小根蒜萃取物對葡聚醣硫酸鈉誘導小鼠急性腸炎之保護效應

Protective effect of Allium macrostemon Bunge extract against acute colitis in dextran sulfate sodium-treated mice

毛語葳(Mao Yu-Wei)

指導教授 ： 蔡帛蓉 ； 侯又禎

國立臺灣師範大學/理學院/營養科學碩士學位學程/碩士(2022年)

https://doi.org/10.6345/NTNU202201368

摘要

發炎性腸道疾病 (inflammatory bowel disease) 是反覆發炎的慢性疾病，其致病因子涉及基因、環境、腸道上皮損傷、腸道菌群失衡與異常的免疫反應等。T細胞及其相關細胞激素的調節與平衡，為維持腸道免疫恆定之重要因子。本研究首先對於台灣花蓮原住民作物「火蔥」進行植株特徵比對及基因序列分析，結果鑑定此「火蔥」為小根蒜 (Allium macrostemon Bunge) 的鱗莖。小根蒜屬於石蒜科蔥屬之藥食兩用的植物，將小根蒜的鱗莖進行蒸煮、乾燥後做為傳統中藥的「薤白」。本研究以葡聚醣硫酸鈉 (dextran sulfate sodium, DSS) 誘導C57BL/6J小鼠急性腸炎模式，探討小根蒜的新鮮鱗莖萃取物是否具緩解腸炎之能力。首先製備小根蒜的水 (aqueous extract, AE) 與50%乙醇萃取物 (50% hydro-ethanolic extract, HEE) 進行實驗一，結果發現預先管餵小根蒜HEE (200 mg/kg/day)，可降低DSS引致急性腸炎小鼠的血液之嗜中性白血球次群、減少大腸組織免疫細胞浸潤、維持完整腸道上皮，具緩解急性腸炎的潛力；然而AE則無此效用。接續進行實驗二，探討補充小根蒜HEE減輕急性腸炎之效用與機轉。C57BL/6J小鼠分為4組：正常控制 (NC) 組、腸炎控制 (DC) 組，與腸炎並介入250 mg HEE/kg/day (DL) 組或500 mg HEE/kg/day (DH) 組。NC和DC組預先管餵水，而DL和DH組預先管餵HEE，經一週後再給予3% DSS飲水引致急性腸炎並持續管餵HEE，經一週後犧牲。實驗結果當與DC組比較，DL和DH組顯著降低血漿haptoglobin濃度；在血液嗜中性白血球、發炎性單核球和調節性T細胞之比率皆降低，以及淋巴球比率上升；脾臟調節型T細胞與輔助型T細胞17相關細胞激素與轉錄因子forkhead box p 3、interleukin (IL)-10、transforming growth factor-1、IL-17A和retinoic acid receptor-related orphan receptor gamma-t之mRNA表現量降低；大腸組織促發炎相關基因C-C chemokine ligand 2、tumor necrosis factor-和C-X-C motif chemokine ligand-1與腸道黏膜屏障相關保護因子trefoil factors 3之mRNA表現量，以及大腸沖洗液Immunoglobulin G的濃度皆降低，並發現DL和DH組有較低的疾病活動指數與大腸水腫程度，另於大腸組織切片觀察其腸道免疫細胞浸潤程度較低，且具有相對較完整的隱窩與腸道上皮。本研究結果顯示，預防性給予小根蒜HEE可透過免疫調節與抑制發炎反應，具有緩解急性腸炎的潛力。

關鍵字

小根蒜 ； 發炎性腸道疾病 ； 葡聚醣硫酸鈉 ； 抗發炎

並列摘要

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestine. The pathogenesis of IBD is complex, including gene, environment, epithelial barrier disruption, gut microbiota dysbiosis and immune response dysregulation. T cells play an important role in maintaining intestinal immunity underling IBD pathogenesis. Allium macrostemon Bunge is a traditional crop from the indigenous people in Taiwan. The purpose of this study was to evaluate whether the bulb extract of A. macrostemon Bunge extract attenuates dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6J mice. Aqueous extract (AE) and hydro-ethanolic extract (HEE; 50% ethanol) of A. macrostemon bulb were prepared. The preliminary experiment showed that HEE supplementation (200 mg/kg/day) alleviated DSS-induced murine acute colitis, but AE did not. Subsequently, the protective effect and mechanism of HEE against DSS-induced acute colitis were investigated. C57BL/6J mice were divided into normal control (NC) group, DSS-treated control (DC) group, DSS+ 250 mg/kg HEE (DL) group and DSS+ 500 mg/kg HEE (DH) group. All animals were given free access to standard chow diet. Mice in the DC, DL, and DH groups received drinking water containing 3% (w/v) DSS for 7 days to induce acute colitis. The DL and DH groups received HEE by daily oral gavage for one week before starting the DSS treatment and throughout the entire experimental period. Results showed that the disease activity index, plasma haptoglobin, immunoglobulin G of colon lavage fluid, the percentage of circulating neutrophils, inflammatory monocyte and regulatory T cells levels in blood were lower, whereas the lymphocyte populations in blood were higher in the DL and DH groups, as compared with the DC group. HEE supplementation reduced mRNA levels of forkhead box p 3, interleukin (IL)-10, transforming growth factor-1, IL-17A and retinoic acid receptor-related orphan receptor gamma-t in spleens and also down-regulated the C-C chemokine ligand-2, tumor necrosis factor-C-X-C motif chemokine ligand-1 and trefoil factors 3 in colons. The histological observation of the colon further implied that HEE could reduce inflammatory cell infiltration and restore intestinal epithelial barrier integrity. In conclusion, HEE could significantly alleviate the signs and symptoms of the DSS-induced acute colitis by modulating immune responses and suppressing expression of inflammatory mediators.

並列關鍵字

Allium macrostemon Bunge ； inflammatory bowel disease ； dextran sodium sulfate ； anti-inflammatory

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