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. 2017 May;174(9):867-879.
doi: 10.1111/bph.13760. Epub 2017 Mar 23.

Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti-inflammatory effects of eucalyptol

Ana I Caceres  1 , Boyi Liu  1   2 , Sairam V Jabba  1 , Satyanarayana Achanta  1 , John B Morris  3 , Sven-Eric Jordt  1   4
Affiliations

Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti-inflammatory effects of eucalyptol

Ana I Caceres et al. Br J Pharmacol. 2017 May.

Abstract

Background and purpose: Eucalyptol (1,8-cineol), the major ingredient in the essential oil of eucalyptus leaves and other medicinal plants, has long been known for its anti-inflammatory properties. Eucalyptol interacts with the TRP cation channels among other targets, but it is unclear which of these mediates its anti-inflammatory effects.

Experimental approach: Effects of eucalyptol were compared in wild-type and TRPM8 channel-deficient mice in two different models: footpad inflammation elicited by complete Freund's adjuvant (CFA) and pulmonary inflammation following administration of LPS. Oedema formation, behavioural inflammatory pain responses, leukocyte infiltration, enzyme activities and cytokine and chemokine levels were measured.

Key results: In the CFA model, eucalyptol strongly attenuated oedema and mechanical allodynia and reduced levels of inflammatory cytokines (IL-1β, TNF-α and IL-6), effects comparable with those of ibuprofen. In the LPS model of pulmonary inflammation, eucalyptol treatment diminished leukocyte infiltration, myeloperoxidase activity and production of TNF-α, IL-1β, IFN-γ and IL-6. Genetic deletion of TRPM8 channels abolished the anti-inflammatory effects of eucalyptol in both models. Eucalyptol was at least sixfold more potent on human, than on mouse TRPM8 channels. A metabolite of eucalyptol, 2-hydroxy-1,8-cineol, also activated human TRPM8 channels.

Conclusion and implications: Among the pharmacological targets of eucalyptol, TRPM8 channels were essential for its anti-inflammatory effects in mice. Human TRPM8 channels are more sensitive to eucalyptol than rodent TRPM8 channels explaining the higher potency of eucalyptol in humans. Metabolites of eucalyptol could contribute to its anti-inflammatory effects. The development of more potent and selective TRPM8 agonists may yield novel anti-inflammatory agents.

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Figures

Figure 1
Figure 1
Effects of eucalyptol on CFA‐induced paw swelling in mice. (A) Schedule of treatment and experimental procedures. Paw oedema (% increase in paw volume) was measured by plethysmography directly (0 h), 2, 4, 24 and 48 h after injection of CFA (10 μL per paw) into the hind paw. Vehicle (corn oil), eucalyptol (30, 100 or 300 mg·kg−1) or ibuprofen (30 mg·kg−1) were injected (i.p.) 30 min before testing time points at 0, 24 and 48 h and in between at 6 and 30 h after CFA injection (total of five injections). (B) Percent increase in paw volume of CFA‐injected mice treated (i.p.) with eucalyptol (Euc) doses of 30, 100 or 300 mg·kg−1 or vehicle (Veh). The control group (Control) received saline injection into the hind paw instead of CFA and treatment with vehicle (corn oil) thereafter. n = 6–8 mice per group. # P < 0.05, significantly different from control group; *P < 0.05, significantly different from CFA + Veh group, one‐way ANOVA.
Figure 2
Figure 2
Comparison of the effects of eucalyptol on CFA‐induced paw oedema in wild‐type and Trpm8−/− mice, with animals treated as in Figure 1A. (A) Representative photographs of CFA‐induced paw oedema in wild‐type (top row) or Trpm8−/− mice (bottom row) injected i.p. with vehicle (Veh, corn oil) or 300 mg·kg−1 eucalyptol (Euc), taken 50 h after intraplantar CFA injection. (B) Time course of CFA‐induced paw oedema in wild‐type mice, treated with vehicle, eucalyptol (300 mg·kg−1) or the reference drug ibuprofen (Ibu, 30 mg·kg−1). Black arrows indicate the time points of treatment. (C) Time course of CFA‐induced paw oedema in Trpm8−/− mice. (D) Comparisons of weights of paw biopsies from wild‐type and Trpm8−/− mice of control group (no CFA), CFA groups treated with vehicle, eucalyptol or ibuprofen respectively. Tissues were collected 50 h after CFA injection from mice tested in Figure 2B, C. n = 6–8 mice per group. # P < 0.05, significantly different from control group, *P < 0.05, significantly different from CFA + Veh group, NS, no significance (P > 0.05), one‐way ANOVA.
Figure 3
Figure 3
Effects of eucalyptol on CFA‐induced mechanical allodynia in wild‐type and Trpm8−/− mice, measured by von Frey hair analysis (administration as in Figure 1A). Testing was performed 26 h after CFA injection, 2 h after the mice received the third treatment of vehicle (Veh, corn oil), eucalyptol (Euc, 300 mg·kg−1) or ibuprofen (Ibu, 30 mg·kg−1). # P < 0.05, significantly different from control group; *P < 0.05, significantly different from CFA + Veh group, NS, no significance (P > 0.05), one‐way ANOVA. n = 6–12 mice per group.
Figure 4
Figure 4
Influence of eucalyptol treatment on CFA‐induced inflammatory cytokines in hind paw skin of wild‐type and Trpm8−/− mice. Mice were treated (i.p.) with vehicle (Veh, corn oil), eucalyptol (Euc, 300 mg·kg−1) or ibuprofen (Ibu, 30 mg·kg−1) as in Figure 1A. Hind paw biopsies were collected 50 h after CFA injection, and levels of IL‐1β, IL‐6 and TNF‐α were determined by ELISA in tissue homogenates. # P < 0.05, significantly different from control group; *P < 0.05, significantly different from CFA + Veh group, NS, no significance (P > 0.05), one‐way ANOVA. n = 6–12 mice per group.
Figure 5
Figure 5
TRPM8‐dependent suppression of LPS‐induced pulmonary inflammation by eucalyptol. (A) LPS administration and eucalyptol treatment timeline. (B) Comparison of cell differential counts (total cells, neutrophils, lymphocytes and macrophages) in BALF of wild‐type or Trpm8−/− mice exposed to vehicle (saline) or LPS and injected with treatment vehicle (corn oil) or eucalyptol (200 mg·kg−1). (C) Cytokine levels in BALF of mice in Figure 5B, tested by bead array multiplex analysis. Protein concentrations are shown for TNF‐α, IFN‐g, KC, IL‐6 and G‐CCSF. (n = 4–8 mice per group). *P < 0.05, WT + LPS significantly different from WT + LPS + Eucalyptol.
Figure 6
Figure 6
Cytokine transcripts analysed by quantitative real time qPCR in cDNA from whole lungs of LPS‐ and vehicle‐exposed mice treated and untreated with eucalyptol. (A) Impaired induction of cytokines in Trpm8−/− mice treated with eucalyptol before and after intranasal LPS administration. (B) Unaltered expression of cytokines in Trpm8−/− mice treated with eucalyptol before and after LPS exposure. (n = 4–6 mice per group). *P < 0.05, significantly different as indicated.
Figure 7
Figure 7
Comparison of TRPV4 and TRPM8 expression in lung and sensory ganglia. Averaged relative quantities (RQ) of TRPV4 and TRPM8 transcript levels were measured by real‐time Taqman PCR of cDNA from whole lung, DRG and TG of wild‐type mice. We used one TRPV4 Taqman probe (mTRPV4: Mm00499025_m1, 25) and two mTRPM8 probes covering different cDNA segments (mTrpm8: Mm01299593_m1, 93 and mTrpm8: Mm00454566_m1, 66). Actin was used as reference gene (n = 4 mice per group).
Figure 8
Figure 8
Comparison of eucalyptol sensitivities of TRPM8 channel species orthologues (human, mouse and rat) and determination of sensitivity of human TRPM8 channels to a eucalyptol metabolite, measured by Ca2+ fluorimetry in HEK293T cells. (A) Concentration response analysis of eucalyptol activation of human TRPM8 channels,with EC50 values as shown. (B) Concentration–response relationship of human TRPM8 channel activation by 2‐hydroxy‐1,8‐cineol. (C) Dose–response relationship of eucalyptol activation of human, mouse and rat TRPM8. The estimated EC50,values are shown in the Figure.
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References

    1. Alexander SPH, Catterall WA, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015). The Concise Guide to PHARMACOLOGY 2015/16: Voltage‐gated ion channels. Br J Pharmacol 172: 5904–5941. - PMC - PubMed
    1. Barr A (1988). Traditional Bush Medicines: An Aboriginal Pharmacopoeia: Aboriginal Communities of the Northern Territory of Australia. Greenhouse Publications: Richmond, Victoria, Australia.
    1. Bastos VP, Brito TS, Lima FJ, Pinho JP, Lahlou S, Abreu Matos FJ et al. (2009). Inhibitory effect of 1,8‐cineole on guinea‐pig airway challenged with ovalbumin involves a preferential action on electromechanical coupling. Clin Exp Pharmacol Physiol 36: 1120–1126. - PubMed
    1. Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, Stucky CL et al. (2007). The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448: 204–208. - PubMed
    1. Bautista DM, Pellegrino M, Tsunozaki M (2013). TRPA1: a gatekeeper for inflammation. Annu Rev Physiol 75: 181–200. - PMC - PubMed

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