於高果糖餵食誘導模式下以蛋白質體學方法探討龍眼花水萃物改善大鼠代謝症候群之機制

Abstract

代謝症候群(metabolic syndrome)盛行於已開發國家中，為數種代謝異常症狀的綜合臨床徵兆。根據前人研究，龍眼花水萃物於高果糖餵食誘導模式中可減緩大鼠之代謝症候群症狀，然而，其分子機制路徑尚未得知。因此，本實驗室取基礎飲食組(A)、高果糖飲食組(B)、龍眼花水萃物灌食組(C)之大鼠肝臟進行蛋白質體學方法分析，利用差異性螢光標記二維電泳膠體(2-dimensional differential gel electrophoresis, 2D-DIGE)及電噴霧串聯質譜法（electrospray ionization-tandem mass spectrometry, ESI-MS-MS）鑑定出在三種實驗條件下表現量具有差異的蛋白質，透過文獻探討與實驗確認，試圖建立各蛋白質間所依循的分子路徑，提出龍眼花水萃物對於代謝症候群改善作用之假說。本實驗室在分析蛋白質體學資料後，發現數種具有結合細胞內鈣離子能力且受內質網壓力所調控之蛋白質，分別為78 kDa glucose-regulated protein (GRP78)、94 kDa glucose-regulated protein (GRP94)、calreticulin (CRT)、 protein disulfide-isomerase A3 (PDIA3)，其中GRP78蛋白被眾多文獻用於作為偵測內質網壓力之指標；數據已透過即時定量聚合酶連鎖反應作進一步確認，因此我們推斷龍眼花水萃物可能透過抑制內質網壓力來減緩代謝症候群。以HepG2細胞模式進行驗證，結果顯示龍眼花水萃物可抑制tunicamycin所誘發的內質網壓力，使上述四種蛋白質表現量降低，證明龍眼花水萃物具有抑制內質網壓力之效果，而可佐證龍眼花水萃物改善代謝症候群之分子機制可能部分來自抑制內質網壓力。

Metabolic syndrome (MetS) is prevalent in the developed countries, consisting of several metabolic abnormal symptoms. According to the previous study, the water extract of Longan (Dimocarpus longan Lour.) flower (LFWE) could attenuate the symptoms of MetS in fructose-fed rat model. Nevertheless, the molecular mechanism remains unknown. In this study, we employed proteomic analysis by 2-dimensional differential gel electrophoresis (2D-DIGE) and electrospray ionization-tandem mass spectrometry (ESI-MS-MS) to investigate the possible molecular pathway. Through the differential protein patterns displayed in the livers among control, high fructose, and high fructose plus LFWE treated rats, the mechanism underlying the preventive function of LFWE and the pathogenic signaling pathways MetS might be explored. Through proteomic results, we have identified the differential expression of several endoplasmic reticulum (ER) stress related proteins, including 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), calreticulin (CRT), protein disulfide-isomerase A3 (PDIA3). The real time PCR results further confirmed the involvement of the above proteins after LFWE treatment. We thus hypothesize that endoplasmic reticulum(ER) stress may be the target for LFWE mediated prevention of MetS. Utilizing HepG2 cell as cell model, we observed that LFWE inhibited ER stress and the mRNA expression of those ER stress marker proteins identified from our proteomics results. The result indicates LFWE might attenuate MetS at least partially through inhibiting ER stress.