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. 2019 Feb 19;26(1):21.
doi: 10.1186/s12929-019-0513-1.

Effects of glucosamine against morphine-induced antinociceptive tolerance and dependence in mice

Faezeh Basiri  1 , Abolfazl Rad  2 , Davood Mahdian  2 , Mehdi Molavi  3 , Bahareh Amin  4
Affiliations

Effects of glucosamine against morphine-induced antinociceptive tolerance and dependence in mice

Faezeh Basiri et al. J Biomed Sci. .

Abstract

Background: The most important limitations of morphine in pain therapy are its tolerance and dependence. In this study, we evaluated the protective effect of glucosamine against morphine-induced tolerance and dependence in mice.

Methods: Mice received twice daily morphine (20 mg/kg, s.c.) alone, or along with orally administered glucosamine (500, 1000 and 2000 mg/kg), for 9 continuous days. To assess antinociceptive effect of morphine, percentage of maximal possible effect (%MPE) of animals exposed to thermal stimulus was measured in the hot plate test, 30 min after morphine administration. Test was performed on days 1, 3, 5, 7 and 9. The effect of glucosamine on the naloxone (5 mg/kg, i.p.)-precipitated morphine withdrawal, was also evaluated. Changes in brain gene expression levels of induced nitric oxide synthase (iNOS), enzyme responsible for nitric oxide generation, as well as pro-inflammatory mediator, tumor necrosis alpha (TNF-α) were measured in morphine tolerated animals, as well as after withdrawal by real-time polymerase chain reaction (RT-PCR). Protein content of TNF-α was evaluated via ELISA assay.

Results: Tolerance to antinociceptive effect of morphine was developed after 7 days of morphine treatment. The concurrent administration of glucosamine (500, 1000 and 2000 mg/kg) with morphine, significantly inhibited tolerance development, on days 7 and 9. In addition, glucosamine ameliorated the naloxone-precipitated opioid withdrawal symptoms (tremor, jumping, teeth chattering, grooming). However, diarrhea was significantly improved only with the dose of 500 mg/kg. Increased mRNA expression of iNOS as well as TNF-α mRNA expression and protein, after both morphine tolerance and withdrawal, were considerably reduced by glucosamine (1000 mg/kg) in the morphine withdrawal animals.

Conclusion: These data support the utility of glucosamine in attenuating both tolerance to nociceptive effects of morphine as well as withdrawal-induced behavioral profile. Anti-oxidant and anti-inflammatory effects are responsible, at least in part, for the protective effects of this drug.

Keywords: Dependence; Glucosamine; Mice; Morphine; Tolerance.

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Conflict of interest statement

Ethics approval and consent to participate

All applied procedures were approved by the Animal Care and Use Committee of the Sabzevar University of Medical Sciences, Sabzevar, Iran (ir.medsab.rec.v1394.126).

Consent to publication

N/A

Competing interests

The authors declare that there are no competing of interest.

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Figures

Fig. 1
Fig. 1
Time course effect of glucosamine on the development of tolerance to morphine (20 mg/kg, s.c., twice daily), during 9 days in mice. a: maximum possible effect (%), b: Paw withdrawal latency. Glucosamine was administered via gavage (500, 1000 and 2000 mg/kg, twice daily, for 9 days). Analgesia was measured 30 min after the treatment, on the hot plate apparatus. Values are expressed as mean ± SEM (n = 6). Statistical analyses was performed by mixed model ANOVA with repeated measure and post hoc bonferroni for evaluating antinociceptive activity. *p < 0.05, **p < 0.01 comparison between morphine and control (normal saline) groups. #p < 0.05, ##p < 0.01, ###p < 0.001 treated glucosamine+morphine as compared to morphine treated group. +p < 0.05, ++p < 0.01, treated glucosamine+morphine as compared to control (normal saline) group
Fig. 2
Fig. 2
Effect of glucosamine on the development of naloxone (5 mg/kg, i.p.)-induced morphine withdrawal symptoms, 2 h after last injection of morphine. Glucosamine was administered via gavage (500, 1000 and 2000 mg/kg, twice daily, for 9 days). a: Duration of tremor; b: Number of jumping; c: Number of teeth chattering; d: Number of grooming; e: Percent of animals with diarrhea. Values are expressed as means ± SEM. One way ANOVA and post hock sidak was used. *p < 0.05, ***p < 0.001 as compared to control group; #p < 0.05, and ###p < 0.001 as compared to morphine treated group
Fig. 3
Fig. 3
Effect of gavage glucosamine (1000 mg/kg, twice daily, for 9 days), on the mRNA levels of iNOS (induced nitric oxide synthase) in both morphine tolerated mice and morphine-dependent mice received naloxone (5 mg/kg, i.p.) challenge. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a reference gene and was used for normalization as an internal control. Results (mean ± SEM) are expressed in terms of relative quantification (fold-change) using the 2-ΔΔCT method for n = 4. One way ANOVA and post hock Tukey was used *p < 0.05, ***p < 0.001 vs. normal saline control group; #p < 0.05 morphine+glucosamine vs. morphine treated group. $p < 0.05 morphine+glucosamine+naloxane vs. morphine+naloxane treated group
Fig. 4
Fig. 4
Effect of gavage glucosamine (1000 mg/kg, twice daily, for 9 days), on the A: mRNA levels of the TNF-α (tumor necrosis alpha) in both morphine tolerated and morphine-dependent mice received naloxone (5 mg/kg, i.p.) challenge. B: Protein levels of the TNF-α in morphine tolerated and morphine-dependent mice received naloxone (5 mg/kg, i.p.) challenge by ELISA assay. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a reference gene and was used for normalization as an internal control. Results (mean ± SEM) are expressed in terms of relative quantification (fold-change) using the 2-ΔΔCT method for n = 5. One way ANOVA and post hock Tukey was used. **p < 0.01, ***p < 0.001 vs. normal saline control group; #p < 0.05, ##p < 0.01 morphine+glucosamine vs. morphine treated group. $p < 0.05, $$p < 0.01 morphine+glucosamine+naloxane vs. morphine+naloxane treated group
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