紫檀芪透過抑制發炎與腸纖維化減緩高脂飲食和葡聚糖硫酸誘導C57BL/6J小鼠結腸炎

Abstract

由於飲食習慣與生活作息改變，近幾年全世界肥胖人口逐漸增加，且近年研究顯示肥胖是發炎性腸道疾病的風險因子之一。攝取過多的油脂會影響腸道屏障功能與腸道發炎，甚至造成代謝症候群、胰島素阻抗或透過腸腦循環造成神經相關疾病的發生。紫檀芪 (Pterostilbene, PTS) 是 stilbenoid 之一，已有研究顯示 PTS 有抗發炎、抗氧化、抗纖維化、抗肥胖、調節血脂與抗癌等功效。因此，本次實驗研究目的為利用高脂飲食與葡聚糖硫酸 (Dextran sodium sulfate, DSS) 誘導結腸炎動物模式探討 PTS 預防效果的相關機轉。依劑量的不同，將 PTS 組別分成兩組，分別為添加 0.005% PTS 於飼料中的低劑量組 (CO-PTS (0.005%)) 及添加 0.025% PTS 於飼料中的高劑量組 (CO-PTS (0.025%))。於第九週和第十一週給予2.5% DSS 開始每天紀錄 Disease Activity Index (DAI) 值並於第十二週犧牲，收集血液與臟器，進行後續實驗分析。本次實驗結果顯示，PTS 在高脂飲食肥胖小鼠結腸炎中，顯著降低血漿中促發炎細胞激素 IL-6。在 H&E 與 Aberrant crypt foci (ACF) 的組織染色中，誘導組中可觀察到免疫細胞浸潤、壞死性結腸炎、隱窩損失 (Crypt loss) 與黏膜下層及肌肉層因潰瘍性壞死呈纖維化，然給予 PTS 的組別有效減少免疫細胞浸潤、壞死性結腸炎、Crypt loss 與黏膜下層及肌肉層因潰瘍性壞死呈纖維化等情況。另外，以西方墨點法分析相關分子機制，PTS 顯著減緩高脂飲食與 DSS 誘導結腸炎所產生的內質網壓力指標如 CHOP、發炎細胞酵素或激素如 COX-2 與 IL-1β，並維持腸道上皮屏障功能蛋白如 Muc2 與 E-cadherin 表現量，推測可增加腸道完整性，使通透性下降，減緩 DSS 或腸道中 LPS 引發的發炎反應，與抑制發炎相關併發症腸纖維化 TGF-β1/Smad2 的路徑，而減少細胞外基質堆積。因此進一步探討，高脂飲食誘導肥胖後，在給予 DSS 時介入 PTS 的機制，實驗結果發現也可顯著減少單位腸道長度重量比值與維持 Muc2 的表現，且顯著抑制 IL-1β、MMP2 和 TGF-β1 的表現。綜合上述結果，攝取 PTS 的組別顯著減緩高脂飲食肥胖小鼠結腸炎腸道屏障改變、發炎與腸纖維化，可能在高脂飲食造成腸道發炎與通透性改變時扮演調節的功效，甚至可預防腸發炎進一步產生腸纖維化，預防腸道功能損失或腸狹窄 (Intestinal stenosis) 的發生。

The worldwide prevalence of obesity has dramatically increased over the past few decades. It is currently believed that obesity is a risk factor for developing inflammatory bowel disease. Pterostilbene (PTS), a naturally occurring stilbene from blueberries, is known to have anti-cancer, anti-inflammation, anti-fibrosis and anti-obesity effects. The aim of this study was to investigate the impact of PTS supplementation on the susceptibility of high-fat diet (HFD)-fed mice to dextran sulfate sodium (DSS)-induced colitis. Beginning at 5 weeks of age, mice were fed normal diet, 50% HFD alone or containing PTS, and were given DSS (2.5%, w/v) in drinking water for week 9 and week 11 of the experiment. Disease activity index (DAI) was scored daily during the duration of the DSS treatment. After the second DSS treatment, all mice were sacrificed. Body weight, colon length, and colon weight-to-length ratio were measured directly. Plasma levels of pro-inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected using a commercial ELISA kit. Histological study was performed using a hematoxylin and eosin (H&E) staining assay. Colons were evaluated for aberrant crypt foci (ACF) formation and intestinal fibrosis and for C/EBP homologous protein (CHOP), mucin 2 (Muc2), E-cadherin, interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and TGF-β1/Smad signaling expression. It was found that PTS significantly attenuated HFD and DSS-induced plasma IL-6 accumulation and DAI, which implied that it suppressed weight loss, diarrhea, gross bleeding, and the infiltrations of immune cells. PTS suppressed HFD and DSS-induced formation of ACF and reduced colon weight-to-length ratio in HFD and DSS-induced colitis. Moreover, histological examinations indicated that PTS suppressed inflammation, necrotizing colitis, fibroblast cell infiltration and the loss of crypts induced by HFD and DSS. Furthermore, PTS inhibited CHOP, IL-1β, COX-2 and TGF-β1/Smad2 expression and maintain Muc2, E-cadherin expression. In addition, post-treatment with PTS also decreased colon weight-to-length ratio and loss of Muc2. CHOP, IL-1β, MMP2, TGF-β1 expression was significantly decreased in HFD and DSS-induced colitis after post-treatment with PTS. In conclusion, the result of the present study suggest that PTS is of great interest for the prevention of HFD and DSS-induced colitis in C57BL/6J mice.