傳統中藥樟芝萃取物對棕櫚酸處理血管內皮細胞脂質累積的緩解作用

高脂血症是動脈粥樣硬化最主要的危險因素，過程涉及動脈粥樣硬化患者在早期時脂質於動脈內膜中的累積，而形成動脈粥樣硬化的過程中，血管內皮細胞、巨噬細胞的氧化壓力、細胞凋亡、CD36以及Kruppel-like factor 4(KLF4)蛋白質的表現均扮演重要角色。本研究嘗試探討牛樟芝萃取物對於動脈粥樣硬化的調節潛力，使用棕櫚酸(Palmitic acid)處理血管內皮細胞SVEC4-10與RAW264.7巨噬細胞作為高脂累積細胞模式，藉由胞外實驗(in vitro)評估牛樟芝萃取物對於高脂累積的緩解作用。本研究採用由牛樟芝萃取出的化合物，利用HPLC確定牛樟芝萃取物的化學指標活性成份，並用DPPH測定牛樟芝萃取物的自由基清除能力，再以油紅O染色法(Oil Red O Stain)檢視牛樟芝萃取物可否有效降低高脂累積內皮細胞SVEC4-10的油滴脂質累積，以酵素結合免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA) 檢測牛樟芝萃取物可否有效降低炎性細胞因子TNF-α (tumor necrosis factor-α)、IL-1β(interleukin-1β)的表現量；利用細胞免疫螢光染色方法檢測牛樟芝萃取物是否有效降低CD36蛋白質表現以及增強KLF4蛋白質表現；利用遷移試驗(transwell migration assay)檢測牛樟芝萃取物是否增加RAW264.7巨噬細胞的移動能力來清除血管內皮細胞的脂質累積。本實驗結果顯示：此牛樟芝萃取物具備很好的自由基清除能力，可以顯著降低血管內皮細胞的油滴脂質累積，減少TNF-α、IL-1β以及CD36的表現量，但是增強KLF4蛋白質表現。此外，牛樟芝萃取物可以促進RAW264.7巨噬細胞的遷移能力。綜合上述結果，我們認為牛樟芝萃取物可以透過減少血管內皮細胞的油脂累積，抑制巨噬細胞的氧化壓力與細胞凋亡，來達到緩解高脂累積，以及調節動脈粥樣硬化的潛力。

關鍵字

高脂血症；動脈粥樣硬化；牛樟芝；血管內皮細胞；巨噬細胞；氧化壓力；細胞凋亡

並列摘要

Hyperlipidemia is the most important risk factor for atherosclerosis (AS). The process involves the accumulation of lipids in the arterial intima in the early stage of AS patients, and oxidative stress and apoptosis, and expressions of CD36 and Kruppel-like factor 4 (KLF4) proteins in vascular endothelial cells and macrophages may play important roles in AS formation. This study attempted to investigate the therapeutic potential of Antrodia camphorata extract (ACE) for AS patients. In this experiment, palmitic acid (Palmitic acid; PA)-treated vascular endothelial SVEC4-10 cells and RAW264.7 macrophages were used as a cell model with lipid accumulation. Then alleviation effect of ACE on lipid accumulation was evaluated by in vitro experiment. We first extracted high-purity of extract from Antrodia camphorata, used high performance liquid chromatography (HPLC) to determine the chemical index active components of ACE, used α,α-diphenyl- β-pricrylhydrazyl (DPPH) assay to determine the free radical scavenging ability of ACE, used oil red dye staining to check whether ACE could effectively reduce oil droplet lipid accumulation in PA-treated SVEC 4-10 cells, used enzyme-linked immunosorbent assay (ELISA) to determine whether ACE can effectively reduce expressions of inflammatory cytokines of TNF-α and IL-1β of PA-treated SVEC 4-10 cells, used immunohistochemistry (IHC) staining to determine whether ACE could effectively reduce the expression of CD36 protein and enhance the expression of KLF4 protein of PA-treated SVEC 4-10 cells, and used migration assay to determine whether ACE could increase the mobility of RAW264.7 macrophages to clear hyperlipidemia SVEC4-10 cells. Our results showed that ACE has a good free radical scavenging ability, which could significantly alleviate the lipid accumulation in oil droplets, expressions of TNF-α, IL-1β and CD36 protein, but enhance KLF4 protein expression in hyperlipidemia SVEC4-10 cells. In addition, ACE could promote the migration ability of RAW264.7 macrophages to hyperlipidemia SVEC4-10 cells. We suggested that ACE may alleviate lipid accumulation and treat AS by reducing oil droplet lipid accumulation and alleviating oxidative stress and apoptosis in vascular endothelial cells and macrophages.

並列關鍵字

hyperlipidemia ； atherosclerosis ； Antrodia camphorata ； vascular endothelial cells ； macrophages ； oxidative stress ； apoptosis.

參考文獻

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