不同品系山苦瓜及其內源性酵素水解衍生物對肝臟脂質代謝的影響

Abstract

肥胖以及代謝異常相關慢性疾病，如糖尿病、高血壓、非酒精性脂肪肝等，為目前極受關注的危害健康議題。研究指出苦瓜能有效控制血糖以及改善脂質代謝。山苦瓜的特色活性成分中，三萜類化合物已被報導能夠活化 AMPK 以促進葡萄糖汲取， 9c, 11t, 13t-conjugated linolenic acid (CLN) 則能透過活化 PPARα 進而調節脂質代謝。本研究第一部分首先就花蓮改良場近年雜交育種的幾種山苦瓜品系，檢測比較其三萜類化合物與 CLN 含量，並分析其活化 PPARα 與 LXRα 之轉錄活性，以初步篩選出改善代謝異常效果最好之品系。結果顯示，品系 55M 、 H4 (花蓮四號) 與 N81 之三萜類化合物較高，而 CLN 含量以 55M 與 N81 較高。活化 PPARα 之轉錄活性以 CKP55 、 1758 與 V81 較高。另外，本研究首次觀察到山苦瓜萃物具有拮抗 LXRα 轉錄活性，可能具有預防肝中脂質堆積之功效。各品系中以 55M 之拮抗 LXRα 最高， N81 次之。由於此二品系之 EA 萃物中 CLN 含量也是最高，推測拮抗 LXRα 之活性可能與 CLN 有關。進一步以 CLN 進行實驗，結果顯示 CLN 拮抗 LXRα 之活性，且具劑量效應。本研究進一步以高脂飲食誘導 C57BL/6J 小鼠代謝異常之動物模式，探討數種品系山苦瓜於體內模式之效應。研究結果顯示各苦瓜品系皆可改善代謝異常之指標。其中又以品系 H4 於改善血糖、血脂及脂肪組織重量等之幅度，優於其他品系。所有品系山苦瓜皆能有效降低肝臟中三酸甘油酯含量，而品系 55M 、 1758 與 P81 改善肝膽固醇 (mg/liver) 之效果最佳。有趣的是測試品系中，以品系 55M 可促進肝中 LXRα 下游基因 Srebf1 、 Fasn 、 Abcg1 及 Cyp7a1 之表現，且並未促進肝中脂肪堆積。 過去研究指出苷元形式葫蘆烷型三萜類化合物相較於帶醣基之形式有更佳活化 AMPK 之效果。而本實驗室先前研究發現 H4 山苦瓜具有內源性 β-glucosidase ，且在特定催化條件下有效增加山苦瓜 EA 萃物中苷元或帶有短鏈醣基形式三萜類化合物。經動物實驗證實，餵食 37℃ 水解後的山苦瓜凍乾粉末可有效促進肝臟粒線體相關基因表現並減少肝臟脂質堆積。接續先前研究，本部分實驗初步探討各品系山苦瓜水解後 EA 萃物組成分與生物活性之改變。結果顯示品系 H4 山苦瓜有最佳內源性 β-glucosidase活性，經過 37℃ 與 60℃ 水解後 H4 之 EA 萃物以及 60℃ 水解後 CKP55 之 EA萃物中三萜類化合物顯著增加。然而水解後 EA 萃物中 CLN 含量與活化 PPARα 轉錄活性顯著低於未水解萃物。不過，研究結果顯示水解後 EA 萃物拮抗 LXRα 活性顯著高於未水解。接續以上研究結果，最後以 T0901317 誘導 HepG2 細胞三酸甘油酯堆積之模式，探討未水解及水解 H4 山苦瓜 EA 萃物能否透過拮抗 LXRα 轉錄活性抑制肝臟三酸甘油酯合成。研究結果顯示較低劑量的未水解以及60℃ 水解山苦瓜 EA 萃物皆能夠降低 T0901317 誘導的三酸甘油酯增加量，而最後研究也發現 H4 山苦瓜 EA 萃物能夠降低 HepG2 細胞中 LXRα 下游脂肪酸合成相關基因表現。 綜合以上，本研究結果顯示未水解及水解後的山苦瓜皆能夠調節肝臟脂質代謝。而以 LXRα 細胞轉錄活性分析篩選出的品系 55M ，在動物體中也能夠調控 LXRα 目標基因。值得一提的是，這是第一個探討山苦瓜萃物能夠調控 LXRα 轉錄活性的研究，且根據研究結果而推論山苦瓜及 CLN 可能是 LXRα 的選擇性調節劑。

Obesity and its related chronic diseases, such as type 2 diabetes, hypertension, and non-alcoholic fatty liver disease, become a key issue in public health. Previous studies indicated that bitter gourd can ameliorate hyperglycemia and dyslipidemia. The bioactive compound in bitter gourd have been identified as cucurbitane-type triterpenoids, 9c, 11t, 13t-conjugated linolenic acid (CLN) and peptide, etc. Particularly, triterpenoids can improve hyperglycemia via AMPK to enhance glucose uptake, and CLN can regulate lipid metabolism by PPARα activation. Multiple cultivars of wild bitter gourd (WBG) have been crossbred by Hualien District Agricultural Research and Extension Station. The first part of this study aims to examine triterpenoids and CLN content in various cultivars of bitter gourd, as well as PPARα and LXRα transactivation activities of bitter gourd ethyl acetate (EA) extract. The results showed that cultivars 55M, H4 and N81 have the highest triterpenoids content. 55M and N81 have the highest CLN content. In the cell-based transactivation assay, EA extracts of CKP55, 1758 and V81 showed the highest transactivation activities of PPARα. Interestingly, WBG EA extracts dose-dependently inhibited the transactivation of T0901317, a synthetic LXRα ligand, and 55M showed the highest inhibition. Based on results from CLN content and LXRα transactivation assay, we speculate that CLN in WBG might modulate transcriptional activities of LXRα. The first part of this study also examined effects of various cultivars of WBG in an animal study. WBG was supplemented to a high-fat diet and fed C57BL/6J mice for 4 weeks. Among the cultivars, H4 performs outstandingly in ameliorating hyperglycemia and dyslipidemia, as well as fat accumulation in WAT. 55M, 1758 and P81 display advantages in reducing cholesterol accumulation in mice liver. Intriguingly, cultivar 55M up-regulated LXRα-related genes in the liver, including Srebf1, Fasn, Abcg1 and Cyp7a1. Previous studies reported that triterpenoid aglycones had higher activities than glycoside froms in enhancing glucose uptake of adipocytes. Previous studies in our lab have developed a hydrolysis procedure using the endogenous β-glucosidase of WBG. Previous animal studies in our lab also showed that 37℃ hydrolyzed WBG powder ameliorates the obesity-induced fatty liver more effectively than the non-hydrolyzed WBG. The second part of this study further examined effects of hydrolysis on composition and biological activities in various cultivars of WBG. Among various cultivars tested, H4 has the highest β-glucosidase activity. Triterpenoids content in EA extract of H4 WBG increases sharply under 37℃ and 60℃ hydrolysis as expected. In contrast, CLN content and PPARα transactivation activities of EA extracts decrease after hydrolysis. Surprisingly, antagonistic effect on LXRα significantly elevated after hydrolysis of WBG. The antagonistic effect is further examined in HepG2 cells. The T0901317-induced triglyceride accumulation in these cells was suppressed by lower dose of H4 EA extract. In addition, the mRNA expression levels of genes related to fatty acid synthesis were also down-regulated. In conclusion, WBG and hydrolyzed WBG both can improve hepatic lipid metabolism. Cultivar selected by the cell-based LXRα transactivation assay, i.e. 55M, also modulate LXRα target genes in animal model. Noticeably, this is the first study that demonstrates WBG extracts can modulate the transactivation activities of LXRα. Results of this study implies that WBG/CLN might act as a “selective modulator” of LXRα.