Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

Yi Zhang,1,2,* Hui Guo,1,* Brian Chi-Yan Cheng,1 Tao Su,1 Xiu-Qiong Fu,1 Ting Li,1 Pei-Li Zhu,1 Kai-Wing Tse,1 Si-Yuan Pan,3 Zhi-Ling Yu1,3,4 1Centre for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong; 2Department of Pharmacology, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 3Research and Development Centre for Natural Health Products, HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, People’s Republic of China; 4Consun Chinese Medicines Research Centre for Renal Diseases, Hong Kong Baptist University, Hong Kong, People’s Republic of China *These authors contributed equally to this work Background: Dingchuan tang (asthma-relieving decoction), a formula of nine herbs, has been used for treating respiratory inflammatory diseases for >400 years in the People’s Republic of China. However, the mechanisms underlying the anti-inflammatory action of dingchuan tang is not fully understood. This study aims to investigate the effects of Dingchuan tang essential oil (DCEO) on inflammatory mediators and the underlying mechanism of action.Materials and methods: DCEO was extracted by steam distillation. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. Production of nitric oxide (NO) was determined by the Griess test. Protein secretion and mRNA levels of inflammatory mediators were measured by the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Protein levels were examined by Western blot. Nuclear localization of nuclear factor-kappa B (NF-κB) was detected using immunofluorescence analyses.Results: DCEO significantly reduced LPS-triggered production of NO and prostaglandin E2 (PGE2) and decreased protein and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). LPS induced upregulation of protein and mRNA levels of cytokines (interleukin-1β [IL-1β], interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), and chemokines (monocyte chemoattractant protein-1 [MCP-1], chemokine [C-C motif] ligand 5 [CCL-5], and macrophage inflammatory protein [MIP]-1α) were suppressed by DCEO treatment. Phosphorylation and nuclear protein levels of transcription factors (activator protein-1 [AP-1], NF-κB, interferon regulatory factor 3 [IRF3]) were decreased by DCEO. Protein levels of phosphorylated IκB-α, IκB kinase α/β (IKKα/β), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), TGF β-activated kinase 1 (TAK1), TANK-binding kinase 1 (TBK1), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) were lowered by DCEO. Moreover, degradation of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 induced by LPS was inhibited by DCEO treatment.Conclusion: Suppression of the interleukin-1 receptor-associated kinase (IRAK)/NF-κB, IRAK/AP-1 and TBK1/IRF3 pathways was associated with the inhibitory effects of DCEO on inflammatory mediators in LPS-stimulated RAW264.7 macrophages. This study provides a pharmacological justification for the use of dingchuan tang in managing inflammatory disorders. Keywords: traditional Chinese medicine, respiratory diseases, anti-inflammation, molecular pathway