十字花科蔬菜衍生物對Lipopolysaccharide活化巨噬細胞所誘導之血管新生作用的影響

Effects of cruciferous vegetable derivatives in lipopolysaccharide activated macrophage induced angiogenesis

王美琳(Wang Mei Lin)

指導教授 ： 陳玉華

臺北醫學大學/公共衛生暨營養學院/保健營養學研究所/碩士(2006年)

摘要

本研究主要探討十字花科蔬菜衍生物benzyl isothiocyanate (BITC) 、phenylethyl isothiocyanate (PEITC) 與indole-3-carbinol (I3C) 對於活化巨噬細胞所誘導之血管內皮細胞血管新生之影響。將細菌內毒素lipopolysaccharide (LPS) 加入巨噬與血管內皮共同培養之細胞顯示，以Greiss reagent 所測得培養液中一氧化氮之生成與以matrigel 所分析之血管內皮細胞類血管生成皆有增加的現象，而BITC、PEITC與I3C的添加則可抑制NO與類血管的生成。為探討此抑制之作用 是否因影響巨噬細胞之故，因而使用LPS與BITC、PEITC或I3C共同處理之巨噬細胞condition medium (Co-BITC-CM, Co-PEITC-CM, Co-I3C-CM) 投予血管內皮細胞，結果指Co-BITC-CM, Co-PEITC-CM與Co-I3C-CM中含較低濃度之一氧化氮，且Co-I3C-CM可抑制血管內皮細胞之類血管生成的作用，且此抑制伴隨著較低血管內皮生長因子vascular endothelial growth factor (VEGF)的釋放與較低基質金屬蛋白?MMP-9 (matrix metalloproteinase-9) 活性，此外投與NO抑制劑L-NAME亦有相類似之結果。然而Co-BITC-CM與Co-PEITC-CM雖可抑制血管內皮細胞類血管的生成與MMP- 9的活性，反而可以促進VEGF之釋放。此外BITC、PEITC、I3C與L-NAME對於活化之血管內皮細胞影響亦被分析，因而以LPS活化巨噬細胞之condition medium (CM) 處理血管內皮細胞，並於BITC、PEITC與I3C之存在下偵測血管新生相關因子的影響。結果顯示，I3C與 L-NAME亦可抑制經CM誘導血管內皮細胞培養基內一氧化氮之生成、血管內皮細胞類血管生成、MMP- 9之活性與VEGF之分泌，而BITC與PEITC亦可抑制經CM誘導血管內皮細胞培養基內一氧化氮之生成、血管內皮細胞之類血管生成與MMP- 9之活性，但亦增加血管內皮生長因子生成。總而言之，十字花科蔬菜衍生物BITC、PEITC與I3C可同時藉由抑制LPS所誘導巨噬細胞活化，以及影響血管內皮細胞的作用而具有抗血管新生之作用，且其中I3C之抑制作用主要藉由NO之抑制，而BITC與PEITC之抑制作用則與NO無關。

關鍵字

十字花科蔬菜衍生物 ； 血管新生 ； 巨噬細胞 ； 血管內皮細胞 ； 共同培養 ； 一氧化氮

並列摘要

The aim of this study was to investigate the roles of cruciferous vegetable derivatives, BITC (benzyl isothiocyanate), PEITC (phenylethyl isothiocyanate), and indole-3-carbinol (I3C), in activated macrophage-induced angiogenesis. After co-culturing macrophages and vascular endothelial cells, we observed that the nitric oxide (NO) production and tube formation were significantly enhanced by LPS (lipopolysaccharide), and cotreatment with BITC, PEITC, and I3C significantly inhibited such enhancement. To clarify the inhibitory roles of BITC, PEITC, and I3C on macrophages, LPS and BITC-, PEITC-, I3C-, or NO inhibitor- (Nitro-L-arginine methyl ester, L-NAME) treated macrophage condition medium was used to cultivate vascular endothelial cells. The tube formation, vascular endothelial growth factor (VEGF) secretion, and matrix metalloproteinase-9 (MMP-9) activity were suppressed by I3C and L-NAME, and tube formation and MMP-9 activity were suppressed but VEGF secretion was enhanced by BITC and PEITC in vascular endothelial cells. To understand whether BITC or PEITC or I3C or L-NAME also affected vascular endothelial cells, we used LPS-activated macrophages condition medium (CM) to treat to vascular endothelial cells. The results showed that addition of I3C and L-NAME inhibited CM-induced NO production, tube formation, VEGF secretion and, MMP-9 activity in vascular endothelial cells. BITC and PEITC inhibited CM-induced NO production, tube formation, and MMP-9 activity in vascular endothelial cells, but stimulated CM-induced VEGF secretion. In summary, we demonstrate that the cruciferous vegetable derivatives, BITC, PEITC, and I3C, not only inhibits LPS stimulated macrophage activation but also affects vascular endothelial cells to inhibit macrophage-induced angiogenesis. Additionally, the inhibitory effect of I3C is dependent upon NO production, whereas the effects of BITC and PEITC are irrelevant to NO.

並列關鍵字

cruciferous vegetable derivative ； angiogenesis ； macrophages ； vascular endothelial cells ； co-culture ； nitrite

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