The Anti-inflammatory Effect of Gnaphalium affine Through Inhibition of NF-κB and MAPK in Lipopolysaccharide-Stimulated RAW264.7 Cells and Analysis of Its Phytochemical Components.
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.
Department of Microbiology, College of Natural Sciences, Pukyong National University, 45, Yongso-ro, Nam-Gu, Busan, 608-737, Korea.
Yeong-Ae
YA
Seong
Department of Microbiology, College of Natural Sciences, Pukyong National University, 45, Yongso-ro, Nam-Gu, Busan, 608-737, Korea.
Dukhyun
D
Hwang
Department of Microbiology, College of Natural Sciences, Pukyong National University, 45, Yongso-ro, Nam-Gu, Busan, 608-737, Korea. gundokim@pknu.ac.kr.
Gun-Do
GD
Kim
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D015232:Dinoprostone / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D008401:Gas Chromatography-Mass Spectrometry; D036622:Gnaphalium / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D064209:Phytochemicals / Q000737:chemistry / Q000494:pharmacology*; D000067996:RAW 264.7 Cells; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*
D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D016328:NF-kappa B; D064209:Phytochemicals; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone
Anti-inflammatory effect; Cyclooxygenase-2 (COX-2); Gnaphalium affine; Mitogen-activated protein kinase (MAPK); Nitric oxide (NO); Nuclear factor-κB (NF-κB); Prostaglandin E2