Re-Du-Ning inhalation solution exerts suppressive effect on the secretion of inflammatory mediators via inhibiting IKKα/β/IκBα/NF-κB, MAPKs/AP-1, and TBK1/IRF3 signaling pathways in lipopolysaccharide stimulated RAW 264.7 macrophages

Abstract

Background: Re-Du-Ning inhalation solution (RIS) is a novel preparation derived from the Re-Du-Ning injection, which has been clinically used to treat respiratory diseases such as pneumonia for more than twenty years in China. However, scant reports have been issued on its anti-inflammatory mechanisms. Aim: we investigated the suppressive effect of RIS on inflammatory mediators and explored the underlying mechanism of action. Methods: RIS freeze dried powder was characterized by HPLC analysis. Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage was selected as the cell model. The cell viability was determined by using the MTT assay. Moreover, the production of nitric oxide (NO) was measured by the Griess reaction. The protein secretions from inflammatory mediators were determined by the enzyme-linked immunosorbent assay (ELISA). The protein levels and enzyme activities were examined by Western blotting. The nuclear translocation of nuclear factor-kappa B (NF-κB), AP-1, and IRF3 was further explored by immunofluorescence assay. Results: the viability of the RAW 264.7 cells was not significantly changed after 24 h incubation with RIS concentration up to 400 μg mL⁻¹. The RIS remarkably reduced the production of NO and prostaglandin E₂ (PGE₂), and downregulated the expression of iNOS and COX-2. The concentrations of cytokines (IL-1β, IL-6, and TNF-α) and chemokines (MCP-1, CCL-5, and MIP-1α) in the culture medium were significantly decreased by the RIS treatment. Furthermore, the phosphorylation of IκB-α, IKKα/β, TBK1, ERK, p38, JNK, NF-κB, AP-1, and IRF3 was downregulated by the RIS treatment. The nuclear translocation of NF-κB, AP-1, and IRF3 was also inhibited after the RIS treatment. Conclusion: the suppressive effect of RIS is associated with the regulated NF-κB, AP-1, and IRF3 and their upstream proteins. This study provides a pharmacological basis for the application of RIS in the treatment of inflammatory disorders.