山苦瓜葉萃取物對高果糖飲食誘導小腸損傷之保護效果

Abstract

我們的日常生活中充斥著高果糖食物，高果糖食物在攝入後首先到達小腸，造成腸道菌相改變、引起腸道發炎並破壞腸道屏障，進而造成代謝性疾病、慢性發炎、非酒精性脂肪肝等，因此保護小腸為高果糖飲食下的首要關鍵。在先前研究中發現山苦瓜葉萃取物中富含三萜類化合物 (Triterpenoid-enriched extract, TEE)能降低DSS (Dextran sulfate sodium)誘導的慢性腸炎，並且對於腸道屏障也有恢復的作用，因此本研究利用TEE減輕果糖所誘導的腸道損傷，探討TEE保護腸道免於高果糖傷害的機制為何。 在動物實驗中以週齡12週大的C57BL/6公鼠作為模式動物以固態和液態兩種果糖添加於飲水或飼料中的模式作為誘導14週，同時給予TEE進行比較，發現在腸道菌相中TEE能有效恢復兩種果糖對腸道菌相的豐富度，並增加Akkermansia spp.在腸道菌中所佔比例，對腸道菌叢有正面的影響。在分析與腸道共生菌直接接觸的黏膜屏障及分泌黏液(mucin)的杯狀細胞數量上，給予TEE的組別能有效恢復杯狀細胞數量且增加MUC2的表現，顯示TEE能促進杯狀細胞生長及mucin分泌以保護腸道。果糖引起的腸道發炎在給予TEE的組別中提升了NLRP6的表現量，且有效降低p-P65, TLR4造成的發炎反應。最後結果發現TEE能保護固態果糖飼料組別中腸道上皮細胞屏障的完整性，進而降低血清中內毒素和肝指標，而液態果糖中，TEE在腸道上皮細胞屏障和內毒素入侵方面則沒有發揮保護效果。 在單獨培養Akkermansia muciniphila的實驗中發現TEE對於腸道菌叢中Akkermansia spp.的增加並非直接促進其生長，可能是經由提高杯狀細胞分泌的mucin提供碳源，而使其數量增加。因此探討了TEE提升杯狀細胞分泌mucin的途徑，實驗中利用HT29細胞進行分化為類杯狀細胞後，發現TEE經由K-Ras, Raf-1, MEK1/2, ERK1/2最終提高MUC2的表達量，我們更近一步發現在分化過程中給予TEE會提升Wnt3a, Wnt5a, ESE1的表現量，表示TEE可能促進細胞分化的過程產生較多腸道幹細胞(Intestinal stem cells, ISC)，並使ISC趨向於分化成為具有分泌mucin能力的杯狀細胞。另外TEE作用於果糖所引發的IEC-6腸細胞發炎，不僅提高NLRP6與其下游訊號分子IL-18的表現量，同時降低NLRP3, IL-1β, TNF-α等促發炎因子的表現量下降，並利用NLRP6 shRNA knockdown的腸細胞進行反證，確認TEE降低腸道發炎的機制是經由NLRP6主導的途徑達成，綜合上述結果TEE對高果糖所引起的腸道損傷確實具有保護效果。

Foods contained high fructose are rich in our daily life, these foods reach the small intestine first, cause intestinal flora alteration, induce intestinal inflammation, damage intestinal barrier and further lead to disease, such as metabolic syndrome, chronic inflammation, and non-alcoholic fatty liver disease (NAFLD). Therefore, fiinding an effective therapy to protect small intestine from fructose-induced damages is necessary. Previous study showed that triterpenoid enrich extract (TEE) obtained from wild bitter melon leaves can alleviate DSS induced intestinal inflammation, and recover colon epithelial barrier. In this study we aim to find out whether TEE can protect the small intestine under high fructose diet and explore the under lying mechanisms. In in vivo model, 12-week-old C57BL/6 male mice were fed with fructose in two forms, solid or liquid for 14 weeks, and TEE was given at the same time. Microbiota diversity altered by fructose was restored by TEE, and the ratio of beneficial bacteria, Akkermansia spp. was increased in both TEE fed group, indicating TEE had a positive impact on intestinal flora. With the direct interaction with bacteria, mucin and its producer, goblet cell were investigated. TEE promoted mucin secreion with higher expression of MUC2 and elevated number of goblet cells, which revealed that TEE protect intestinal mucus barrier under high fructose diet. Intestinal inflammation is another damage caused by high fructose, TEE alleviated the inflammation by increasing NLRP6 and reducing p-P65, TLR4 expessions. At last, the intestinal epithelial barrier integrity was examined, TEE protected the disruption of epithelial junction to reduc the serum endotoxin level in solid, but not liquid fructose fed group. The reduced liver inflammation was observed in both groups. In the in vitro models, the growth curve of Akkermansia muciniphila (A. muciniphila) was studied with supplements of fructose and TEE. TEE didn’t accelerate the growth of A. muciniphila, and thus TEE was speculated to elevate Akkermansia spp. ratio in intestinal flora by increasing mucin sectretion, which is the major carbon source of A. muciniphila. In addition, TEE induced mucin secreion through K-Ras/Raf-1/MEK1/2/ ERK1/2 pathway and MUC2 expression in HT29-differentiated goblet cell. TEE not only promoted mucin secreion in goblet cells, but also stimulated the differentiation of intestinal stem cells. Next, TEE protected intestinal cells from fructose-induced inflammation through increasing NLRP6 and its downstream product IL-18, and decreasing NLRP3, IL-1β, and TNF-α. With the shRNA targeting NLRP6, NLRP6 was proved to be the key for the protective effect of TEE in the intestinal cells. In summary, TEE effectively protected fructose-induced intestinal injuries, and further rescue the systemic inflammation.