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Oleanolic Acid Acetate Exerts Anti-Inflammatory Activity via IKKα/β Suppression in TLR3-Mediated NF-κB Activation

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Oleanolic Acid Acetate Exerts Anti-Inflammatory Activity via IKKα/β Suppression in TLR3-Mediated NF-κB Activation

Hyung Jin Lim et al. Molecules.

Abstract

Oleanolic acid acetate (OAA), a major triterpenoid compound of Vigna angularis (azuki bean, V. angularis), has been shown to downregulate inflammatory responses in macrophages. Here, we show the molecular basis for the effect of OAA on Toll-like receptor (TLR) downstream signaling. OAA treatment significantly inhibited the secretion of embryonic alkaline phosphatase (SEAP) induced by polyinosinic acid (poly(I), TLR3 ligand) in a dose-dependent manner and without cytotoxicity in THP1-XBlue cells. In addition, OAA downregulated the gene expression of poly(I) induced pro-inflammatory cytokines and chemokines genes such as MCP-1, IL-1β, IL-8, VCAM-1 and ICAM-1. Furthermore, we found that the inhibition activity of OAA was accompanied by decreased activation of not only nuclear factor-kappa B (NF-κB) signaling but also mitogen-activated protein kinase (MAPK) signaling upon stimulation with the TLR3 agonist. Interestingly, the interaction of OAA with IκB kinase α/β (IKKα/β) strongly attenuated the production of certain proteins and inflammatory cytokines in the TLR3 signaling pathway, such as nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), extracellular regulated kinases (ERK), and p38, in an in vitro model. The action of OAA was regulated by TLR3, demonstrating that TLR3 plays a critical role in mediating the physiologically-relevant anti-inflammatory action of OAA and that the interaction with IKKα/β is modulated through TLR3. These results reveal new insight into the understanding of the regulatory mechanisms of the downstream TLR3 signaling pathway and consequent inflammatory responses that are involved in the development and progression of inflammatory diseases.

Keywords: IκB kinase α/β; Vigna angularis; inflammatory cytokines; oleanolic acid acetate; toll-like receptors.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
OAA inhibits poly(I)-induced NF-κB/AP-1 activation and cytokine expression in human monocytes. (A) Chemical structure of oleanolic acid 3-acetate. (B) Cells were incubated with OAA (0–100 μM) for 24 h, and cell viability was determined by the WST-1 assay. (C) Cells were pretreated with OAA (0–60 μM) for 1 h before stimulation with poly(I:C) or poly(I) for 18 h (50 μg/mL), and the secretion of SEAP was measured by QUANTI-Blue. Values are presented as ± SD of three individual experiments. * p < 0.05 and ** p < 0.01 compared with the untreated or Poly I:C and Poly I-treated group.
Figure 2
Figure 2
Cells were pretreated with OAA at the indicated concentrations for 1 h before treatment with poly(I) (50 μg/mL) for 12 h, and then total RNA was isolated. The expression levels of inflammatory chemokines or cytokines such as MCP-1 (A), IL-1β (B), IL-8, (C), VCAM-1 (D), and ICAM-1 (E) were analyzed by quantitative real-time PCR. Values are presented as ± SD of three individual experiments. * p < 0.05 and ** p < 0.01 compared with the Poly I-treated group.
Figure 3
Figure 3
OAA downregulates poly(I)-induced TLR3 signaling. Cells were pretreated with the indicated concentrations of OAA for 1 h and then stimulated with poly(I) (50 μg/mL) for 30 min to 3 h. Cell lysates were prepared for Western blotting analysis with the indicated antibodies. OAA inhibits the poly(I)-induced nuclear translocation of NF-κB and c-fos (A), and phosphorylation of IKKα/β, IκB (B), ERK, and p38 (C) and IRF3 (D). Values are presented as ± SD of three individual experiments. * p < 0.05 and ** p < 0.01 compared with the Poly I-treated group.
Figure 4
Figure 4
OAA targets IKKα/β. Cell lysates were treated with biotin-OAA or OAA for 1 or 6 h at a concentration of 30 μM, and the lysates were then subjected to pull-down experiments using streptavidin agarose beads. (A) The precipitates were analyzed by immunoblotting with the indicated antibodies. (B) Proposed model of OAA anti-inflammatory activity in poly(I)-induced TLR3 signaling.

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