Effect of silymarin on insulin resistance, GLP-1 and Sirt1 in high fat diet-induced rat model of metabolic syndrome

High-calorie and high fat diet has been regarded as the major cause of increased prevalence of the metabolic syndrome. Insulin resistance (IR) is the typical symptom of metabolic syndrome, which may lead to type II diabetes mellitus (T2DM). Under IR condition, decreased protein level of sirtulin 1 (Sirt1) and dysregulation of secretion of the gut hormone glucagon-like peptide-1 (GLP-1) which modulate blood glucose balance are observed, which leading to metabolic imbalance and injury of liver and pancreas, and cause the associated complications. Silymarin (SM), flavonoids of milk thistle (Silybum marianum) seed and fruit extracts, has a variety of biological activity, and the most known is protective effect on liver and pancreas. Therefore, the aim of the study was to investgate the effect of SM on abnormalities of blood biochemistry and IR in response to high-fat diet, as well as modulation of GLP-1 and Sirt1. In this study, SD rats were fed with high-fat diet to induce metabolic syndrome symptoms, and intervention of SM (100/200/400 mg/kg b.w.) from 0 week or 8th week of the time course of the experiment, to assess its effect on prevention and improvement of metabolic injury in rats. Results showed that serum TG, LDL, VLDL and other indicators of metabolic syndrome, as well as insulin secretion significantly increased in high-fat diet-induced rats. Besides, HOMA-IR indicated that long-term high-fat diet lead to IR in rats. Interestingly, SM intervention from 0 week or 8th week of experimental period can significantly improve the IR and metabolic syndrome in rats fed with high-fat diet. We also found that SM can improve the impaired glucose tolerance in these rats. Notably, long-term consumption of high-fat diet rats would lead to reduced GLP-1 secretion in rats, while SM intervention from 0 week or 8th week of experimental period, may recover and further enhance GLP-1 secretion in rats fed with high-fat diet. Histopathological analysis showed that high-fat diet may contribute to hepatic steatosis and pancreas fibrosis in rats, and intervention of SM from 0 week can prevent the liver damage caused by high-fat diet. Western blot indicated that decreased Sirt1 protein levels in pancreas and liver were observed in high-fat diet-induced rats, while SM intervention from 0 week of experimental period can recover high-fat diet-impaired Sirt1 protein expression in liver and pancreas of rats. In conclusion, SM can prevent and ameliorate high-fat diet-induced phenomena of IR and dyslipidemia, and can improve the secretion of GLP-1 in rats. SM can also improve fatty liver caused by high-fat diet and enhance Sirt1 protein expression of liver and pancreas in rats. Taken together, SM can prevent and retard the metabolic injury caused by high-fat diet, suggesting that SM may be a potential food factor to improve symptoms of metabolic syndrome.隨著時代演進，人民攝取高熱量食物比率大增，進而導致代謝症候群 (metabolic syndrome) 盛行。代謝症候群典型之病徵為胰島素阻抗 (insulin resistance, IR) 現象，其可能進一步引發第二型糖尿病 (type 2 diabetes)，並造成動物體中重要保護性蛋白 sirtulin 1 (Sirt1) 之表現下降，以及調控體內血糖平衡之腸道荷爾蒙 glucagon-like peptide-1 (GLP-1) 分泌異常，導致體內代謝失衡及肝臟與胰臟損傷，並產生相關之併發症。已知從水飛薊 (Silybum marianum) 種子及果實中萃取之類黃酮 (flavonoid) 化合物－水飛薊素 (silymarin, SM) 具有多種生物活性，其對肝臟與胰臟均具有保護效果。因此，本研究將探討 SM 對高脂飲食誘導大鼠血清生化指標異常及 IR 現象等代謝性損傷之影響，以及 GLP-1 及 Sirt1 在SM介入後之改變情形。 本實驗以高脂飼料誘導 SD 大鼠使其產生代謝症候群相關症狀，實驗進行 16 週，並分別在實驗時程第 0 週及第 8 週後介入低、中、高劑量之 SM (100/200/400 mg/kg b.w.)，個別探討 SM 對高脂飲食誘發大鼠代謝性損傷之預防與改善效果。血清生化值分析結果指出，餵食高脂飼料，能夠使大鼠血清中的 TG、LDL、VLDL 等代謝症候群的指標顯著提高，同時亦會使其胰島素的分泌量增加；經過 HOMA-IR 的公式計算，長期高脂飲食確實會使大鼠產生 IR 現象。然而，於實驗第 0 週及第 8 週介入 SM 可顯著改善上述高脂飲食誘發之 IR 及代謝症候群血清生化指標。口服葡萄糖耐受性試驗 (oral glucose tolerance test, OGTT) 結果證實，SM 能改善高脂飲食引起大鼠葡萄糖耐受性降低之情形。另外，實驗結果亦發現大鼠長期攝取高脂飼料將導致其 GLP-1 分泌量降低，而於實驗第 0 週及第 8 週介入 SM 均可顯著提升其 GLP-1 分泌量。組織病理分析顯示，高脂飲食會引起大鼠肝臟油滴堆積及其胰臟部分纖維化，而於實驗第 0 週即介入 SM 則可減少肝臟受損之情形，證實 SM 確實對肝臟與胰臟具有保護的效果。西方墨漬法 (western blot) 結果說明，高脂飲食會使大鼠肝臟及胰臟之 Sirt1 蛋白表現顯著降低，而於實驗第 0 週即介入 SM 則可提升其肝臟及胰臟之 Sirt1 蛋白表現量。 綜合上述，SM 可預防及改善高脂飲食誘發之 IR 及血脂異常現象，且有助於提升 GLP-1 之分泌。SM 更可改善高脂飲食引起之脂肪肝及提升高脂誘導大鼠之肝臟及胰臟 Sirt1 蛋白表現量。由此可知，SM 具有防止及減緩高脂飲食引起代謝性損傷之功效，證實 SM 可作為一種具改善代謝症候群病徵潛力的輔助食品。目次 全文摘要..........................................i Abstract.......................................iii 目次.............................................v 圖次..........................................viii 表次.............................................x 縮寫表...........................................xi 第一章 文獻整理...................................1 壹、代謝症候群 (Metabolic syndrome)...............2 貳、胰島素阻抗 (Insulin resistance)...............4 一、胰島素之訊息傳遞...............................4 二、胰島素阻抗 (Insulin resistance)...............4 參、糖尿病 (Diabetes mellitus, DM)................9 一、糖尿病簡介....................................9 二、糖尿病主要類型.................................9 三、糖尿病併發症..................................11 肆、腸泌素 (Incretin)............................13 一、類升糖素胜肽 (Glucagon-like peptide -1, GLP-1) ................................................13 二、胃抑素 (Gastric inhibitory peptide, GIP).....13 三、腸泌素效應 (Incretin effect)..................14 四、腸泌素及第二型糖尿病之治療......................14 伍、沉默訊息調節因子 (Silent information regulator 1, Sirt1)..........................................18 一、Sirt1 介紹...................................18 二、Sirt1 之生理活性..............................18 三、Sirt1 與 AMP kinase (AMPK) 能量代謝途徑........18 陸、第二型糖尿病之動物模式..........................22 一、飲食誘導......................................22 二、藥物處理......................................23 三、基因改造......................................23 柒、研究材料......................................25 一、水飛薊 (Milk thistle).........................25 二、水飛薊素 (Silymarin)..........................25 捌、研究目的......................................29 玖、研究架構......................................30 第二章...........................................31 前言.............................................32 材料與方法........................................34 壹、實驗材料......................................34 一、實驗樣品......................................34 二、藥品.........................................34 貳、實驗方法......................................34 一、動物飼料配製...................................34 二、實驗動物飼養...................................35 三、實驗動物分組及實驗設計..........................35 四、血清生化值測定 .................................36 五、大鼠血糖測定...................................37 六、口服葡萄糖耐受試驗 (Oral Glucose Tolerance Test, OGTT)............................................37 七、HOMA-IR 之計算.................................37 八、組織切片染色....................................37 九、GLP-1 分泌試驗.................................37 十、組織蛋白質萃取 .................................38 十一、蛋白質定量...................................38 十二、聚丙烯醯胺膠體電泳分析 (SDS-PAGE)..............39 十三、西方墨漬法 (Western blot)....................39 十四、統計分析.....................................40 結果..............................................41 一、水飛薊素對高脂飲食之 SD 大鼠體重變化之影響........41 二、水飛薊素對高脂飲食之 SD 大鼠攝食量、熱量與食物利用率之影響 .................................................41 三、水飛薊素對高脂飲食之 SD 大鼠臟器重量之影響........41 四、水飛薊素對高脂飲食之 SD 大鼠血清生化參數之影響....42 五、水飛薊素對高脂飲食之 SD 大鼠血糖、胰島素阻抗之影響 .................................................42 六、水飛薊素對高脂飲食之 SD 大鼠葡萄糖耐受性之影響....43 七、水飛薊素對高脂飲食之 SD 大鼠腸道荷爾蒙 GLP-1 分泌之影響 .................................................44 八、水飛薊素對高脂飲食 SD 大鼠脂肪組織重量與體脂比率之影響 .................................................44 九、水飛薊素對高脂飲食之 SD 大鼠肝臟、腎臟與胰臟組織病理之影響 .................................................45 十、水飛薊素對高脂飲食之 SD 大鼠組織中Sirt1 蛋白表現量 之影響 .................................................45 討論.............................................47 第三章 總結論.....................................77 參考文獻..........................................7