice in each group. B: Intracellular ROSdetection using the fluorescent probe(DCFH-DA) in VA treatment group andML385 inhibitor group. C: IntracellularROS detection by flow cytometry in VAtreatment group and ML385 inhibitorgroup. D: Western blotting for detectingcolonic expression levels of Nrf2/HO-1 ofthe mice in each group. E: Western blottingfor detecting esxpression levels of Nrf2/HO-1 in Caco-2 cells in each group. *P<0.05vs (WT/VA-C/control) group. #PP<0.05 vs(DSS/H₂O₂) group.

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