沒食子酸藉由調控miR-199a-3p抑制糖尿病性血管內皮發炎及凋亡之研究

動脈粥狀硬化是一種慢性發炎疾病，為全球性的臨床問題，開發有效的動脈粥狀硬化治療標靶有益於人類福祉，許多研究已證實miRNA在動脈粥狀硬化中扮演重要角色。糖尿病小鼠(db/db mice)以高油脂飼料連續餵食12週誘導產生糖尿病性動脈粥狀硬化發生，其中加藥組輔以100 mg/kg沒食子酸餵食，觀察沒食子酸的抑制效果。人類內皮細胞在高糖高脂(25 mM high glucose + 200 uM palmitic acid)的環境下，以150 uM的沒食子酸處理24小時，並利用MTT和流式細胞儀測定人類內皮細胞的存活率和凋亡率，再藉由西方墨點法分析細胞凋亡相關蛋白變化，包含Bax, cleaved caspase-3, cleaved caspase-8，並進一步確立高糖高油脂造成內皮損傷的NF-kB蛋白路徑。高糖高脂模擬糖尿病環境的細胞實驗中，證實沒食子酸可改善高油脂誘導的凋亡相關蛋白變化，使Bax, cleaved caspase-3, cleaved caspase-8的表現量下降，而NF-kB路徑中的p-IkB, NF-kB和TNF-a蛋白表現，亦在沒食子酸處理後減少。經過12週誘導後之動物數據中，觀察到沒食子酸有效降低體重增加速率，改善高脂飲食導致的血清數值異常，減緩心血管內的脂肪堆積，降低NF-kB和TNF-a的表現量。接著，透過動物實驗檢體進行microarray分析，搜尋涉及調控NF-kB的可能標的miRNA。自microarray的分析報告中，篩選出miR-199a-3p，作為沒食子酸抑制NF-kB導致發炎反應和細胞凋亡的調控標的，在高糖高脂的誘導之下可以觀察到內皮細胞內miR-199a-3p表現量明顯地降低，而經沒食子酸處理後miR-199a-3p的表現量顯著提升。綜合上述實驗結果，沒食子酸可藉由調控miR-199a-3p，達到抑制NF-kB訊號傳導之路徑，使高糖高脂造成的內皮細胞發炎反應和凋亡現象減少，進而趨緩糖尿病性動脈粥狀硬化的病程。

Atherosclerosis is a chronic inflammatory disease, and it is a global clinical problem. The development of new and effective therapeutic targets for atherosclerosis is necessary. A number of microRNAs (miRNAs) have been demonstrated to serve a crucial role in atherosclerosis. Diabetic db/db mice were fed either a high fat diet (HFD) for 12 weeks to induce diabetic atherosclerosis, and the drug group was supplemented with 100 mg/kg gallic acid. The effect of gallic acid (150 uM) treated for 24 h in EA.hy926 cells with high glucose (HG, 25 mM) and palmitic acid (PA, 200 uM) was evaluated. MTT and flow cytometry assays were performed to evaluate cell proliferation and cell death, respectively. Then, western blot was used to quantify the apoptosis-related proteins including Bax, cleaved caspase-3, cleaved caspase-8, and further established the NF-kB/TNF-a pathway for high glucose and palmitic acid-induced endothelial damage. In the animal data after 12 weeks of induction, it was observed that gallic acid effectively reduced the rate of weight gain, improved the abnormal serum values caused by high fat diet, slowed down cardiovascular lipid accumulation, and reduced the expression of NF-kB and TNF-a. The cell experiments simulating diabetic environment by high glucose and palmitic acid have confirmed that gallic acid can down-regulate the changes of apoptosis-related proteins. The protein expression of p-IkB/NF-kB/TNF-a signaling pathway was also reduced after gallic acid treatment. Furthermore, microarray analysis was performed on animal samples to search for candidate miRNAs involved in the regulation of NF-kB. From the analysis report of microarray, miR-199a-3p was screened as a target for gallic acid to inhibit NF-kB. High glucose and palmitic acid inhibited the expression of miR-199a-3p in human endothelial cell. After gallic acid treatment, the level of miR-199a-3p was significantly elevated. Based on the above experimental results, gallic acid can inhibit the NF-kB signaling pathway by regulating miR-199a-3p, which can reduce the inflammation and apoptosis of endothelial cells caused by high glucose and palmitic acid, and then slow down the course of atherosclerosis.