白藜蘆醇影響人類臍靜脈內皮細胞與小鼠胸主動脈中表現黏附因子的效果與分子機轉

Abstract

心血管疾病已經成為全球最主要的健康問題之一，而且已連續三年成為台灣十大死因前三名，不僅如此，根據世界衛生組織統計，全球每年約有1650萬人死於心血管疾病。心血管疾病已被證實屬於一種慢性的發炎反應，當血管受到刺激時，會促使內皮細胞釋放細胞激素與表現ICAM-1等黏附因子。Resveratrol是可以經由葡萄、紅酒與許多植物中萃取出的天然物質，它是一種非黃酮的多酚類化合物(natural nonflavonoid polyphenolic compound)。近年來許多研究指出resveratrol有抗氧化及抗發炎的功能，進而防止心血管疾病的發生，但其中的機制仍尚未明瞭，因此我們的研究主軸是探討resveratrol對血管內皮細胞抗發炎的效果與機制。我們使用人類臍靜脈內皮細胞作為in vitro的實驗材料，實驗證明，在人類臍靜脈內皮細胞中TNF-α會顯著誘發ICAM-1表現，而先給予resveratrol處理再投予TNF-α則可以有效抑制由TNF-α誘發的ICAM-1表現。在探討機制的研究中發現，TNF-α會促使p-ERK、p-JNK、p-p38、p-IκB、p-p65及Ac-p65的表現量上升，而預先以resveratrol處理的細胞會抑制p-ERK、p-p38、p-IκB、p-p65及Ac-p65的表現。實驗進一步發現加入JNK、p-38與p-65的抑制劑，再加入TNF-α刺激會降低ICAM-1的表現，換句話說，在人類臍靜脈內皮細胞中，TNF-α是經由JNK、p38與NF-κB這三個路徑去誘發ICAM-1的表現，由此推論resveratrol可以藉由影響p38與NF-κB進而抑制TNF-α所誘發的ICAM-1表現。不僅如此，我們也發現resveratrol可以有效降低單核球與內皮細胞黏附的情形。在轉錄因子部分，resveratrol可以顯著抑制NF-κB p65在細胞核的表現及其與ICAM-1基因的結合活性(binding activity)。我們的實驗也發現，resveratrol可以活化AMPK，並且再加入AMPK活化劑後，可以有效降低TNF-α所誘發的ICAM-1的表現，顯示，resveratrol也可以藉由活化AMPK而抑制ICAM-1的表現。除此之外我們也發現當AMPK被活化時會誘發p-p38表現，而p-p38會誘發p-IκB的表現，綜合以上結論，說明resveratrol對於p38、AMPK與IκB可以藉由直接或間接的方式進行調控並影響ICAM-1的表現。在動物實驗方面，我們使用8週的成年雄性C57BL/6J小鼠，將其分為四組:DMSO控制組、TNF-α刺激組、resveratrol治療組與resveratrol組。給予resveratrol、TNF-α與DMSO後，取其胸主動脈進行實驗，結果顯示，在小鼠的胸主動脈中，resveratrol也可以抑制ICAM-1的表現。我們也使用Apo-E缺陷小鼠進行實驗，實驗發現有給予resveratrol組的血液中sICAM-1、三酸甘油脂與總膽固醇的表現皆比未給予resveratrol組低，並且在動脈硬化病灶處ICAM-1與TNF-α的表現也較低。綜合以上實驗結果，我們認為resveratrol對TNF-α誘發的ICAM-1有良好的抑制效果，顯示也許resveratrol可以藉由抗發炎進而達到預防心血管疾病的發生。

Resveratrol, a natural polyphenolic compound, is isolated from grapes, peanuts and other plants. Accumulating evidence indicates that resveratrol exerts its beneficial effects on cardiovascular system by its antioxidative and anti-inflammatory properties. However, the detailed mechanisms of its effects need to be investigated. Therefore, we focused on its effects on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-treated human umbilical vein endothelial cells (HUVECs) and its related mechanisms. TNF-α significantly induced ICAM-1 mRNA and protein expression in HUVECs and the effect was inhibited by pretreatment with resveratrol. TNF-α rapidly induced the phosphorylation of extracellular-regulated kinase (ERK), p38 MAPK, c-Jun N-terminal kinase (JNK) , inhibitory protein kappa B (IκB), nuclear factor kappa B (NF-κB) and NF-κB acetylation in HUVECs. Pretreatment with SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor) and Bay-117082 (a NF-κB inhibitor) significantly reduced TNF-α-induced ICAM-1 expression, whereas PD98059 (an ERK1/2 inhibitor) has no effect. Furthermore, pretreatment with resveratrol effectively reduced the phosphorylation of ERK, p38 MAPK, NF-κB and NF-κB acetylation, but not JNK phosphorylation in TNF-α-treated HUVECs. In addition, the nuclear translocation and DNA binding activity of NF-κB was also induced by TNF-α and the effects were inhibited with resveratrol pretreatment as demonstrated by immunostaining and EMSA. Moreover, resveratrol can also reduce monocyte adhesion to TNF-α-treated HUVECs. AMP-activated protein kinase (AMPK) can suppress inflammation. We also found that resveratrol can induce phosphorylation of AMPK in HUVECs. Moreover pretreatment with AICAR (an AMPK activator) significantly reduced TNF-α-induced ICAM-1 expression. In animal study, the mice were randomly divided into four groups: (1) the DMSO group, (2) the TNF-α group, (3) the TNF-α plus resveratrol group, and (4) the resveratrol group. DMSO and resveratrol (10 mg/kg) were given by intraperitoneal injection for five days and then mice were challenged with TNF-α (10 μg/kg) for the next three days. The thoracic aortas were isolated for paraffin section and the expression of ICAM-1 and CD31 was detected on serial sections by immunohistochemistry. We observed that administration of resveratrol effectively attenuated ICAM-1 expression in ECs in thoracic aortas of TNF-κ-treated mice. Our data showed that resveratrol inhibits TNF-κ-induced activation of NF-κB and p38MAPK, thereby suppresses ICAM-1 expression. Taken together, these results suggest that resveratrol may prevent the development of atherosclerosis and inflammation.