利用白藜蘆醇調控 Nrf2 減緩氧化性損傷以改善糖尿病症狀之研究

Abstract

糖尿病 (diabetes) 是現今全球罹患率增長最快的慢性疾病，而肇因於糖尿病的併發症不勝計數，造成沉重的醫療負擔。近年來研究顯示，造成糖尿病的原因及其危險因子，不乏與最終糖化終產物 (advanced glycation end products, AGEs) 及其前驅物甲基乙二醛 (methylglyoxal, MG) 的生成與堆積有關。醣類代謝異常是糖尿病最顯著的特徵之一，通常伴隨著胰島素阻抗 (insulin resistance) 的生理變化。而高血糖會引起內源性 (endogenous) 的 MG 與AGEs 的生成，MG是一種糖化反應 (glycation) 過程所產生的高活性二羰基 (dicarbonyl) 化合物。體內 MG 含量的增加會促使活性氧 (reactive oxygen species, ROS) 生成，進而形成氧化壓力 (oxidative stress) 及發炎反應 (inflammation)，造成胰島素阻抗。而 nuclear factor erythroid 2-related factor 2 (Nrf2) 是組織細胞對抗氧化性損傷的防衛轉錄因子。因此，找尋具活化 Nrf2 的天然抗氧化物質，評估其改善及預防糖尿病的潛力就顯得格外重要。虎杖 (Plygonum cuspidatum Sieb. et. Zucc.) 乾燥根莖是一味中國歷代普遍使用的藥材，是已知白藜蘆醇 (resveratrol) 含量最高的植物。白藜蘆醇是一種植物防禦素 (phytoalexin)，可透過多種途徑，從而改善胰島素的敏感性。於是，研究採用選育自南投仁愛鄉梅峰附近山區富含白藜蘆醇之虎杖單株，經無性繁殖成 051111 選系，種植於梅峰二年之虎杖植株地下部之 100 g 乾燥粉末，經分離純化得 230 mg 之白藜蘆醇。更進一步評估白藜蘆醇活化 Nrf2 減緩 MG 誘導細胞氧化性損傷的相關分子機制。結果顯示，MG 明顯誘導 Hep G2 肝細胞產生胰島素阻抗。此外，白藜蘆醇經由活化 extracellular signal regulated kinase (ERK) 路徑，進而導致 Nrf2 的磷酸化轉位進入細胞核，提升抗氧化酵素 heme oxygenase-1 (HO-1) 和 glyoxalase 的轉錄表現，從而改善 Hep G2 細胞的胰島素阻抗和提升葡萄糖的攝取能力。另外，MG 亦能誘導胰臟 RINm5F β 細胞凋亡的訊號分子表現，進而降低胰島素分泌。然而，細胞經白藜蘆醇預處理後，發現白藜蘆醇正向調控peroxisome proliferator-activated receptor gamma (PPARγ) 和 pancreatic-duodenal homeobox-1 (PDX-1) 的活性，抑制負向調控因子 CCAAT/enhancer-binding protein β (C/EBPβ) 的活化，促進 Nrf2 的磷酸化，進而減緩氧化壓力增進胰島素分泌。總之，補充天然抗氧化物質抑制蛋白質修飾所產生的糖化氧化壓力 (glycoxidative stress) 作用，在糖尿病的病理生理研究有其重要的意義。本研究證實白藜蘆醇經由活化 Nrf2 轉錄因子，能有效的抑制 MG 誘導細胞產生的氧化壓力，減緩 Hep G2 肝臟細胞胰島素阻抗，並促進 RINm5F β 細胞的胰島素分泌。顯然的，白藜蘆醇調控 Nrf2 促進 MG 的代謝，相對抑制 AGEs 的生成與累積，減緩細胞氧化性損傷，從而改善糖尿病症狀。

Diabetes is the world's fastest growing chronic disease that has caused many serious complications, resulting in a heavy burden of medical care. Recently studies have shown that causes of diabetes are all related to advanced glycation end products (AGEs) as well as the production and accumulation of its precursor-methylglyoxal (MG). Disorders of carbohydrate metabolism are among the most significant features of diabetes which is often accompanied by insulin resistance. Hyperglycemia will cause the production of the two endogenous – MG and AGEs. MG is a dicarbonyl produced during glycation which higher levels in the human body result in faster reactive oxygen species (ROS) which causes oxidative stress and inflammation. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a defense mechanism against oxidative damage in cells. Because of this, it is especially important that we find the natural antioxidants via the activation of Nrf2 and evaluate its potential for the prevention of diabetes. It is known that the dry rhizomes of Plygonum cuspidatum Sieb. et. Zucc. (knotweed) contain a higher level of resveratrol, a phytoalexin which through various pathways can improve insulin sensitivity. The resveratrol rich knotweed plant was planted in the mountains of the Mei-Feng Area, Ren-Ai Township, Nantou County for two years. Via asexual reproduction, the final product of the selected strain produced ‘no.051111’. The rhizomes of the knotweed were converted into dry powder (100 g) and were then purified to produce a high yield of resveratrol (230 mg). The aim of this study is to evaluate the use of resveratrol in the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) to attenuate MG-induced oxidative damage in cell lines. The results suggested that MG-induced insulin resistance in Hep G2 cells. However, resveratrol activated via the extracellular signal regulated kinase (ERK) pathway lead to Nrf2 nuclear translocation as well as the elevation of HO-1 and glyoxalase expression levels. Moreover, resveratrol significantly elevated glucose uptake and protected against MG-induced insulin resistance in Hep G2 cells. Furthermore, MG treatment induced the expression of apoptotic signaling molecules and decreased insulin production. Pretreatment with resveratrol increased insulin synthesis via upregulation of peroxisome proliferator-activated receptor γ (PPARγ) and pancreatic-duodenal homeobox-1 (PDX-1), while inhibiting MG-mediated expression of CCAAT/enhancer-binding protein β (C/EBPβ), a negative regulator of insulin production. Resveratrol strongly activated Nrf2 expression, which resulted in the attenuation of oxidative stress and increased insulin secretion. In conclusion, dietary antioxidants inhibit glycoxidative stress generated by protein modification, an important and significant property in research regarding diabetes pathophysiology. This study demonstrated that resveratrol upregulates Nrf2 expression to attenuate methylglyoxal-induced insulin resistance in Hep G2 cells, and protects RINm5F pancreatic cells from methylglyoxal-induced apoptosis. It has been made clear that resveratrol upregulates Nrf2 to promote MG metabolism, causing relative inhibition of AGEs formation and accumulation, which ultimately attenuates cellular oxidative damage, thereby improving the symptoms of diabetes.