荷葉萃取物及主要成分miquelianin抑制糖尿病環境下誘發血管內皮細胞損傷之作用

Nelumbo nucifera leaves extract and principal component miquelianin reduced vascular endothelial cell injured in diabetic environment

蔡欣蓓(Hsin-Pei Tsay)

指導教授 ： 王朝鐘 ； 張雲菁

中山醫學大學/醫學院/生化微生物免疫研究所/碩士(2019年)

https://doi.org/10.6834/CSMU201900233

摘要

糖尿病患者副病變成心血管疾病的機率高於正常人的2-4倍之多，最常見的就是血管病變促使糖尿病患者死亡率提高。於2018年台灣衛生署公布的年度十大死因排行中，糖尿病則是位居第五，而心臟心血管疾病高居第二。過去的研究已知，荷葉對於降低血脂、抗氧化、抑制癌症等功效，但於第二型糖尿病副病變心血管相關研究甚少。本研究目的為探討荷葉水萃物、多酚萃取及其主要成分Miquelianin (Q3GA)對於人類正常內皮細胞於以高糖(high glucose, HG)與高油脂(palmitic acid, PA)模擬糖尿病環境下，觀察具有達到保護細胞並減少細胞死亡的現象。先是利用細胞存活率分析篩選合適的高糖(25 mM HG)與高油脂(150 μM PA)劑量後，並找出具有保護作用的Q3GA劑量。利用PI stain，Annexin V/PI染色與Caspase-3/CPP32活性比色分析結果證明HG+PA確實誘導內皮細胞產生損傷，並走向凋亡。並觀察到抗細胞凋亡蛋白Bcl-2表現量的上升與細胞凋亡蛋白Bak，Bax的表現量下降，再次證實HG+PA誘導內皮細胞的凋亡，上述的試驗結果也發現Q3GA具有減緩細胞受損死亡之作用，並發現能夠增加NF-κB的表現，利用lipopolysaccharide (LPS)活化NF-κB路徑之特性，也驗證活化NF-κB確實能夠降低HG+PA誘導內皮細胞死亡率，增加細胞存活量，再次說明，Q3GA是透過NF-κB達到保護內皮細胞的作用。將糖尿病小鼠心臟組織利用微陣列分析小分子核糖核酸(microRNA)變化的結果，找到與NF-κB表現相關的miR-29b-2-5p，透過聚合酶連鎖反應證實，於糖尿病環境誘導下內皮細胞miR-29b-2-5p表現量會上升，且隨Q3GA劑量增加而降低miR-29b-2-5p表現量。另外，利用高油脂飲食餵食B6.BKS(D)-Leprdb/J小鼠誘導第二型糖尿病副病變發展模式證實，富含Q3GA之荷葉水萃取物(NLE)和荷葉多酚萃取物(NLPE)，NLPE具有減緩糖尿病小鼠體重增加的趨勢。
綜合以上結果，在細胞模式中說明Q3GA對於誘導糖尿病性之血管內皮細胞損傷具有改善的現象，而富含G3GA的荷葉萃取物在動物實驗中目前發現具有調控體重上升的作用。研究結果可以證明荷葉萃取物其中主要作用成分Q3GA具有減緩細胞傷害之作用，在未來的預防醫學領域中具有其探討的價值。

關鍵字

糖尿病 ； 心血管疾病 ； Miquelianin ； 荷葉 ； 細胞凋亡 ； 小分子核糖核酸

並列摘要

Diabetes can lead to a buildup of sugars in the blood, which can increase the risk of dangerous complications, especially cardiovascular disease (CVD). CVD occurs with higher than normal frequency in diabetes and is the major cause of morbidity and death in diabetic patients worldwide. Ministry of Health and Welfare (MOHW) estimate that diabetes was the fifth and CVD was the second leading cause of death in 2018. Previous studies have shown that Nelumbo nucifera leaves are effective in reducing blood lipids, anti-oxidation, and even in cancer, but there are few cardiovascular-related studies in type 2 diabetic complications.
The aim of this study was to investigate the protective effect of Nelumbo nucifera leaves extract, polyphenols extract, and their main component, miquelianin (Q3GA) in human normal endothelial (EA.hy926) cells under the simulation environment of diabetes. In this study, we found that significant cell death induction was observed when cells were cotreated with palmitic acid (PA, 150 μM) and glucose (HG, 25 mM) in EA.hy926 cells. Moreover, PA+HG-induced cell death was suppressed by the pretreatment of Q3GA. Q3GA inhibited PA+HG-induced apoptotic cell death, as evidenced by annexin V/PI staining. The results of western blot analysis indicated that apoptosis protein Bcl-2 (anti-apoptosis) was increased and Bak, Bax (pro-apoptosis) was decreased in high dose of Q3GA under diabetic-like environment. We also found that NF-κB was inhibited by PA+HG treatment, and LPS restored the activation of NF-κB in PA+HG-induced EA.hy926 cells. On the other hand, microarray data shown that miR-29b-2-5p was one of the up-regulated miRNA in diabetic mice.
Taken together, these results indicate that Nelumbo nucifera leaves and and its major component Q3GA has an improved in endothelial cells effect in a diabetic-like environment. The Nelumbo nucifera leaves extract has the potential to protect the cardiovascular system from diabetic-like environment, and the main component of Q3GA could be considered in the future for dietary supplements.

並列關鍵字

Diabetes mellitus ； Cardiovascular disease ； Nelumbo nucifera leaves ； Miquelianin ； Cell apoptosis ； microRNA

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