阿司匹林丁香酚酯对体外脂多糖诱导小鼠巨噬细胞炎症反应的抑制效应

Inhibiting Effect of Aspirin Eugenol Ester on Lipopolysaccharide-induced Inflammatory Response in Mouse Macrophages in vitro

本文旨在研究阿司匹林丁香酚酯(aspirin eugenol ester,AEE)对脂多糖(lipopolysaccharide,LPS)诱导小鼠巨噬细胞(RAW264.7细胞)炎症反应的抑制作用及其潜在作用机制。LPS刺激RAW264.7细胞诱导其炎症模型,细胞分为对照组、LPS组、阿司匹林组(aspirin,Asp,150μmol·L^(-1))、丁香酚组(eugenol,Eug,150μmol·L^(-1))、AEE低(75μmol·L^(-1))、中(150μmol·L^(-1))、高剂量(300μmol·L^(-1))组。CCK-8法检测AEE对小鼠巨噬细胞RAW264.7的细胞毒性;ELISA法检测各组细胞中IL-1β、IL-6、IL-8、TNF-α炎症因子的含量变化;RT-PCR法检测细胞中花生四烯酸(arachidonic acid,AA)代谢通路关键酶PLA2、COX-2、COX-1、CYP450和5-LOX的mRNA表达量;分子对接研究Asp、Eug、AEE与AA代谢通路关键酶的相互作用。结果表明:1)CCK-8结果显示,AEE浓度在0~350μmol·L^(-1)时,对细胞无毒性;2)ELISA结果表明,与空白对照组比较,LPS组炎症因子IL-1β、IL-6、IL-8、TNF-α表达极显著升高(P<0.01);经不同剂量的AEE处理能极显著降低炎症因子IL-1β、IL-8、TNF-α表达水平(P<0.01);在炎症因子IL-1β、IL-6、IL-8中,与Asp和Eug组比较,等摩尔的AEE与其差别不显著(P>0.05),表明其抗炎效果相似;不同剂量的AEE在炎症因子IL-1β、IL-6、IL-8之间差异不显著(P>0.05);3)RT-PCR结果表明,与空白对照组比较,LPS组中AA代谢通路关键酶表达极显著升高(P<0.01);经不同剂量的AEE处理能显著降低AA代谢通路关键酶的mRNA表达量(P<0.05);与Asp和Eug组比较,等摩尔量的AEE能够显著降低AA代谢通路关键酶PLA2的表达量(P<0.05);不同剂量的AEE在COX-1、COX-2、5-LOX和CYP450关键酶表达量之间差异不显著(P>0.05);4)分子对接结果显示,AEE与靶蛋白的结合能均低于-20.9 kJ,表明AEE与靶蛋白结合稳定且质量高,且AEE能够与PLA2、COX-2、COX-1、CYP450和5-LOX之间形成氢键。综上,AEE能够抑制LPS诱导的RAW264.7细胞炎症反应,并且与前体化合物Asp和Eug抗炎作用相似,其可能通过AA代谢通路发挥抗炎作用。

The purpose of this study was to investigate the inhibitory effects and potential mechanism of aspirin eugenol ester(AEE) on lipopolysaccharide(LPS)-induced inflammatory response in mouse macrophage(RAW264.7 cells). Inflammation in RAW264.7 cells was induced by LPS. Cells were divided into control group, LPS group, aspirin group(Asp, 150 μmol·L), eugenol group(Eug, 150 μmol·L), AEE low-(75 μmol·L), medium-(150 μmol·L) and high-dose(300 μmol·L) groups. CCK-8 assay was used to detect the cytotoxicity of AEE in RAW264.7 cells. The levels of IL-1β, IL-6, IL-8 and TNF-α in each group were detected by ELISA. The mRNA expression levels of COX-2, COX-1, PLA2, CYP450 and 5-LOX, the key enzymes in arachidonic acid(AA) metabolism pathway, were detected by RT-PCR. Meanwhile, the interaction of Asp, Eug and AEE with key enzymes related to arachidonic acid metabolism pathway was studied by molecular docking. The results were showed as follows: 1) CCK-8 results indicated that AEE with the concentration of 0 to 350 μmol·Lwas non-toxic to cells;2) The results of ELISA showed that, compared with the control group, the expression levels of IL-1β, IL-6, IL-8 and TNF-α in the LPS group were extremely significantly increased(P<0.01);After treated with different doses of AEE extremely significantly reduced the expression levels of IL-1β, IL-8, and TNF-α(P<0.01);Compared with the Asp and Eug groups, no significant difference was observed for the equimolar AEE in inflammatory factors of IL-1β, IL-6, IL-8(P>0.05), indicating the similar anti-inflammatory effects of AEE with Asp and Eug;And no significant difference of the levels of IL-1β, IL-6 and IL-8 among different doses of AEE was observed(P>0.05);3) RT-PCR results showed that, compared with the control group, the expression of key enzymes in the AA metabolic pathway in the LPS group was extremely significantly increased(P<0.01);After treated with different doses of AEE significantly reduced the mRNA expression of key enzymes in the AA metabolic pathway(P<0.05);Compared with the Asp and Eug groups, the equimolar amount of AEE significantly reduced the expression of PLA2(P<0.05);There was no significant difference of the expression levels of COX-1, COX-2, 5-LOX and CYP450 was observed among different doses of AEE(P>0.05);4) The results of molecular docking showed that the binding energy of AEE to the target proteins was lower than-20.9 kJ, indicating the binding of AEE to target proteins was stable and high quality. Meanwhile, AEE could form hydrogen bonds with PLA2, COX-2, COX-1, CYP450 and 5-LOX. In summary, AEE could inhibit the inflammatory response of RAW264.7 cells induced by LPS, and the anti-inflammatory effects of AEE were similar to the precursor compounds Asp and Eug, which might be associated with the regulation of AA metabolic pathway.