| Abstract: | 代謝症候群（metabolism syndrome)是一種包含肥胖、血壓偏高、血糖偏高以及血脂異常等多項危險 因子綜合表現的臨床徵候，伴隨著這些危險因子的增加，罹患心血管疾病、糖尿病和脂肪肝也相對提升。 這三個代謝性疾病已被證明可用飲食來控制。因此利用健康食品來預防或改善代謝症候群應有其發展的可 行性。蓮蓬（Lotus seedpod)味苦性澀溫，自古即被用來行氣除脹、益補脾胃、止血化瘀、清熱、降肝火 及止血功效。過去研究顯示蓮蓬原花青素（procyanidins of lotus seedpod，LSPCs)具有抗氧化以及免疫調 節之活性。目前我們已進行蓮蓬水萃取物(lotus seedpod extract，LSE)之分離，初步已完成HPLC成份鑑定 (附件Table 1以及Fig. 1)，發現LSE主成份為類黃酮（flavonoid) ： catechin及EGC (兩者為原花青素之裂 解產物），後續亦積極進行原花青素之精萃。此類黃酮具有抗氧化、抗發炎、降血脂、防癌及抑制心血管 疾病，顯示LSE具有預防及改善代謝症候群的潛力。近來我們實驗室研究顯示LSE具有抑制脂質過氧化、 LDL氧化（附件Fig. 2)及泡沫細胞生成（附件Fig. 3A)之作用，可以減少單一高脂誘導紐西蘭大白兔動脈 硬化斑沉積表現（附件Fig. 4)。故本研究計劃進一步探討LSE及LSPCs對於代謝症候群（包含胰島素阻抗、 脂質異常及動脈粥狀硬化多項危險因子）是否具預防及保護效果，並釐清其中多重調控的分子機轉。 第一年之計劃擬以高脂合併高糖飲食誘發代謝症候群之動物模式’評估LSE以及LSPCs可否改善代 謝症候群的症狀。在評估代謝症候群症狀方面以體重及腹部脂肪堆積作為肥胖指標；以血糖、血清胰島素、 OGTT及ITT血糖變化評估是否胰島素阻抗；並量測血脂及血壓。另測量血管和肝臟?脂質堆積表現、肝 臟脂質代謝相關酵素活性；以及分析抗氧化酵素、血液脂肪細胞激素（包括adiponectin和leptin)及發炎 激素（TNFa、IL-1p、IL-6)濃度；並以免疫組織染色觀察各組織當中異常蛋白質表現變化。第二年工作’ 以研究LSE及LSPCs增加細胞對胰島素敏感性’以及促進胰臟|3-細胞存活之影響’作為改善代謝症候群 以避免第2型糖尿病的發生。觀察當胰臟P-細胞處於高糖、ox-LDL或TNFa刺激的環境下，細胞葡萄糖 代謝異常及細胞凋亡情形是否因此增加而導致P-細胞衰竭、降低P-細胞的質量使得胰島素分泌量下降無法 發揮正常功能。另外其相關調節胰島素訊息傳遞路IRS-1/PI3K/Akt路徑的活化是否受到影響將一併討論。 當在此不利的生理壓力條件下，於細胞培養液中添加LSE以及LSPCs之後，是否對P-細胞具有調節及保 護的作用。第三年工作，以研究LSE及LSPCs促進肝細胞?脂肪代謝之影響’以及抗氧化和抗發炎活性 評估’作為改善代謝症候群以避免脂肪肝的發生。透過探討高糖、ox-LDL或TNFa刺激肝細胞HepG2脂 肪堆積表現以及脂肪代謝相關酵素活性、蛋白和mRNA (包含HMG-CoA reductase、FAS、PPARs、SREBPs 和LDLR等）表現改變來釐清LSE及LSPCs排除肝臟脂肪之機轉。另一方面，評估其細胞?抗氧化酵素 活性、以及脂肪細胞激素和發炎激素之含量，以證實LSE及LSPCs為抗氧化及抗發炎物質，可減輕因氧 化壓力與發炎反應，修正細胞激素分泌異常的狀況。第四年工作，以研究LSE及LSPCs對?皮細胞失能’ 以及平滑肌細胞增生及遷移作用之影響，作為改善代謝症候群以避免動脈粥狀硬化的發生。利用高糖、 ox-LDL或TNFa誘導血管?皮細胞HUVEC死亡（附件Fig. 5 ：預備實驗比較不同inducer之功效），以及 血管平滑肌細胞A7r5不正常增生與遷移現象。第一部份探討LSE及LSPCs對於HUVEC細胞之保護作用， 觀察其是否具有抗凋亡以及分析其相關的分子機轉。第二部份則以LSE及LSPCs調控A7r5細胞週期停滯 以及抑制細胞遷移為方向，釐清其中的分子調控路徑；由於我們預備實驗也發現CTGF與A7r5細胞的增 生遷移相關（附件Fig. 3B)。因此，實驗第四年的另一工作焦點，則放在血管病灶中特有的CTGF上。擬 利用CTGF-mediated atherosclerosis之基因轉殖細胞模式，藉此媒介其後的訊息傳遞機轉與A7r5細胞增生 遷移等變化，並測試LSE及LSPCs是否能藉由調節CTGF而產生效用。 從以上動物和細胞實驗結果可以預期LSE以及LSPCs具有下列作用：（1)降血糖、抑制胰島素阻抗、 降血脂、排除肝臟脂肪堆積及減少動脈粥?化變灶；（2)增加細胞對胰島素敏感性及促進胰臟P-細胞存 活；（3)促進肝細胞脂質代謝、具抗氧化和抗發炎作用，以及修正細胞激素分泌異常的狀況；（4)保護 ?皮細胞免於損傷、促進血管平滑肌細胞週期停滯達到抑制細胞增生，並有效地調控細胞遷移作用；以及 (5)我們亦將釐清牽涉於血管病變中大量表達之CTGF相關訊息傳遞路徑，以及CTGF是否可作為一項分 子標的。因此，本研究可以說明蓮蓬萃取物LSE及其功能性成份LSPCs發揮改善代謝症候群之功效。由於 蓮蓬在國?容易栽培，但過去一直被視為無經濟價值的植物部位；而LSE以及LSPCs也容易分離，因此， 本研究成果除了可應用於發展新的保健食品，也能推廣蓮蓬為可食用之資源。
Metabolic syndrome is characterized by obesity, hypertension, hyperglycemia and dyslipidemia as well as a cluster of risk factors. Along with these risk factors rising, the probability of suffering from cardiovascular disease, diabetes and fatty liver would be increased. Nelumbo nucifera Gaertn., commonly known as lotus, is a perennial aquatic plant, grown and consumed throughout Asia. Almost all parts of lotus are eaten as vegetables and are also used for various medicinal purposes in oriental medicine. Lotus seedpod is usually discarded, except when sometimes used as a traditional medicine with hemostasis function and for eliminating bruise. Previous studies have demonstrated that a procyanidin-rich fraction (LSPCs) could be isolated from the lotus seedpod, which exhibited some powerful biological effects, including antioxidant and anti-cancer activities. It might have possessed the potential of anti-metabolic syndrome. Recently, our laboratory has determined that lotus seedpod aqueous extract (LSE) has been initially demonstrated to be rich in flavonoid compounds, including catenchin and (-)-epigallocatechin (EGC), using HPLC assay (supplementary Table 1 and Fig. 1). We also demonstrated that LSE could inhibit lipid peroxidation, the oxidative modification of LDL induced by copper (supplementary Fig. 2), and the formation of foam cells (supplementary Fig. 3A). In supplementary Fig. 4, LSE was demonstrated exhibit hypolipidemia and antiatherosclerotic effects in rabbits with experimental atherosclerosis. In the present study, we aimed to investigate the anti-metabolic syndrome effects of LSE and LSPCs, including improvement of insulin resistance, regulation of lipid metabolism, and inhibition of atherosclerosis. First year, the study was designed to investigate the effect of LSE on the animal model, a high fat diet (HF) and sucrose-containing drinking water (SW)-induced metabolic syndrome and the possible molecular mechanisms involved were also studied. For assessing metabolic syndrome, body weight and visceral fat accumulation were used as an indicator of obesity. Fasting serum glucose and insulin, oral glucose tolerance (OGTT) and insulin tolerance test (ITT) were used as an indicator of insulin resistance. Serum lipid levels including triglyceride and cholesterol and blood pressure were also measured. For exploring the possible mechanism for diet-induced metabolic syndrome, serum levels of adipocytokines (leptin and adiponectin) and proinflammatory factors (TNFα, IL-1β and IL-6), lipid accumulation in liver and the enzyme activity and protein expression levels of factors participating in lipid metabolism in liver were all measured. Second year, we will demonstrate whether LSE and LSPCs could ameliorate insulin resistance and pancreatic β-cell failure, and to study its molecular mechanisms. This study was designed to investigate the inhibitory effects of both extracts on high glucose, oxidative LDL (ox-LDL), or TNFα-induced cellular insulin resistance and apoptosis via regulation of insulin signaling, IRS-1/PI3K/Akt pathway. Third year, we will demonstrate whether LSE and LSPCs could eliminate hepatic cholesterol accumulation and inhibit the oxidative stress generation, and to study its molecular mechanisms. This study was designed to investigate the inhibitory effects of both extracts on high glucose, ox-LDL, or TNFα-induced hepatic lipid accumulation via regulation of hepatic fat metabolism-related proteins, including HMG-CoA reductase, FAS, PPARs, SREBPs and LDLR, in HepG2 cells. On the other hand, the antioxidant and anti-inflammatory activities of LSE and LSPCs could ameliorate the generation of oxidative stress and abnormal secretion of adipocytokines. Fourth year, we will demonstrate whether LSE and LSPCs could protect human umbilical vein endothelial cells (HUVEC) from death, and inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and to study its molecular mechanisms. This study was designed to investigate the inhibitory effects of both extracts on high glucose, ox-LDL, or TNFα-induced HUVEC death (supplementary Fig. 5) via regulation of apoptosis signaling, and VSMC proliferation and migration via cell cycle arrest induction and MMPs signaling blockage. Additively, our prepared data shows connective tissue growth factor (CTGF) involve in VSMC cell proliferation and migration (supplementary Fig. 3B). Hence, the goal of fourth year is to elucidate the role of CTGF on atherosclerosis lesion. We are going to investigate if over-expression CTGF can mediate the downstream signals of VSMC proliferation and migration, and to test if LSE and LSPCs regulate CTGF thus transuding the protective effects. In summary, our results will show that the effects, (1) LSE and LSPCs improved metabolic syndrome in vivo via (2) An increase in cellular insulin sensitivity and β-cell survival, and the regulation of insulin signaling; (3) Promotion of hepatic lipid metabolism, and antioxidant and anti-inflammatory activity; (4) Protection of endothelial cell from dysfunction, and inhibition of VSMC proliferation and migration, and (5) The signal pathways involved in the process and the role of the molecular target CTGF will also be elucidated. Also, we hope these observations will be useful for clinical implications in the prevention of metabolic syndrome. |
