Synergistic inhibition by sesamol and aspirin on LPS-induced COX-2 in RAW 264.7 macrophages

Abstract

摘要 前列腺素合成酶 (prostaglandin endoperoxide H synthase , PGHS ) 由cyclooxygenase (COX) 與peroxidase組成，依據表現部位、產物功能不同，可將PGHS分為常態表現型COX-1與誘導型COX-2。在發炎的組織中，吞噬細胞受到前致發炎細胞素、細菌的脂多醣體(Lipopolysaccharide, LPS)等刺激物質的刺激，可誘導COX-2基因大量表現，產生PGE2。阿斯匹靈(aspirin, ASA)是非固醇類藥物(Non-steroidal anti-inflammatory drugs, NSAIDs)之一，其作用方式主要是乙醯化COX-2活性部位，造成COX-2活性下降，以及直接抑制COX-2 gene表現，但長期、高劑量服用阿斯匹靈會抑制COX-1活性，容易引起胃部消化性潰瘍副作用。有研究顯示，維生素E可抑制COX-2；另有學者發現將阿斯匹靈與維生素E共同培養巨噬細胞株時，能協同抑制COX-2及PGE2表現。 芝麻酚(sesamol)為一種具抗氧化及抗發炎性質的物質，動物試驗顯示其可能抑制COX-2活性，本試驗目的為探討sesamol對COX-2的影響，以及探討sesamol與ASA是否有協同作用。做法為：以sesamol單獨(30, 100 M)或與ASA (300)共同預培養RAW 264.7細胞3小時，其後加入LPS培養6小時。結果顯示Sesamol不會抑制COX-2的表現，但可與ASA協同抑制COX-2蛋白表現。本研究的另一目標為探討sesamol是否會影響COX-1，結果顯示sesamol本身或與ASA共同使用時並不影響COX-1蛋白表現量。在COX-2活性方面(以PGE2濃度為指標)，本研究發現ASA或sesamol具有輕微降低PGE2含量的作用，但sesamol與ASA共同使用並不會協同抑制PGE2含量，但。 以上結果說明，在不影響細胞存活率的實驗條件下，Sesamol +ASA可協同抑制COX-2蛋白表現；而sesamol並不影響COX-2和COX-1蛋白質表現，但可輕微抑制COX-2活性，而sesamol＋ASA對PGE2並無協同抑制作用。

Abstract Prostaglandin endoperoxide H synthase (PGHS) consists of cyclooxygenase and peroxidase. Based on the difference of expression site and functions, PGHS is divided into two forms: COX-1 and COX-2. During inflammation, COX-2 is up-regulated by pro-inflammatory agents such as cytokines and lipopolysaccharide (LPS) leading to synthesis of PGE2. Aspirin (ASA) is one of the non-steroidal anti-inflammatory drugs (NSAIDs) and it can either inhibit COX-2 activity by acetylating the COX-2 active site or down-regulating COX-2 gene expression. ASA is associated with perforation of gastrointestinal ulcer because of inhibition of COX-1. Some studies show that vitamin E can inhibit COX-2 activity and can synergistically inhibit the enzyme when combined with ASA. Sesamol is an antioxidant and anti-inflammatory substance. Animal studies suggest that sesamol can inhibit COX-2 activity. The purpose of this study was to evaluate the effect of sesamol on COX-2 activity and its possible synergism with ASA. We pre-incubated RAW 264.7 cells with 30 M and 100 M sesamol with or without 300 M ASA for 3 hr followed by incubation with LPS for 6 hr. The results showed that sesamol itself did not inhibit COX-2 expression but synergistically inhibited COX-2 expression when combined with ASA. Another purpose of this study was to investigate whether sesamol affects COX-1. The result showed that sesamol alone or in combination with ASA did not affect COX-1 expression. Regarding COX-2 activity (assayed as PGE2 concentration), we found that, although ASA or sesamol alone reduced PGE2 contents slightly, the combination of sesmaol and ASA did not enhance the inhibition of the PGE2 level. In summary, under the experimental conditions that cell viability is not affected, we demonstrate that sesamol itself only slightly inhibits COX-2 activity and does not affect the expression of COX-2 and COX-1 proteins, but the combination of sesamol with ASA synergistically inhibit COX-2 but not COX-1 protein expression. However, sesamol in combination with ASA exhibits no additive inhibition of PGE2 synthesis.