白皮杉醇與3′-羥基紫檀芪維持腸黏膜屏障完整性緩解發炎性腸道疾病之功效

Effects of piceatannol and 3′-hydroxypterostilbene on alleviating inflammatory bowel disease by maintaining intestinal epithelial integrity

闕岱伶(Tai-Ling Chueh)

指導教授 ： 潘敏雄

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2022年)

https://doi.org/10.6342/NTU202203694

摘要

發炎性腸道疾病已被視為一項全球性的疾病，主要受到工業化國家生活方式西化及環境因子的影響。腸道屏障損傷是造成腸炎持續惡化的主因，而腸道屏障主要受到上皮緊密結合蛋白 (tight junction, TJ) 的支持。普遍而言，TJ 的損傷早於疾病進程，而腸道屏障功能喪失也受到促發炎細胞激素分泌及腸道菌相紊亂的影響，因此維持腸道屏障的完整性被視為腸炎的治療標的。雖然目前已有許多藥物用於治療腸炎，然而長期使用卻會引發諸多副作用，因此許多植化素已被作為腸炎的疾病預防策略。芪類化合物 (stilbenoids) 是廣泛存在於葡萄、百香果、花生等植物中的酚類物質，已被證實具有抗氧化、抗發炎、抗癌等生物活性，其中又以白皮杉醇 (piceatannol. PIC) 及三羥基紫檀芪 (3′-hydroxypterostilbene, HPSB) 被認為具有相對良好的生物活性，然而兩者改善腸道屏障受損的相關機制仍有待釐清。因此本實驗旨在探討 PIC 及 HPSB 是否具有保護腸道屏障以緩解腸炎的能力，首先利用 TNF-α 誘導的細胞模型模擬腸道發炎環境，評估 PIC 及 HPSB 抗發炎及保護腸道屏障之潛力，再藉由 dextran sulfate sodium (DSS) 誘導的腸炎模式評估兩者調控分子機制、腸道菌相以改善腸炎的效果。細胞實驗結果顯示，PIC 與 HPSB 皆顯著下調 p-p65/p65 表現量，並且降低腸道上皮細胞單層的通透性。而在 DSS 誘導之 ICR 小鼠，則發現 PIC 與 HPSB 皆能顯著抑制 TNF-α/NF-κB/MLC 路徑及降低 NLRP3 發炎小體活化緩解發炎反應，但僅有 PIC 顯著調節 TJ 組成，推測可能與 PIC 更可顯著抑制腸道上皮細胞凋亡蛋白 bax/bcl-2 及 caspase-3 活化有關。從腸道菌相分析結果則觀察到 PIC 與 HPSB 皆能調節腸道菌相，提高 Akkermansiaceae、Lactobacillus intestinalis 等益菌，並下調 Spiroplasmataceae、Acholeplasmataceae 等害菌。由 LEfSe 分析則顯示介入 PIC 的組別，以丁酸鹽產生菌作為標識菌種，由此推測 PIC 尚具有增加短鏈脂肪酸合成之潛力，而可提供上皮細胞能量來源，以維持腸道屏障之構造。綜上所述，PIC 顯著降低發炎反應、抑制細胞凋亡，並調節腸道菌相，而相較 HPSB 更能夠維持腸道屏障的完整性，在未來具有作為腸炎化學預防製劑之潛力。

關鍵字

結腸炎 ； 腸道屏障 ； 緊密結合 ； NLRP3 發炎小體 ； 白皮杉醇 ； 三羥基紫檀芪

並列摘要

Inflammatory bowel disease (IBD) has emerged as a global disease, which is attributed to westernized lifestyle and other environmental factors in industrialized countries. Research has indicated that the disruption of gut barrier plays a crucial role in the development of colitis, and it is mainly supported by epithelial tight junctions (TJs). Commonly, TJs are impaired substantially precede the development of the disease. Besides, gut barrier dysfunction also affected by pro-inflammatory cytokines production and gut microbiota dysbiosis. Thus, maintaining gut barrier integrity is considered as the therapeutic target in colitis. Many synthetic drugs are currently in use to treat IBD; however, long-term usage of these drugs results in many complications. Hence, lots of phytochemical compounds have been proven as preventive strategy for IBD treatment. Stilbenoids are a group of phenolic compounds present in various plants, such as grape, passionfruit, and peanuts, which exert diverse bioactivities ranging from anti-oxidant, anti-inflammation, and cancer prevention. Among them, piceatannol (PIC) and 3′-hydroxypterostilbene (HPSB) are considered to have relatively good bioactivities. However, the mechanisms of PIC and HPSB on ameliorating gut barrier dysfunction haven’t been fully investigated. Therefore, we aimed to evaluate the ability of PIC and HPSB on gut barrier protection to reduce the severity of colitis. Firstly, we used TNF-α induced cell model to examine the anti-inflammatory and gut protection effect of PIC and HPSB, then used a DSS-induced colitis mice model to clarify the effect of both compounds on underlying mechanisms and microbiota modulation. Results obtained from in vitro experiments showed that, both of PIC and HPSB downregulated p-p65/p65 expression and decreased the permeability of intestinal epithelial monolayer. In contrast, in DSS-induced colitis ICR mice, both of PIC and HPSB could attenuate inflammation through inhibiting TNF-α/NF-κB/MLC pathway and reducing NLRP3 inflammasome activation. However, PIC was comparably effective in TJs modulation. The results may attribute to the effect of PIC on reducing cell apoptosis associated protein expression, including bax/bcl-2 and caspase-3 activation. Furthermore, the result of microbiota analysis showed that both of PIC and HPSB could increase the representative probiotics, including Akkermansiaceae and Lactobacillus intestinalis. On the other hand, supplementation of PIC and HPSB exhibited inhibitory effects on the several bacteria species (at family level) with undesired effect, including Spiroplasmataceae and Acholeplasmataceae. Based on LEfSe analysis, butyrate-producing bacteria was regarded as a biomarker in PIC group, so we speculated that PIC has the capability to increase short chain fatty acid production and can be used as an energy source for epithelial cell. In conclusion, the results demonstrated that PIC reduced inflammation, inhibited cell apoptosis, and regulated microbiota composition. Consequently, PIC is more effective on maintaining gut barrier integrity, and it will be a promising ingredient applied to the development of functional food for colitis prevention.

並列關鍵字

colitis ； gut barrier ； tight junctions ； NLRP3 inflammasome ； piceatannol ； 3′-hydroxypterostilbene

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