Inhibition of Spinal Oxidative Stress by Bergamot Polyphenolic Fraction Attenuates the Development of Morphine Induced Tolerance and Hyperalgesia in Mice - PubMed (original) (raw)

. 2016 May 26;11(5):e0156039.

doi: 10.1371/journal.pone.0156039. eCollection 2016.

Luigino Antonio Giancotti 1, Sara Ilari 1, Concetta Dagostino 2 3, Micaela Gliozzi [ 4](#full-view-affiliation-4 "Institute of Research for Food Safety & Health (IRC_FSH), Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy."), Chiara Morabito [ 5](#full-view-affiliation-5 "Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy."), Valentina Malafoglia 6, William Raffaeli 6, Maurizio Muraca [ 7](#full-view-affiliation-7 "Research Laboratories, Children's Hospital "Bambino Gesù" Research Institute, Rome, Italy."), Bianca M Goffredo [ 7](#full-view-affiliation-7 "Research Laboratories, Children's Hospital "Bambino Gesù" Research Institute, Rome, Italy."), Vincenzo Mollace [ 4](#full-view-affiliation-4 "Institute of Research for Food Safety & Health (IRC_FSH), Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy."), Carolina Muscoli [ 4](#full-view-affiliation-4 "Institute of Research for Food Safety & Health (IRC_FSH), Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.")

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Inhibition of Spinal Oxidative Stress by Bergamot Polyphenolic Fraction Attenuates the Development of Morphine Induced Tolerance and Hyperalgesia in Mice

Filomena Lauro et al. PLoS One. 2016.

Abstract

Citrus Bergamia Risso, commonly known as Bergamot, is a fruit whose Essential Oil and Bergamot Polyphenolic Fraction have numerous medicinal properties. It is also an excellent antioxidant and in this study, for the first time, its potential effect on morphine induced tolerance in mice has been investigated. Our studies revealed that development of antinociceptive tolerance to repeated doses of morphine in mice is consistently associated with increased formation of superoxide, malondialdehyde and tyrosine-nitrated proteins in the dorsal horn of the spinal cord such as the enzyme glutamine synthase. Nitration of this protein is intimately linked to inactivation of its biological function and resulting increase of glutamate levels in the spinal cord. Administration of Bergamot Polyphenolic Fraction (5-50 mg/kg) attenuated tolerance development. This effect was accompanied by reduction of superoxide and malondialdehyde production, prevention of GS nitration, re-establishment of its activity and of glutamate levels. Our studies confirmed the main role of free radicals during the cascade of events induced by prolonged morphine treatment and the co-administration of natural derivatives antioxidant such as Bergamot Polyphenolic Fraction can be an important therapeutic approach to restore opioids analgesic efficacy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist. The commercial affiliation does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1

Fig 1. Morphine dose-response curve analysis.

Acute injection of morphine (3 mg/kg), in mice, produced a significant near-maximal antinociceptive response until 60 minutes. Results are expressed as mean ± SEM for 15 mice;*P<0.001 vs morphine 0 mg/kg.

Fig 2

Fig 2. The development of morphine antinociceptive tolerance is prevented by substances with antioxidant properties.

(A-B) A significant loss to the antinociceptive effect of the acute injection of morphine was observed in animals that received repeated administration of morphine over 4 days. (A) Co-administration of morphine over 4 days with BPF (5–50 mg/kg) inhibited the development of tolerance in a dose-dependent manner. When tested alone, the highest dose of BPF (50 mg/kg) did not have antinociceptive effects. (B) In morphine-treated mice, the effect of BPF (25 mg/kg) was comparable to L-NAME (10 mg/kg) or MnTBAP (10 mg/kg). Results are expressed as mean ± SEM for 15 mice. *P<0.001 vs Naive; †P<0.01 vs morphine; ††P<0.001 vs morphine.

Fig 3

Fig 3. Chronic morphine administration is associated with increased superoxide formation in the L4-L5 portion of spinal cord.

Chronic morphine administration induced an overproduction of O2·- in the spinal cord compared with mice naïve group as demonstrated by HE oxidation. BPF (25 mg/kg; n = 15) or MnTBAP (10 mg/kg) co-administration was able to reduce O2·- chronic morphine-induced increase. Original magnification, ×10. Micrographs are representative of at least 3 from different animals in experiments performed on different days.

Fig 4

Fig 4. MDA assay demonstrated increased lipid peroxidation in tolerant mice.

Spinal cord extract from morphine group demonstrated a significant increase in MDA that was reduced by BPF (25 mg/kg) as well as by MnTBAP (10 mg/kg). Results are expressed as mean ± SEM for 15 rats. *P<0.001 vs Naive; †P<0.01 vs morphine.

Fig 5

Fig 5. Morphine antinociceptive tolerance is associated with nitroxidative stress which is blocked by BPF.

(A) When compared with naive mice, morphine group lead to significant nitration of GS in the L4-L5 portion of the spinal cord tissues as measured by immunoprecipitation. Co-administration of morphine over 4 days with BPF (25 mg/kg) prevented GS nitration. Total protein levels did not change among groups as measured by Western blotting analysis. (B) Posttranslational nitration of GS led to functional enzymatic inactivation as evidenced by increased amount of glutamate. Co-administration of morphine with BPF (25 mg/kg) restored the enzymatic activity of GS. Gels shown are representative of gel results obtained from six animals and of at least three experiments performed on different days. ***P<0.001 vs Naive; †††P<0.001 vs morphine.

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This work was supported by grants from: Programma Operativo Nazionale "Ricerca e Competitività" 2007-2013 (PON "R&C") PON03PE_00078_1 to Vincenzo Mollace; Programma Operativo Nazionale "Ricerca e Competitività" 2007-2013 (PON "R&C") PON03PE_00078_2 to Vincenzo Mollace; Giovani Ricercatori GR-2010-2318370 to Carolina Muscoli. The funder provided support in the form of salaries for authors [FL, LAG, SI], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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