山苦瓜葉萃取物對葡聚醣硫酸鈉誘導小鼠腸炎的保護效應與免疫調節之影響

發炎性腸道疾病 (inflammatory bowel disease, IBD)包括克隆氏症與潰瘍性腸炎，為一種慢性反覆發炎的免疫媒介疾病。在腸道免疫系統，輔助型T細胞 (helper T cell, Th)與調節型T細胞(regulatory T cell, Treg)的失衡被認為與IBD有關。山苦瓜葉(wild bitter melon leaf, WBML)具有抗發炎、調節免疫、抗氧化等生理活性。本研究以葡聚醣硫酸鈉(dextran sulfate sodium, DSS)誘導小鼠腸炎模式，探討介入WBML對於腸炎疾病的可能保護功效與其作用機轉。首先觀察預先給予WBML (4種山苦瓜品系：HL-2、HL-3、#1621、#8123) 預防性減緩腸炎的效應。將C57BL/6J小鼠分為正常控制組、腸炎組(DSS組)和DSS+EE (山苦瓜葉乙醇萃取物, ethanol extract)組，先管餵EE經1週後停止介入，再以3 % DSS誘導急性腸炎經5天後進行犧牲觀察，發現4種品系EE均能顯著降低血液中B cell、T cell以及Th1、Th2、Th17比率，推測WBML具免疫調節和緩解腸炎的潛力，其中以HL-3的效果較佳因此選用該品系進行後續實驗。實驗二，將小鼠分為正常控制組、DSS組及DSS+WBML組(分別為餵食山苦瓜全葉乾燥粉末(whole leaf, WL)組、EE組和富含三萜類之區分物 (triterpenoid enriched-extract, TEE)組)，管餵1週後再以3.5 % DSS 誘導急性腸炎並持續給予WL、EE、TEE經7天後犧牲。結果發現TEE顯著減緩腸炎小鼠體重減輕、疾病活動指數 (DAI)、脾臟重量增加情形和降低糞便lipocalin-2、大腸組織ICAM-1和腸腔沖洗液CCL2 (MCP-1)濃度。從組織病理切片觀察，TEE顯著降低DSS誘導腸炎的嚴重程度。而EE顯著減少腸腔沖洗液TNF-α和CCL2濃度。WL顯著降低腹腔沖洗液(PCW)中neutrophil、B cell 及血液中macrophage比率，EE能顯著減少PCW中neutrophil、macrophage、B cell 及血液中neutrophil、B cell 比率。TEE顯著抑制PCW中B cell及血液中neutrophil比率。進一步分析血液中T細胞次群，發現EE和TEE均顯著抑制 Th1比率，EE 顯著抑制Th2比率，而WL、EE、TEE皆能顯著抑制血液中Th17 比率並提高Treg比率。與DSS組比較，WL、EE、TEE組的腸繫膜淋巴結T-bet (Th) mRNA表現量皆較低，而Foxp3 (Treg) mRNA表現量皆增加，其中TEE組達顯著差異。本研究結果推測WBML可藉由調控DSS誘發腸炎造成之Th/Treg 失衡，具有緩解腸炎的潛力。

關鍵字

發炎性腸道疾病；山苦瓜葉；腸道免疫；調節型T淋巴細胞；輔助型T淋巴細胞；調節免疫

並列摘要

Inflammatory bowel disease (IBD), a dysregulated immune inflammatory state of the gastrointestinal tract, is classified into two archetypal phenotypes: Crohn's disease (CD) and ulcerative colitis (UC). A dysregulated intestinal T cell response is presumed in patients with IBD. An altered balance between Treg cells and T effector cells in the intestinal microenvironment might contribute to the pathogenesis of IBD. Wild bitter melon (WBM; Momordica charantia L.var.abbreviata Seringe), a variety of bitter melon, possesses several biological activities. The aim of this study was to investigate the protective potential of WBM leaf (WBML) against dextran sulfate sodium (DSS)-induced murine colitis. First, C57BL/6 mice were randomly divided into six groups: one normal control (NC) group and five DSS-treated groups, including DSS group (colitis model) and the ethanol extract (EE) of WBML (WBM cultivars: HL-2, HL-3, #1621, and #8123) treatment groups. The results showed that 1-week pre-treatment of all of four EE significantly reduced B cell and T cell populations, and ratios of Th1, Th2, Th17 in blood. Our data suggested that WBML might ameliorate DSS-induced colitis, especially the HL-3 cultivar. Hence, WBML from HL-3 was used for subsequent experiments. Next, we prepared WBML samples including whole leaf (WL) and its extracts (EE and triterpenoid enriched-extract, TEE). C57BL/6 mice were randomly divided into five groups: one normal control (NC) group and four DSS-treated groups, including DSS group (colitis model), WL group (DSS+ WL), EE group (DSS+EE) and TEE group (DSS+TEE). The DSS-treated groups drank distilled water containing 3.5% DSS for 7 d to induce colitis, while NC group drank distilled water. WL, EE, and TEE groups was respectively administrated by oral gavage with WL (700 mg/kg BW), EE (100 mg/kg BW), and TEE (70 mg/kg BW) for 7 days before DSS-induced colitis and subsequently received WL, EE, or TEE after DSS-induced colitis for 7 days. Our results showed that TEE significantly ameliorated DSS-induced mice weight loss, disease activity index (DAI), colon shortening, colonic CCL2 (MCP-1), colonic intercellular cell adhesion molecule-1 (ICAM-1), and fecal lipocalin-2 levels. In addition, TEE reduced the severity of DSS-induced histopathology in the colon. WL supplementation significantly reduced the proportion of peritoneal neutrophil and B cell and the proportion of macrophage in blood. EE supplementation significantly reduced the proportion of peritoneal neutrophil macrophage, and B cell and the proportion of neutrophil in blood. TEE supplementation significantly reduced the proportion of peritoneal B cell and the proportion of neutrophil and B cell in blood. All three WBM treatments significantly decreased Th17 proportion and enhanced Treg proportion. In addition, TEE significantly lowered Th2. All three groups significantly inhibited T-bet mRNA expression (Th1) in spleen and mesenteric lymph nodes. Moreover, TEE supplementation significantly increased foxp3 mRNA expression (Treg) in spleen and mesenteric lymph nodes. In conclusion, these results suggested that WBML treatment regulated Th/Treg-mediated immunity and may consequently attenuate inflammatory responses, thus improving the symptoms of colitis in DSS-treated mice.