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Eucalyptol alleviates inflammation and pain responses in a mouse model of gout arthritis - PubMed

. 2020 May;177(9):2042-2057.

doi: 10.1111/bph.14967. Epub 2020 Feb 12.

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Eucalyptol alleviates inflammation and pain responses in a mouse model of gout arthritis

Chengyu Yin et al. Br J Pharmacol. 2020 May.

Abstract

Background and purpose: Gout arthritis, which is provoked by monosodium urate (MSU) crystal accumulation in the joint and periarticular tissues, induces severe pain and affects quality of life of the patients. Eucalyptol (1,8-cineol), the principal component in the essential oils of eucalyptus leaves, is known to possess anti-inflammatory and analgesic properties. We aimed to examine the therapeutic effects of eucalyptol on gout arthritis and related mechanisms.

Experimental approach: A mouse model of gout arthritis was established via MSU injection into the ankle joint. Ankle oedema, mechanical allodynia, neutrophil infiltration, oxidative stress, NLRP3 inflammasome, and TRPV1 expression were examined.

Key results: Eucalyptol attenuated MSU-induced mechanical allodynia and ankle oedema in dose-dependently, with effectiveness similar to indomethacin. Eucalyptol reduced inflammatory cell infiltrations in ankle tissues. Eucalyptol inhibited NLRP3 inflammasome activation and pro-inflammatory cytokine production induced by MSU in ankle tissues in vivo. Eucalyptol reduced oxidative stress induced by MSU in RAW264.7 cells in vitro as well as in ankle tissues in vivo, indicated by an increase in activities of antioxidant enzymes and reduction of ROS. Eucalyptol attenuated MSU-induced up-regulation of TRPV1 expression in ankle tissues and dorsal root ganglion neurons innervating the ankle. The in vivo effects of eucalyptol on ankle oedema, mechanical allodynia, NLRP3 inflammasome, IL-1β, and TRPV1 expression were mimicked by treating MSU-injected mice with antioxidants.

Conclusion and implications: Eucalyptol alleviates MSU-induced pain and inflammation via mechanisms possibly involving anti-oxidative effect. Eucalyptol and other antioxidants may represent promising therapeutic options for gout arthritis.

© 2019 The British Pharmacological Society.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1

The structure of eucalyptol and experimental protocols. (a) The molecular structure of eucalyptol. (b) in vitro experiment protocol: RAW264.7 cells were seeded in six‐well plates. Eucalyptol (0.1–10 μM) was applied 20 min before MSU stimulation (0.5 mg·ml−1). Cells were incubated for 4 hr, and then MTT or ROS assay was performed. (c) in vivo experiment protocol: MSU (0.5 mg/20 μl) or PBS (20 μl) was injected into the ankle joint to establish the gout arthritis model or control group. Mechanical hypersensitivity and ankle oedema were measured at 0, 2, 4, 6, 8, 24, and 48 hr after model establishment. Eucalyptol (30, 100, 300, or 600 mg·kg−1), indomethacin (10 mg·kg−1), or vehicle (corn oil) were injected i.p., 1 hr before MSU injection and 5, 23, and 47 hr after model establishment for a total of four times. For tissue analysis, mice were killed 24 hr after model establishment, and ankle joint pathological analysis, MPO assay, qPCR, western blot, and antioxidant activity were assayed

Figure 2
Figure 2

Effects of eucalyptol on inflammation and pain in MSU‐induced gout arthritis in mice. (a) Representative photographs of ankles 24 hr after MSU injection. Black arrow indicates the injected ankle. (b) Time course of the effects of indomethacin (Indo) and different dosages of eucalyptol (Euca) on ankle oedema. n = 6 mice per group. (c) Normalized AUC of (b). (d) Time course of the effects of indomethacin and different doses of eucalyptol on mechanical allodynia of the hind paw. n = 6 mice per group. (e) Normalized AUC of (d). *P < .05, significantly different from Control group; # P < .05, significantly different from MSU + Veh group; NS: not significantly different from MSU + Veh group; one‐way or two way ANOVA followed by Tukey's post hoc test

Figure 3
Figure 3

Effects of eucalyptol on inflammatory cell infiltration in ankle joint tissues of MSU‐induced gout arthritis mice. (a) Representative microscopic photos of mice ankle tissue sections from Control, MSU + Veh, MSU + Eucalyptol (MSU + Euca), and MSU + Indomethacin (MSU + Indo) groups. (b) Summarized data showing the number of infiltrated inflammatory cells per observation field. Control group was taken as 100%. (c) Summarized data showing the myeloperoxidase (MPO) activity determined in ankle tissue samples. n = 6 mice per group. *P < .05, significantly different from Control group; # P < .05, significantly different from MSU + Veh group; one‐way ANOVA followed by Tukey's post hoc test

Figure 4
Figure 4

Eucalyptol inhibited NLRP3 inflammasome activation in ankle joint tissues of MSU‐induced gout arthritis mice. (a–c) Summarized data showing the expression of Nlrp3 (a), Caspase‐1 (b), and Il‐1β (c) genes in Control, MSU + Veh, MSU + Euca, and MSU + Indo groups determined by qPCR in mice ankle tissues 24 hr after MSU injection. n = 6 mice per group. (d–f) NLRP3 (d), caspase‐1 (e), and IL‐1β (f) protein expressions determined by western blotting in mice ankle tissues 24 hr after MSU injection. n = 5 mice per group for NLRP3 group. n = 6 mice for caspase‐1 and IL‐1β groups. Upper panel shows representative images of NLRP3, caspase‐1, IL‐1β, and β‐actin protein expression from Control, MSU + Veh, MSU + Euca, and MSU + Indo groups. Lower panel shows summarized NLRP3, caspase‐1, and IL‐1β protein expression normalized to β‐actin. *P < .05, significantly different from Control group; # P < .05, significantly different from MSU + Veh group;. one‐way ANOVA followed by Tukey's post hoc test

Figure 5
Figure 5

Eucalyptol suppresses MSU‐induced oxidative stress in RAW264.7 cells. (a) Upper panel: representative pictures showing cellular oxidative stress in RAW264.7 cells induced by 0.1, 0.5, and 1.0 mg·ml−1 MSU for 4 hr determined by DCF under fluorescence microscope. Lower panel: summarized results DCF fluorescence intensity measured by microplate reader. *P < .05 significantly different from Control group (no MSU added). NS: not significantly different from Control group. (b) Representative photographs showing the cellular oxidative stress induced by MSU in RAW264.7 cells treated with different dosages of eucalyptol (Euca; 0.1, 1, 5, and 10 μM) determined by DCF. (c) Summarized results of DCF fluorescence intensity changes in groups treated with different dosages of eucalyptol. DCF fluorescence intensity was normalized to that of the vehicle‐treated group (no MSU, no eucalyptol added) and shown as % of relative ROS. (d) RAW264.7 cells were treated with 0.1, 1, 5, or 10 μM eucalyptol for 4 hr, and cell viability was determined by MTT assay; n = 6 wells per group. Scale bar indicates 20 μm. *P < .05, significantly different from Control group (no MSU, no eucalyptol added); # P < .05, significantly different from MSU + Veh group; NS: not significantly different from MSU + Veh group; one‐way ANOVA followed by Tukey's post hoc test

Figure 6
Figure 6

Eucalyptol suppressed oxidative stress in ankle joint tissues of MSU‐induced gout arthritis mice. (a–c) Summarized data showing SOD activity (a), GSH‐Px activity (b), and MDA content (c) determined in mice ankle tissues 24 hr after MSU injection (d) Summarized data showing the expression of Nrf2 gene in Control, MSU + Veh, MSU + Euca, and MSU + Indo groups determined by qPCR in mice ankle tissues 24 hr after MSU injection. (e) Nrf2 protein expression determined by western blotting in mice ankle tissues 24 hr after MSU injection. Upper panel shows representative images of Nrf2 and β‐actin protein expression from Control, MSU + Veh, MSU + Euca, and MSU + Indo groups. Lower panel shows summarized Nrf2 protein expression normalized to β‐actin. n = 5 mice per group. *P < .0,5 significantly different from Control group; # P < .05, significantly different from MSU + Veh group; NS: not significantly different from MSU + Veh group; one‐way ANOVA followed by Tukey's post hoc test

Figure 7
Figure 7

Effects of eucalyptol on pro‐inflammatory cytokine and chemokine expression in ankle joint tissues of MSU‐induced gout arthritis mice. (a–d) Summarized data showing the expression of Tnf‐α (a), Il‐6 (b), Cxcl1 (c), and Cxcl2 (d) genes in Control, MSU + Veh, MSU + Euca, and MSU + Indo groups determined by qPCR in mice ankle joint tissues 24 hr after MSU injection; n = 6 mice per group. *P < .05, significantly different from Control group; # P < .05 , significantly different from MSU + Veh group; NS: not significantly different from MSU + Veh group; one‐way ANOVA followed by Tukey's post hoc test

Figure 8
Figure 8

Effects of eucalyptol on expression of TRPV1 channels in ankle joint tissues and DRG neurons of MSU‐induced gout arthritis mice. (a, b) Western blot determination of TRPV1 protein expression in ankle joint tissues (a) and DRGs (b) of Control, MSU + Veh, and MSU + Euca groups of mice. Upper panel shows representative images of TRPV1 and β‐actin protein expression from Control, MSU + Veh, and MSU + Euca groups. Lower panel shows summarized TRPV1 protein expression normalized to β‐actin. (c) Representative immunofluorescence images indicating TRPV1 staining in DRG neurons from Control, MSU + Veh, and MSU + Euca groups of mice. Areas staining positive for TRPV1 protein are shown in green. DRGs were co‐stained with the pan‐neuronal marker NeuN (red) to identify DRG neurons. Scale bar indicates 100 μm. (d) Summary of the normalized % increase in fluorescence intensity of TRPV1 staining in the observation field as in panel (c). (e) Summary of the % of TRPV1 positively stained neurons (TRPV1+) from each observation field among all NeuN+ neurons. The total number of DRG neurons per field was calculated by positive NeuN staining. n = 5 mice per group. *P < .05, significantly different from Control group; # P < .05, significantly different from MSU + Veh group; one‐way ANOVA followed by Tukey's post hoc test

Figure 9
Figure 9

Effects of the antioxidants NAC and Tempol on NLRP3 inflammasome activation, IL‐1β production, and TRPV1 overexpression in ankle joint tissues of MSU‐treated mice. (a–c) Summarized data showing the MDA content (a), SOD activity (b), and GSH‐Px activity (c) determined in mice ankle tissues 24 hr after MSU injection. (d–g) The protein expression of NLRP3 (d), caspase‐1 (e), IL‐1β (f), and TRPV1 (g) determined by western blotting in mice ankle tissues 24 hr after MSU injection. n = 6 mice per group. Upper panel displays the representative images of NLRP3, caspase‐1, IL‐1β, TRPV1, and β‐actin protein expression from Control, MSU + Veh, MSU + NAC, and MSU + Tempol groups. Lower panel illustrates the corresponding summarized data normalized to β‐actin. *P < .05, significantly different from Control group; # P < .05, significantly different from MSU + Veh group; one‐way ANOVA followed by Tukey's post hoc test

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