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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 To investigate the active components of Liuzhuo and its anti-inflammatory mechanism of synergistic induction of RAW264.7 cells by interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Methods Solvent extraction and D101 macroporous resin were used to prepare the extract and the total flavonoids of Radix Aconiti Lateralis respectively. The content of total flavonoids was determined by ultraviolet spectrophotometry. The model of inflammation was induced by IFN-γ and LPS. Greissreaction was used to determine the content of total flavonoids. Flavonoids on the content of nitrite in the supernatant of the cells, the effect of total flavonoids on the expression of inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) mRNA by RT-PCR, The effect of total flavonoids on the protein expression of iNOS, COX-2 and phosphorylated extracellular signal-regulated kinase (p-ERK) was investigated by Western blotting. Results Compared with other extracts, the ethyl acetate fraction of Liuluo Nong had the highest inhibition rate of nitrite content at the same dosage, and the content of total flavonoids was 53%. The content of nitrite in the cell supernatant was inhibited dose-dependently by the total flavonoids of S.japonicus, and the protective effect of the total flavonoids of S.japonicus on the cell viability was inhibited. The content of iNOS and COX-2 mRNA was decreased, -2, p-ERK protein expression. CONCLUSION: The active-oriented separation suggests that the ethyl acetate fraction is one of the anti-inflammatory components of S. liugout. Thus, the total flavonoid enriched inhibits nitrite content in the cell supernatant in a dose-dependent manner and is partially activated by mitogen The extracellular signal-regulated kinase (ERK) signaling pathway in protein kinases (MAPKs) inhibits the expression of iNOS and COX-2 genes and proteins to achieve anti-inflammatory effects.