AIM OF THE STUDY: As allergy could be mediated by both IgE and IgG, we further evaluated the anti-allergy potential of CNAE in both in vitro model of IgG-induced macrophage activation and in vivo anaphylaxis models to further dissect the mechanism of action underlying the anti-allergic properties of CNAE.
MATERIAL & METHODS: The anti-allergy potential of CNAE was evaluated in in vivo anaphylaxis models of ovalbumin-challenged active systemic anaphylaxis (OVA-ASA) and IgE-challenged passive systemic anaphylaxis (PSA) using Sprague Dawley rats as well as IgG-challenged passive systemic anaphylaxis (IgG-PSA) using C57BL/6 mice. Meanwhile, in vitro model of IgG-induced macrophage activation model was performed using IC-21 macrophages. The release of soluble mediators from both IgE and IgG-mediated pathways were measured using enzyme-linked immunosorbent assay (ELISA). The signaling molecules targeted by CNAE were identified by performing Western blot.
RESULTS: IgG, platelet-activating factor (PAF) and IL-6 was suppressed by CNAE in OVA-ASA, but not IgE. In addition, CNAE significantly suppressed PAF and IL-6 in IgG-PSA but did not suppress histamine, IL-4 and leukotrienes C4 (LTC4) in IgE-PSA. CNAE also inhibited IL-6 and TNF-α by inhibiting the phosphorylation of ERK1/2 in the IgG-induced macrophage activation model.
CONCLUSION: Overall, our findings supported that CNAE exerts its anti-allergic properties by suppressing the IgG pathway and its mediators by inhibiting ERK1/2 phosphorylation, thus providing scientific evidence supporting its traditional use in managing allergy.
METHODS: Interleukin (IL)-6 cytokine production in histamine-induced HaCaT cells were measured using enzyme-linked immunosorbent assay (ELISA) and cytotoxicity effects were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the inhibitory effects of MS65 on nuclear factor-kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways.
RESULTS: Histamine enhanced IL-6 production in HaCaT cells, with the highest production of IL-6 at 97.41 ± 2.33 pg/mL after 24 h of exposure. MS65 demonstrated a promising anti-inflammatory activity by inhibiting IL-6 production with half maximal inhibitory concentration (IC50) value of 4.91 ± 2.50 μM and median lethal concentration (LC50) value of 28.82 ± 7.56 μM. In gene expression level, we found that MS65 inhibits NF-κB and MAPK pathways through suppression of IKK/IκB/NFκB and c-Raf/MEK/ERK inflammatory cascades.
CONCLUSION: Taken together, our results suggest that MS65 could be used as a lead compound on developing new medicinal agent for the treatment of allergic skin diseases.
METHODS: Acute inflammation was produced by subplantar injection of 0.1 mL of 0.1% histamine into the right hind paw of each rat in the control and treatment groups. The degree of edema was measured before injection and at the time points of 30, 60, 120, 180, 240 and 300 min after injection. Changes of peritoneal vascular permeability were studied using Evans blue dye as a detector. Vascular permeability was evaluated by the amount of dye leakage into the peritoneal cavity in rats. To evaluate the inhibitory effect of AEBO on biochemical mediators of vascular permeability, the levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) were determined in histamine-treated paw tissues. The major constituents of AEBO were determined by gas chromatography-mass spectrometry (GC-MS) analysis.
RESULTS: AEBO produced a significant inhibition of histamine-induced paw edema starting at 60 min time point, with maximal percentage of inhibition (60.25%) achieved with a dose of 150 mg/kg of AEBO at 60 min time point. Up to 99% of increased peritoneal vascular permeability produced by histamine was successfully suppressed by AEBO. The expression of biochemical mediators of vascular permeability, NO and VEGF, was also found to be downregulated in the AEBO treated group. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the major constituent in AEBO was acetic acid.
CONCLUSIONS: The experimental findings demonstrated that the anti-inflammatory activity of AEBO was due to its inhibitory effect on vascular permeability, which was suppressed as a result of the reduced expression of biochemical mediators (NO and VEGF) in tissues. Our results contribute towards the validation of the traditional use of Bixa orellana in the treatment of inflammatory disorders.