刘寄奴抗炎组分筛选及其机制研究

Study on Active Constitutes of Artemisia anomala S.Moore and Its Anti-inflammation Mechanism in Stimulated Macrophages

目的考察刘寄奴活性组分及其对干扰素-γ(IFN-γ)和脂多糖(LPS)协同诱导RAW264.7细胞的抗炎机制。方法应用溶剂萃取和D101大孔树脂分别制备刘寄奴各提取物和总黄酮,紫外分光光度法测定总黄酮的含量;IFN-γ和LPS协同诱导细胞炎症模型;Greissreaction法测定各提取物及总黄酮对细胞上清液中亚硝酸盐含量的影响;RT-PCR法考察总黄酮对细胞内诱导型一氧化氮合酶(iNOS)、诱导型环氧合酶(COX-2)mRNA的影响;Westernblot法考察总黄酮对细胞iNOS、COX-2、磷酸化细胞外信号调节激酶(p-ERK)蛋白表达的影响。结果刘寄奴乙酸乙酯部位与其他提取物相比,在相同剂量时对亚硝酸盐含量的抑制率最高,由此富集获得的总黄酮含量达53%。刘寄奴总黄酮呈剂量依赖性抑制细胞上清液中亚硝酸盐的含量,并对刺激后的细胞活力有保护作用;刘寄奴总黄酮呈抑制iNOS、COX-2mRNA表达,抑制iNOS、COX-2、p-ERK的蛋白表达。结论活性导向的分离提示乙酸乙酯部位是刘寄奴抗炎活性组分之一,由此富集的总黄酮呈剂量依赖性抑制细胞上清液中亚硝酸盐含量,部分通过丝裂原活化蛋白激酶(MAPKs)中的细胞外信号调节激酶(ERK)信号通路抑制iNOS和COX-2基因和蛋白的表达而达到抗炎效果。

Objective This study investigates the active constitutes from Artemisia anomala S. Moore and its anti-inflammation mechanism in RAW 264.7 macrophages stimulated with interferon-γ （ IFN-γ）/lipopolysaccharide （LPS）. Methods Four solvent extracts （ ethanol, ethyl acetate, n-butanol, water） were separated and total flavones purified by D101 macroporous resin （TFAS）. Spectrophotometer was applied to quantify the total flavones TFAS. IFN- γplus LPS stimulated excessive expression of nitric oxide in RAW 264.7 macrophages was used as inflammatory experimental model. Inhibition of inducible nitric oxide synthase（ iNOS）, cy- clooxygenase 2 （COX-2） by the TFAS were examined by RTPCR for mRNA expression; Western blot for iNOS, COX-2 and phosphated-extra cellular regulated protein kinases（p-ERK） protein expression and Griess reaction for nitric oxide production. Results TFAS exhibited the strongest suppressing action on NO formation, which purified from ethyl acetate extract of four solvent extracts in active-oriented extraction. TFAS showed dose-dependent inhibitory effect on NO production in stimulated cells without cytotoxicity. The gene and protein expression of iNOS and COX-2 were inhibited by TFAS, the protein expression of p-ERK also suppressed by TFAS. Conclusion These results suggest that TFAS may provide potential protective effects in attenuating excessive NO generation at inflammatory sites. TFAS inhibited iNOS and COX-2 expression may be through extra cellular regulated protein kinases （ERK） pathway partly. Thus, further studies on TFAS may lead to new drug targeted inhibiting iNOS for curing inflammation.