20-Hydroxy-3-Oxolupan-28-Oic Acid Attenuates Inflammatory Responses by Regulating PI3K⁻Akt and MAPKs Signaling Pathways in LPS-Stimulated RAW264.7 Macrophages

Abstract

20-Hydroxy-3-oxolupan-28-oic acid (HOA), a lupane-type triterpene, was obtained from the leaves of Mahonia bealei, which is described in the Chinese Pharmacopeia as a remedy for inflammation and related diseases. The anti-inflammatory mechanisms of HOA, however, have not yet been fully elucidated. Therefore, the objective of this study was to characterize the molecular mechanisms of HOA in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. HOA suppressed the release of nitric oxide (NO), pro-inflammatory cytokine tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in LPS-stimulated RAW264.7 macrophages without affecting cell viability. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis indicated that HOA also suppressed the gene expression of inducible NO synthase (iNOS), TNF-α, and IL-6. Further analyses demonstrated that HOA inhibited the phosphorylation of upstream signaling molecules, including p85, PDK1, Akt, IκBα, ERK, and JNK, as well as the nuclear translocation of nuclear factor κB (NF-κB) p65. Interestingly, HOA had no effect on the LPS-induced nuclear translocation of activator protein 1 (AP-1). Taken together, these results suggest that HOA inhibits the production of cytokine by downregulating iNOS, TNF-α, and IL-6 gene expression via the downregulation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs), and the inhibition of NF-κB activation. Our findings indicate that HOA could potentially be used as an anti-inflammatory agent for medical use.

Keywords: NF-κB; inflammation; lupane-type triterpene; macrophage; nitric oxide.

Figure 1

The structure of 20-hydroxy-3-oxolupan-28-oic acid (HOA).

Figure 2

Anti-inflammatory effect of HOA on LPS-induced RAW264.7 cells. (A) RAW264.7 cells were treated with various concentrations of HOA for 24 h. The cell viability was determined by MTT assay, as described in section of Materials and Methods. (B–D) Cells were pretreated with various concentrations of HOA for 30 min and treated with lipopolysaccharide (LPS) for an additional 24 h. The NO content was determined by Griess reagent and the production of cytokines were measured by cytometric bead array (CBA) kit using the flow cytometry. The data are presented as means ± SD (n = 3). * indicates a significant difference between LPS group and HOA+LPS groups (p < 0.05). ^# indicates a difference between LPS group and the control group (p < 0.05).

Figure 3

Photograph of RAW264.7 cells after incubation with LPS and HOA under scanning electron microscopy (SEM). (a) Control; (b) LPS treatment; (c) LPS and HOA treatment.

Figure 4

The effect of HOA on LPS-induced pro-inflammatory cytokines expression in RAW264.7 cells. (A) Cells were plated at a density of 5 × 10⁶ cells/dish in 60-mm culture dishes and treated with LPS and HOA for 6 h. After preparation of the nuclear fraction, the mRNA expression levels of inducible NO synthase (iNOS), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) were measured using reverse-transcription polymerase chain reaction (RT-qPCR). The data are presented as means ± SD (n = 3). * indicates a significant difference between LPS group and HOA+LPS groups (p < 0.05). ^# indicates a significant difference between LPS group and the control group (p < 0.05). (B) Cells were plated at a density of 5 × 10⁶ cells/dish in 60-mm culture dishes and treated with LPS and HOA for indicated time points. After preparation of the total protein, the expression of iNOS was measured by western blot.

Figure 5

Effect of HOA on translocation of transcription factors in LPS-induced RAW264.7 cells. (A) Cells were plated at a density of 5 × 10⁶ cells/dish in 60-mm culture dishes and treated with LPS and HOA for indicated time points. After preparation of the nuclear fraction, the protein expression levels of p65, c-Jun, and c-Fos were measured. (B) The localization of NF-κB p65 in the cytoplasm and nuclear were visualized by a confocal microscopy.

Figure 6

Effects of HOA on LPS-induced activation of PI3K/Akt and MAPKs signaling pathways. (A) Cells were plated at a density of 5 × 10⁶ cells/dish in 60-mm culture dishes and treated with LPS and HOA for indicated time points. After preparation of the total protein, the phosphorylated and total forms of PDK1, p85, and Akt were measured by western blot. (B) Cells were plated at a density of 5 × 10⁶ cells/dish in 60-mm culture dishes and treated with LPS and HOA for indicated time points. After preparation of the total protein, the phosphorylated and total forms of ERK, JNK, and p38 were measured by western blot. (C) Inhibitory effects of LY294002 on LPS-induced NF-κB-luc activity in RAW264.7 cells. Results are representative of three experiments. * indicates a significant difference between LPS group and LY294002 + LPS groups (p < 0.05). ^# indicates a significant difference between LPS group and the control group (p < 0.05).