α-香附酮通过调控TLR4/NF-κB信号通路拮抗肠黏膜炎症反应缓解小鼠克罗恩病样结肠炎

张诺; 张敏; 宋雪; 张小凤; 耿志军; 王炼; 葛思堂; 李静; 左芦根; 胡建国

doi:10.12182/20240960104

α-香附酮通过调控TLR4/NF-κB信号通路拮抗肠黏膜炎症反应缓解小鼠克罗恩病样结肠炎

α-Cyperone Antagonizes Intestinal Mucosal Inflammatory Response Through Modulation of TLR4/NF-κB Signaling Pathway to Alleviate Crohn's Disease-Like Colitis in Mice

摘要

摘要:
目的探讨α-香附酮（α-cyperone, CYP）对2，4，6-三硝基苯磺酸（2, 4, 6-trinitrobenzene sulfonic acid, TNBS）诱导的小鼠克罗恩病（Crohn's disease, CD）样结肠炎的作用效果及可能机制。
方法将小鼠随机平均分为野生（wild type, WT）组、TNBS组、CYP组和5-氨基水杨酸（5-ASA）组，每组10只。检测各组小鼠肠炎症状、肠屏障功能和结构及结肠中炎症因子〔白细胞介素（interleukin, IL）-6、肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、IL-1β及γ-干扰素（gamma-interferon, IFN-γ）〕表达水平。构建脂多糖（lipopolysaccharide, LPS）诱导Caco2细胞炎症模型，分为Control组、LPS组及LPS+CYP组，检测各组细胞紧密连接蛋白及炎症因子表达水平。采用基因本体（Gene Ontology, GO）功能富集分析预测CYP可能的作用途径与潜在的分子机制，并进行体内外验证。
结果体内研究中，与TNBS组相比，CYP组和5-ASA组小鼠体质量和结肠长度增加，而疾病活动度评分和组织学炎症评分降低（P<0.05）；异硫氰酸荧光素-葡聚糖水平、细菌移位率（肝脏、脾脏和肠系膜淋巴结）降低，而跨上皮电阻（transepithelial electric resistance, TEER）值、闭锁小带蛋白-1（zonula occluden protein-1, ZO-1）及闭合蛋白-1（claudin-1）表达增加（P<0.05）；炎症因子表达降低（P<0.05）。体外研究中，与LPS组相比，LPS+CYP组中Caco2细胞的TEER值、ZO-1和claudin-1表达增加（P<0.05）；炎症因子表达降低（P<0.05）。富集分析发现CYP与炎症应答相关（P<0.001）。Western blot结果表明，CYP可显著降低体内外Toll样受体4（toll-like receptor 4, TLR4）/核因子-κB （nuclear factor-κB, NF-κB）信号通路中关键蛋白的表达（P<0.05）。
结论 CYP可能通过调控TLR4/NF-κB信号通路的表达，拮抗肠黏膜炎症反应来保护肠屏障，进而缓解TNBS诱导的小鼠CD样结肠炎。

Abstract:
Objective To investigate the effect and potential mechanisms of α-cyperone (CYP) on Crohn's disease (CD) -like colitis induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) in mice.
Methods The mice were randomly and evenly divided into wild type (WT), TNBS, CYP and 5-aminosalicylic acid (5-ASA) groups, with 10 mice in each group. The symptoms of enteritis, the function and structure of the intestinal barrier, and the expression levels of inflammatory factors, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and gamma-interferon (IFN-γ), in the colon were assessed. The lipopolysaccharide (LPS)-induced inflammation model of Caco2 cells was constructed and the cells were divided into Control, LPS and LPS+CYP groups. The expression levels of tight junction protein and inflammatory factors in each group were assessed. Gene Ontology (GO) functional enrichment analysis was conducted to predict the possible pathways of action and potential molecular mechanisms of CYP, and to verify them in vivo and in vitro.
Results In the in vivo study, compared with those of the TNBS group, the body mass and colon length of mice in the CYP group and the 5-ASA group were significantly increased, while the disease activity scores and histological inflammation scores were significantly decreased (P<0.05). The level of lucifcein-glucan isothiocyanate and the bacterial translocation rate (in the liver, the spleen, and mesenteric lymph nodes) were significantly decreased, while the transepithelial electric resistance (TEER) value and the expression levels of zonula occluden protein-1 (ZO-1), and claudin-1 were significantly increased (P<0.05). The expression of inflammatory factors was significantly decreased (P<0.05). In the in vitro study, compared with those of the LPS group, the TEER value and the expression of ZO-1 and claudin-1 in the Caco2 cells in the LPS+CYP group were significantly increased (P<0.05). The expression of inflammatory factors was significantly decreased (P<0.05). Enrichment analysis showed that CYP was correlated with inflammatory response (P<0.001). Western blot results showed that CYP could significantly reduce the expression of key proteins in toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway in vivo and in vitro (P<0.05).
Conclusion CYP may protect the intestinal barrier by antagonizing the inflammatory response of the intestinal mucosa through regulating the expression of the TLR4/NF-κB signaling pathway, thereby alleviating TNBS-induced CD-like colitis in mice.

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