独活挥发油对N-脂肪酰基乙醇胺水解酶的抑制作用及抗炎作用研究  

Identify nature N-acylethanolamide-hydrolyzing acid amide(NAAA) inhibitor: effect of Angelicae Pubescentis Radix on anti-inflammation

作　　者：孙文畅[1] 杨隆河[1] 邱彦[1] 任杰[1] 黄锐[1] 傅瑾[1] 

机构地区：[1]厦门大学医学院药学系,福建厦门361005

出　　处：《中国中药杂志》2011年第22期3161-3166,共6页China Journal of Chinese Materia Medica

摘　　要：目的:研究独活挥发油对内源性大麻素水解酶N-脂肪酰基乙醇胺水解酶(N-acylethanolamine-hydrolyzing acidamidase,NAAA)水解活性的影响以及对脂多糖(LPS)诱导的小鼠巨噬细胞RAW264.7炎症反应模型的抗炎作用。方法:采用水蒸气蒸馏法提取独活挥发油,GC-MS检测化学成分;采用LC-MS检测NAAA水解活性;采用LPS诱导RAW264.7细胞建立细胞炎症反应模型;采用LC-MS检测细胞内棕榈酸乙醇胺(N-palmitoylethanolamine,PEA)水平;采用实时定量PCR检测肿瘤坏子因子-α(TNF-α)、诱导型一氧化氮合酶(iNOS)和白细胞介素-6(IL-6)mRNA表达;采用酶联免疫吸附法(ELISA)检测TNF-α含量;采用Griess法检测一氧化氮(NO)含量。结果:独活挥发油可抑制NAAA水解活性,升高LPS诱导的RAW264.7细胞内PEA水平;独活挥发油可下调LPS诱导的RAW264.7细胞炎症因子TNF-α,iNOS,IL-6 mRNA表达;独活挥发油可抑制LPS诱导的RAW264.7细胞TNF-α和NO释放。结论:独活挥发油能够抑制NAAA水解活性,升高细胞内PEA水平,降低炎症因子表达,具有一定的抗炎作用。Objective： To investigate the effect of Angelicae Pubescentis Radix（APR） on the activity of endocannabinoid hydrolase and N-acylethanolamine-hydrolyzing acid amidase（NAAA）,and to demonstrate the mechanism of anti-inflammatory effect of APR by in vitro lipopolysaccharide（LPS）-induced inflammation model.Method： APR essential oil was extracted by steam distillation,and the chemical components were identified by GC-MS.Enzymatic activity was performed by using recombinant NAAA-overexpressing protein and detected by LC-MS.Lipids were extracted by methonal/chloroform mixure and analyzed by LC-MS.mRNA and protein expression levels of proinflammatory genes were examined by Real time-PCR and ELISA assay kit,respectively.The content of nitro oxide（NO） was detected by Griess reaction.Result： Twenty active components were identified from APR essential oil which inhibited NAAA activity in a dose-dependent manner.On the LPS-induced RAW264.7 cells,APR essential oil reversed LPS-suppressed N-palmitoylethanolamide（PEA） contents in a dose-dependent manner and reduced LPS-induced proinflammatory genes,TNF-α and IL-6.Moreover,APR essential oil reduced the mRNA expression of iNOS,subsequently reduced the release of NO,a classic inflammatory marker.Conclusion： The research demonstrated that the effect of APR on inflammation is mediated by the inhibition of NAAA activity,which increase the cellular endobioactor PEA levels and decrease proinflammatory factor.The results suggest that APR can serve as a nature NAAA inhibitor.

关 键 词：独活挥发油 炎症 N-脂肪酰基乙醇胺水解酶 棕榈酸乙醇胺 小鼠巨噬细胞 

分 类 号：R285[医药卫生—中药学]