Seong Yeong-Ae, Hwang Dukhyun, Kim Gun-Do
Department of Microbiology, College of Natural Sciences, Pukyong National University, 45, Yongso-ro, Nam-Gu, Busan, 608-737, Korea.
Cell Biochem Biophys. 2016 Sep;74(3):407-17. doi: 10.1007/s12013-016-0744-7. Epub 2016 Jun 21.
Gnaphalium affine is an annual herbaceous plant that is used as a traditional medicine in some Latin American and Asian countries. However, systematic studies on its anti-inflammatory activity and signaling pathways have not yet been reported. In this study, we investigated the anti-inflammatory effect of G. affine methanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells and fractioned the methanol extract into hexane, chloroform, ethyl acetate (EtOAc), butyl alcohol (BuOH), and distilled water (DW) by measuring the generation of nitric oxide (NO). G. affine inhibited the generation of NO and prostaglandin E2. The chloroform-soluble fraction most effectively inhibited LPS-stimulated NO production. We also examined the cytotoxicity of G. affine in three normal cell lines: RAW264.7, HEK293, and HaCaT. Cell viability assays showed that the methanol extract and chloroform-soluble fraction of G. affine had no cytotoxic effect on normal cell lines. The expression of pro-inflammatory mediators was also investigated. Western blotting and immunofluorescence showed that G. affine reduces the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in LPS-stimulated RAW264.7 cells. RT-PCR showed that G. affine also negatively regulates inflammatory cytokines at the gene expression level. Taken together, G. affine exerts its anti-inflammatory activity through inhibition of NO generation as a result of inhibiting NF-κB and MAPKs-related inflammatory signaling pathways. In addition, the result of GC-MS analysis revealed the presence of nineteen different types of constituents including guaiacol in the chloroform-soluble fraction of G. affine.
鼠麴草是一种一年生草本植物，在一些拉丁美洲和亚洲国家被用作传统药物。然而，关于其抗炎活性和信号通路的系统研究尚未见报道。在本研究中，我们研究了鼠麴草甲醇提取物对脂多糖（LPS）刺激的RAW 264.7小鼠巨噬细胞的抗炎作用，并通过测量一氧化氮（NO）的生成将甲醇提取物分离为己烷、氯仿、乙酸乙酯（EtOAc）、丁醇（BuOH）和蒸馏水（DW）。鼠麴草抑制了NO和前列腺素E2的生成。氯仿可溶部分最有效地抑制了LPS刺激的NO产生。我们还检测了鼠麴草在三种正常细胞系RAW264.7、HEK293和HaCaT中的细胞毒性。细胞活力测定表明，鼠麴草的甲醇提取物和氯仿可溶部分对正常细胞系没有细胞毒性作用。我们还研究了促炎介质的表达。蛋白质免疫印迹和免疫荧光显示，鼠麴草降低了LPS刺激的RAW264.7细胞中诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）和丝裂原活化蛋白激酶（MAPKs）的表达以及核因子κB（NF-κB） 的激活。逆转录-聚合酶链反应（RT-PCR）表明，鼠麴草在基因表达水平上也对炎性细胞因子产生负调控作用。综上所述，鼠麴草通过抑制NF-κB和MAPKs相关的炎症信号通路来抑制NO生成，从而发挥其抗炎活性。此外，气相色谱-质谱联用（GC-MS）分析结果显示，鼠麴草氯仿可溶部分中存在包括愈创木酚在内的19种不同类型的成分。
