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.

Fig. 1. HPLC chromatograms of chlorogenic acid, geniposide, and RIS at different wavelengths. (A) HPLC chromatogram of chlorogenic acid at 324 nm. (B) HPLC chromatogram of geniposide at 237 nm. (C) HPLC chromatogram of the RIS sample at 324 nm. (D) HPLC chromatogram of the RIS sample at 237 nm.

Fig. 2. HPLC fingerprint chromatogram of RIS Peak S is geniposide. Except geniposide, other 13 common peaks were detected at 225 nm.

Fig. 3. RIS does not affect cell growth in RAW 264.7 macrophages cells were treated with various concentrations of RIS (12.5–400 μg mL⁻¹) for 24 h. The total number of viable cells was determined by the MTT assay. Values are mean ± SEM of six independent observations.

Fig. 4. RIS inhibits the production of NO and PGE₂ and the expression of iNOS and COX-2 in the LPS activated RAW 264.7 macrophages cells were incubated with indicated concentrations of RIS for 24 h with or without LPS. (A) Levels of NO in the culture supernatants of RAW 264.7 cells activated with LPS (1 μg mL⁻¹) in the presence or absence of RIS were determined by the Griess assay. (B) Levels of PGE₂ in the culture supernatants of RAW 264.7 cells treated with LPS (1 μg mL⁻¹) in the presence or absence of RIS were analyzed by ELISA. (C–E) The expression levels of iNOS, COX-2, and β-actin were analyzed by immunoblotting using whole cell lysates. The data shown are the mean ± SEM of three experiments. **p < 0.01 is significantly different from the control. ^#p < 0.05 and ^##p < 0.01 are different from the LPS alone.

Fig. 5. RIS inhibits the production of pro-inflammatory cytokines and chemokines in the LPS activated RAW 264.7 macrophages cells were incubated with indicated concentrations of RIS for 1 h and then stimulated with LPS for 24 h. The cell-free supernatants were collected to detect (A) IL-1β; (B) IL-6; (C) TNF-α; (D) MCP-1; (E) CCL-5; (F) MIP-1α concentrations via ELISA after the LPS treatment for 24 h. The data shown are the mean ± SEM of four experiments. **p < 0.01 is significantly different from the control. ^#p < 0.05 and ^##p < 0.01 are different from the LPS alone.

Fig. 6. RIS inhibits the nuclear protein levels of NF-κB/p65, AP-1/c-Jun, and IRF3 in the LPS activated RAW 264.7 macrophages cells were incubated with indicated concentrations of RIS for 1 h and then stimulated with LPS for 1 h. (A) The nuclear and cytoplasmic protein levels of NF-κB/p65, AP-1/c-Jun, and IRF3 were determined by Western-blotting. (B) The bar graphs show protein levels of NF-κB/p65, AP-1/c-Jun, and IRF3. Values are the mean ± SEM of four experiments. **p < 0.01 is significantly different from the control. ^#p < 0.05 and ^##p < 0.01 are different from the LPS alone.

Fig. 7. RIS suppressed the nuclear translocation of NF-κB/p65, AP-1/c-Jun, and IRF3 in the LPS activated RAW 264.7 macrophages cells were incubated with indicated concentrations of RIS for 1 h and then stimulated with LPS for 1 h. The nuclear localization of (A) NF-κB/p65, (B) AP-1/c-Jun, and (C) IRF3 were determined by immunofluorescence staining. The bar in each photograph indicates 33 μm.

Fig. 8. RIS inhibited IKKα/β/IκBα/NF-κB, MAPKs/AP-1, and TBK1/IRF3 pathways in the LPS activated RAW 264.7 macrophages cells were incubated with indicated concentrations of RIS for 1 h and then stimulated with LPS for 30 min or 60 min. The phosphorylation and total levels of TBK1, IKKα/β, IκBα, NF-κB, p38, ERK, JNK, c-Jun, IRF3, and p65 were detected.

Fig. 9. RIS affected the molecular components of IKKα/β/IκBα/NF-κB, MAPKs/AP-1, and TBK1/IRF3 pathways in the LPS activated RAW 264.7 macrophages cells were incubated with indicated concentrations of RIS for 1 h and then stimulated with LPS for 30 min or 60 min. The phosphorylation and total levels of TBK1, IKKα/β, IκBα, NF-κB, p38, ERK, JNK, c-Jun, IRF3, and p65 were detected. Values are the mean ± SEM of four experiments. **p < 0.01 is significantly different from the control. ^#p < 0.05 and ^##p < 0.01 are different from the LPS alone.

Fig. 10. Inflammatory signaling cascade targeted by RIS in macrophages.