Xiao Qing Long Tang essential oil exhibits inhibitory effects on the release of pro-inflammatory mediators by suppressing NF-κB, AP-1, and IRF3 signalling in the lipopolysaccharide-stimulated RAW264.7 cells

Abstract

Xiao Qing Long Tang (literally "Minor blue dragon decoction" in Chinese), a traditional Chinese formula, is prescribed to treat respiratory diseases. However, only few studies have been reported on its anti-inflammatory mechanisms. In this study, we investigated the inhibitory effects of Xiao Qing Long Tang essential oil on inflammatory mediators and explored the mechanisms of action of XQEO in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells. XQEO was prepared via steam distillation and characterized by GC-MS analysis. MTT and Griess assays were used to measure cell viability and NO production, respectively. The mRNA expression and the production of LPS-induced pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-10) and chemokines (MCP-1, Rantes, and MIP-1α) were determined by real-time PCR and enzyme-linked immunosorbent assay, respectively. Furthermore, we determined the protein levels of the components of NF-κB, AP-1 and IRF3 signalling by Western blotting. Immunofluorescence assay was used to estimate the nuclear translocation of NF-κB, AP-1 and IRF3. The results showed that XQEO inhibited the secretion of NO and PGE2 and down-regulated the mRNA and protein levels of iNOS and COX-2. We also found that XQEO suppressed the LPS-induced overproduction of pro-inflammatory mediators. Moreover, XQEO inhibited the phosphorylation of NF-κB/p65, AP-1/c-Jun, and IRF3 by suppressing their upstream kinases, such as MAPKs, TBK1, Akt, IKKα/β, and IκB, reducing the LPS-induced NF-κB, AP-1 and IRF3 translocation to the nucleus. These findings suggest that XQEO effectively suppresses the production of pro-inflammatory mediators possibly through the inhibition of NF-κB, AP-1, and IRF3 signalling in the LPS-stimulated RAW264.7 cells.

Fig. 1. The GC chromatogram of XQEO. The volatile constituents in the essential oils were separated using GC chromatogram.

Fig. 2. Effects of XQEO on the LPS-induced production of NO and PGE2 and expression of iNOS and COX-2 in RAW264.7 macrophages. After being treated in the absence or presence of XQEO for 1 h, the cells were treated with or without LPS (1 μg mL⁻¹) for another 24 h. The total number of viable cells was determined by the MTT assay (A). The production of NO and PGE2 was determined by the Griess reaction and ELISA kit, respectively (B and C). Cells were incubated with indicated concentrations of XQEO for 1 h and then stimulated with LPS for 6 h. The mRNA levels of mPGES1, iNOS, and COX-2 were determined by qRT-PCR using specific primers (D–F). The protein expression levels of iNOS and COX-2 were detected by Western blotting (G–I). β-Actin was used as an internal control. Experiments were performed independently at least three times. Values shown are the means ± SEM of three independent. *p < 0.05, **p < 0.01 vs. control group. #p < 0.05, ##p < 0.01 vs. LPS-stimulated group.

Fig. 3. Effects of XQEO on the mRNA expression of pro-inflammatory cytokines and chemokines in the RAW264.7 macrophages. Following treatment with or without XQEO (6–50 μg mL⁻¹) for 1 h, cells were stimulated with LPS (1 μg mL⁻¹) for 6 h. Control cells were not treated with LPS or XQEO. Total RNAs were prepared for RT-PCR analysis, and the mRNA levels of TNF-α (A), IL-1β (B), IL-6 (C), IL-10 (D), MCP-1 (E), Rantes (F), and MIP-1α (G) were determined using specific primers. Results were normalized against GAPDH. Experiments were performed independently at least three times. Values shown are the means ± SEM of three independent. *p < 0.05, **p < 0.01 vs. control group. #p < 0.05, ##p < 0.01 vs. LPS-stimulated group.

Fig. 4. Effects of XQEO on the production of pro-inflammatory cytokines and chemokines in the RAW264.7 macrophages. Following treatment with XQEO (6–50 μg mL⁻¹) for 1 h, cells were stimulated with LPS (1 μg mL⁻¹) for 24 h. The culture medium was collected and the production of TNF-α (A), IL-1β (B), IL-6 (C), IL-10 (D), MCP-1 (E), Rantes (F), and MIP-1α (G) were determined using ELISA assay. Control cells were not treated with LPS or XQEO. Experiments were performed independently at least three times. Values shown are the means ± SEM of four independent. *p < 0.05, **p < 0.01 vs. control group. #p < 0.05, ##p < 0.01 vs. LPS-stimulated group.

Fig. 5. Effect of XQEO on the LPS-induced nuclear translocation of NF-κB, c-Jun, and AP-1 in the RAW264.7 macrophages. The cells were treated with XQEO (12.5–50 μg mL⁻¹) for 1 h prior to treatment with 1 μg mL⁻¹ LPS for 1 h. Control cells were not treated with LPS or XQEO. Nuclear and cytosolic proteins were subjected to 10% SDS-PAGE followed by Western blot analysis using anti-NF-κB/p65, AP-1/c-Jun and IRF3 antibodies. Sp1 and β-actin were used as internal controls for the nuclear and cytosolic fractions, respectively. Experiments were performed independently for three times. Values shown are the means ± SEM of three independent. *p < 0.05, **p < 0.01 vs. control group. #p < 0.05, ##p < 0.01 vs. LPS-stimulated group.

Fig. 6. Effect of XQEO on the LPS-induced nuclear translocation of NF-κB, c-Jun, and AP-1 in the RAW264.7 macrophages. The cells were treated with XQEO (0 or 50 μg mL⁻¹) for 1 h prior to treatment with 1 μg mL⁻¹ LPS for 1 h. Control cells were not treated with LPS or XQEO. The nuclear localizations of NF-κB/p65, AP-1/c-Jun, and IRF3 were detected using the immunofluorescence assay. The bar in each image indicates 33 μm.

Fig. 7. Effect of XQEO on the molecular components of NF-κB, AP-1, and IRF3 signalling in the RAW264.7 macrophages. Total cellular proteins were obtained from the cells stimulated with LPS (1 μg mL⁻¹) for 30 min (A) or 1 h (B). Control cells were not treated with LPS or XQEO. The levels of p-IKKα/β, IKKα/β, p-TBK1, TBK1, p-ERK, ERK, p-p38, p38, p-JNK, JNK, p-Akt, Akt, p-p65, p65, p-IκBα, IκBα, p-c-Jun, c-Jun, p-IRF3, and IRF3 were determined using specific antibodies. β-Actin was used as an internal control (A). The bar charts represent the ratio of p-IKKα/β/IKKα/β, p-TBK1/TBK1, p-ERK/ERK (44 kDa), p-ERK/ERK (42 kDa), p-p38/p38, p-JNK/JNK (54 kDa), p-JNK/JNK (46 kDa), p-Akt/Akt, p-p65/p65, p-IκBα/IκBα, p-c-Jun/c-Jun, and p-IRF3/IRF3 (B). Experiments were performed independently three times. Values shown are the means ± SEM of three independent. *p < 0.05, **p < 0.01 vs. control group. #p < 0.05, ##p < 0.01 vs. LPS-stimulated group.

Fig. 8. The molecular components of NF-κB, AP-1, and IRF3 signalling targeted by XQEO in macrophages.