山苦瓜萃物對 DSS 誘發腸炎小鼠免疫反應的影響

Abstract

發炎性大腸疾病發生率增加，是目前逐受重視的飲食與健康議題。本研究室先前研究顯示山苦瓜能降低腸炎小鼠的病情，in vitro實驗指出山苦瓜乙酸乙酯萃物 (EAE) 具抗發炎功效，正丁醇萃物 (BE) 促進Foxp3+調節型T細胞 (regulatory T cell, Treg) 分化，故本研究進一步探討山苦瓜萃物EAE及BE對dextran sulfate sodium (DSS) 誘發腸炎的影響。方法為將8週齡BALB/c雄鼠分為AIN-93飼料的對照組 (Blank組)、腸炎組 (DSS組)、5%山苦瓜凍乾粉飼料的DSS/WBM組，及管餵相當DSS/WBM組所含2.4 mg/day EAE組 (DSS/EAE組)、和相當DSS/WBM組所含BE的3.2 mg/day BE 1倍組 (DSS/BE1x組)和4.8 mg/day的1.5倍組 (DSS/BE1.5x組)。分組餵飼5週後，所有的DSS組於飲水中添加3% DSS誘發腸炎，測定腸炎疾病指標 (disease activity index, DAI)包括體重流失和糞便評分。誘發7天後犧牲，測大腸長度和進行腸組織切片染色，並分離腸道固有層、皮耶氏體、腸繫膜淋巴結 (MLN)及脾臟細胞，以流式細胞儀分析Treg與CD103+樹突細胞 (dendritic cell, DC) 比率，測定血清、腸道均質液、以及MLN和脾臟細胞分泌的細胞激素含量。結果顯示，DSS/WBM組仍顯著改善腸炎指標，降低發炎反應，並提升MLN內Foxp3+ Treg比率。萃物則以DSS/EAE組顯著改善大腸長度、腸道組織受損與免疫細胞浸潤，血清IL-6以及腸組織IL-6、TNF-α、IL-1β及IFN-γ含量顯著降低，抑制MLN細胞分泌IFN-γ、IL-6與IL-10能力。補充BE在病發初期顯著降低DAI，DSS/BE1x組的腸組織TNF-α、IL-6及IFN-γ量顯著較低，DSS/BE1.5x組血清IL-6含量與MLN細胞IL-17A分泌能力顯著降低，同時，也有顯著較高的MLN與腸道黏膜固有層內CD103+ DC，以及MLN和皮耶氏體內Foxp3+ Treg和MLN內CD25+Foxp3+ Treg比率。綜合以上，山苦瓜乙酸乙酯萃物主要具抗發炎作用，而正丁醇萃物可提升腸道Treg比率，抑制腸道發炎作用。由本研究補充山苦瓜凍乾粉較萃物效果顯著，顯示山苦瓜可能透過不同成分的共同作用減緩腸炎小鼠病情。

The incidence of inflammatory bowel disease ha been increasing worldwide. Previous study found that wild bitter melon (WBM) can ameliorate dextran sodium sulfate (DSS)-induced colitis in mice. Ethyl acetate extract (EAE) of WBM showed significant anti-inflammatory effects in vitro. N-Butanol (BE) of WBM increased the regulatory T (Treg) cell populations in primary mesenteric lymph nodes (MLN) cells. We aimed to further investigate the effects of EAE and BE on inflammatory responses and immuno-regulatory effects in DSS-induced colitis in mice. BALB/c mice were fed with AIN-93diet (Blank and DSS group), 5% WBM powder (DSS/WBM group), 2.4 mg/day EAE (DSS/EAE group), 3.2 mg/day BE (DSS/BE1x group) or 4.8 mg/day BE (DSS/BE1.5x group) by oral avage. After 5 weeks, the Blank group was fed with normal drinking water, the other groups were fed with 3% DSS in sterile water to induce colitis. Disease activity index (DAI), including body weight loss and patterns of stool, were calculated. After 7 days of DSS induction, mice were sacrificed. Colons were collected to measure the length or be stained with haematoxylin and eosin. Moreover, cells in lamina propria, Peyer’s patch, MLN and spleen were isolated. The populations of Treg and CD103+ dendritic cells (DC) was analyzed by flow cytometry. Cytokine levels in serum, colons, or secreted by mitogen-stimulated MLN or spleen cells were detected. The results showed that WBM intake significantly ameliorated colitis through inhibiting inflammation and promoting the percentage of Treg cells in MLN. EAE supplement improve colon shortening and histological evaluation of colon. Serum IL-6, colonic cytokine levels (IL-6, TNF-α, IL-1β and IFN-γ) and cytokines (IFN-γ, IL-6 and IL-10) produced by ConA-stimulated MLN cells were decreased. Oral administration of BE significantly reduced DAI during the beginning phase of DSS induction. Colonic cytokines (TNF-α, IL-6 and IFN-γ) levels were decreased in DSS/BE1x group. Serum IL-6 levels and IL-17A secreted by MLN cells were dropped in DSS/BE1.5x group. The percentage of CD103+ DC in MLN and lamina propria, Foxp3+ Treg in MLN and Peyer’s patch, as well as CD25+Foxp3+ Treg in MLN were elevated. Although both EAE and BE are beneficial to colitis, WBM is the most effective treatment on DSS mice. WBM may ameliorate DSS-induced colitis by multiple bioactive components.