Demethylnobiletin inhibits delayed-type hypersensitivity reactions, human lymphocyte proliferation and cytokine production

Abstract

Background and purpose: Our aim was to examine the effect of demethylnobiletin on various experimental models of delayed-type hypersensitivity (DTH) reactions and to determine its influence on the mediators and enzymes involved in these reactions.

Experimental approach: DTH was induced in mice by oxazolone, dinitrofluorobenzene (DNFB) and sheep red blood cells (SRBC). The effect of demethylnobiletin on the ensuing DTH was studied, especially in relation to oedema formation, cell infiltration and tissue damage. Its activity on different mediators implicated in DTH reactions was also determined and its effect on nitric oxide synthase (NOS)-2 analysed. Finally, its influence on T lymphocyte proliferation, apoptosis and caspase 3 activity was tested.

Key results: DTH reactions were all reduced by demethylnobiletin. The experimental results suggest that the compound may act by reducing cell infiltration and by suppressing mediators such as interleukin-2 (IC50=1.63 microM), interleukin-4 (IC50=2.76 microM), tumour necrosis factor-alpha (IC50=0.66 microM), interferon-gamma (IC50=1.35 microM), and interleukin-1 beta (46% at 2.5 microM) and by concomitantly increasing the production of the anti-inflammatory cytokine, interleukin-10. In addition, while demethylnobiletin affected nitric oxide production, it did not modify NOS-2 expression. Finally, demethylnobiletin inhibited proliferation of T cells and induced their apoptosis.

Conclusions and implications: Demethylnobiletin decreased DTH reactions induced by various agents. This finding, along with the fact that the compound has a low toxicity and exhibits several other interesting properties, could pave the way for other structurally related citroflavonoids to be used as pharmacological agents in complementary therapies.

Figure 1

Structure of demethylnobiletin.

Figure 2

Effects of demethylnobiletin (0.5 mg per ear) and dexamethasone (0.025 mg per ear) on DTH reactions induced by oxazolone (a), DNFB (b) and SRBC (c) in mice; n=6 animals; ear swelling increase in μm (a, b); paw volume increase in μl (c). Each measure represents the mean±s.e.mean of the increase in tissue swelling calculated with respect to the same group on day 0, before application of the irritant. Statistical significance of difference from the control ^**P<0.01, ^*P<0.05 by Dunnett's multiple comparison test.

Figure 3

Haematoxylin–eosin-stained section of ears after application of oxazolone (72 h after challenge) (a), DNFB (72 h after challenge) (b) and paws after sensitization with SRBCs (48 h after challenge) (c). (1) Control: ears and paws treated only with oxazolone (a1) or DNFB (b1), and paws treated only with SRBC (c1). (2) Ears treated with demethylnobiletin (DMN) in the DTH induced by oxazolone (a2) or DNFB (b2), and paws treated with demethylnobiletin in the DTH induced by SRBC (c2). (3) Ear treated with dexamethasone (Dexa) in the DTH induced by oxazolone (a3) or DNFB (b3), and paws treated with dexamethasone in the DTH induced by SRBC (c3).

Figure 4

Immunohistochemical study of ears after sensitization with oxazolone (× 40). (a) Control: ear treated only with oxazolone, (b) ear treated with demethylnobiletin and (c) ear treated with dexamethasone. Cells with cytoplasmic staining for COX-2 are marked by an arrow.

Figure 5

Analysis of genomic DNA fragmentation in human lymphocyte cells. Lane 1 corresponds to the standard. Lane 2 corresponds to the DNA ladder of cells treated with PHA (control) and lane 3 to the DNA ladder of non-treated cells (blank). Cells treated with demethylnobiletin (DMN) 25, 10 and 5 μM (lanes 4, 5 and 6) and cycloheximide 30 μM (CHX, lane 7).