體內和體外研究蓮蓬萃取物成份對於胰島beta細胞免於氧化性損傷之保護作用

代謝症候群(metabolic syndrome)容易引發心臟病、高血壓、糖尿病、肝腎病變等症狀。過去研究顯示，主要是由於飲食中的高脂肪和高糖等對於胰臟和血管造成氧化傷害而誘發此多重病癥。天然蓮蓬萃取物(lotus seedpod extracts，LSE)富含之多酚類(polyphenol)經研究證實是有效的抗氧化劑(antioxidant)，具有抗老化、抗肝損傷等功能。因此，本研究以LSE處理細胞及動物進行代謝症候群之研究。首先以過氧化物(H2O2)處理胰島beta細胞(β-cells)RIN-5F模擬氧化損傷之細胞模式。結果發現H2O2抑制胰島β細胞生長及胰島素分泌下降；LSE及其主要成份epigallocatechin (EGC)皆能有效保護β細胞存活及胰島素分泌。流式細胞儀分析細胞週期與ROS含量發現LSE及EGC也能抗細胞凋亡(apoptosis)並減少其氧化傷害。進一步透過DAPI及AVO螢光染色分析發現處理LSE及EGC確實能抑制H2O2所誘導之細胞凋亡，且能增加細胞自噬(autophagy)作用。透過西方點墨法分析發現，LSE及EGC能夠抑制促凋亡蛋白Caspase 3和Bax表現量；以及誘導抗凋亡蛋白Bcl-2和p-Bad之細胞表現，而細胞自噬蛋白LC3-II、Atg5/12、Beclin-1及class III PI3K的表現量也有上升之趨勢。在動物實驗中，透過高脂肪飼料(high fat diet，HFD)合併鏈佐黴素(streptozotocin，STZ)誘導BALB/c小鼠引發代謝症候群模式，並餵食LSE作為試驗組，最後以胰臟組織進行分析。H&E切片染色結果發現，處理LSE能有效地減緩組織因氧化傷害產生的萎縮現象。西方點墨法分析LSE調控胰組織蛋白表現與細胞實驗分析得到相同之結果。總結，LSE能夠透過細胞自噬機制，有效的保護胰臟組織及胰島細胞免於氧化壓力所造成的傷害，具有抗代謝症候群之潛力。

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. Oxidative stress is considered to be a major factor contributing to development of metabolic syndrome. In previous studies, lotus seedpod has been shown to possess antioxidant, radioprotective, and anticancer activities. In this study, we examined the anti-metabolic syndrome effect of lotus seedpod extracts (LSE), which is rich in flavonoid, on pancreatic β-cells. Firstly, LSE or its main compound epigallocatechin (EGC) dose-dependently improved the survival and insulin secretion of RIN-5F rat pancreatic β-cells from H2O2-induced loss of viability and inhibition of insulin secretion. LSE or EGC showed potential in reducing H2O2-induced occurrence of apoptosis confirmed by morphological and biochemical features, including an increase in the distribution of hypodiploid phase, apoptotic bodies formation and caspase-3 activation. In addition, H2O2-induced formation of acidic vesicular organelles and the upregulation of microtubule associated protein 1 light chain 3-II (LC3-II), a marker of autophagy, were increased by LSE or EGC. Molecular data showed that autophagic and antiapoptotic effects of LSE or EGC might be mediated via class III PI3K/Beclin-1/LC3-II and Bad/Bcl-2 signaling pathway. Finally, LSE improved the metabolic syndrome and pancreatic cell injury in vivo. Our data imply that LSE upregulates the protective autophagic pathway, which in turn led to reduced oxidative stress-induced β-cell injury and apoptosis, and these findings may open interesting perspectives to the strategy in treatment of metabolic syndrome.