Exploring the Role of Bergamot Polyphenols in Alleviating Morphine-Induced Hyperalgesia and Tolerance through Modulation of Mitochondrial SIRT3

Abstract

Morphine is an important pain reliever employed in pain management, its extended utilize is hindered by the onset of analgesic tolerance and oxidative stress. Long-term morphine administration causes elevated production of reactive oxygen species (ROS), disrupting mitochondrial function and inducing oxidation. Sirtuin 3 (SIRT3), a mitochondrial protein, is essential in modulating ROS levels by regulating mitochondrial antioxidant enzymes as manganese superoxide dismutase (MnSOD). Our investigation focused on the impact of SIRT3 on hyperalgesia and morphine tolerance in mice, as evaluating the antioxidant effect of the polyphenolic fraction of bergamot (BPF). Mice were administered morphine twice daily for four consecutive days (20 mg/kg). On the fifth day, mice received an acute dose of morphine (3 mg/kg), either alone or in conjunction with BPF or Mn (III)tetrakis (4-benzoic acid) porphyrin (MnTBAP). We evaluated levels of malondialdehyde (MDA), nitration, and the activity of SIRT3, MnSOD, glutamine synthetase (GS), and glutamate 1 transporter (GLT1) in the spinal cord. Our findings demonstrate that administering repeated doses of morphine led to the development of antinociceptive tolerance in mice, accompanied by increased superoxide production, nitration, and inactivation of mitochondrial SIRT3, MnSOD, GS, and GLT1. The combined administration of morphine with either BPF or MnTBAP prevented these effects.

Keywords: Sirtuin 3; antioxidants; hyperalgesia; mitochondrial dysfunction; morphine-induced tolerance; oxidative stress; polyphenols; therapeutic approach.

Figure 1

Acute morphine injection. Administration of morphine (3 mg/kg), in mice, generated a considerable near-maximal antinociceptive response persisting for 60 min. Values are reported as mean ± SEM, based on 15 mice; * p < 0.0001 vs. morphine 0 mg/kg.

Figure 2

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. Concurrent administration of morphine with BPF (5–50 mg/kg) (A) or MnTBAP (5–30 mg/kg) (B) over a period of 4 days inhibited the development of tolerance in a dose-dependent manner. * p < 0.001 compared to vehicle (Veh); † p < 0.01; †† p < 0.001 compared to vehicle + morphine.

Figure 3

(A) Repeated administration of morphine for 4 days in mice caused an increased production of superoxide in the spinal cord compared to the control group (vehicle), as demonstrated by the oxidation of HE. Co-administration of morphine and BPF (25 mg/kg) or MnTBAP (10 mg/kg) was able to reduce the increase in ethidium and therefore in superoxide. Original magnification, ×10. Scale Bar 100 µm. Micrographs illustrate results from at least three distinct animals. (B) Persistent morphine treatment induced protein nitration in the spinal cord. Co-administration of morphine with BPF (25 mg/kg) and MnTBAP (10 mg/kg) inhibited nitrotyrosine formation. Original magnification, ×10. Scale Bar 100 µm. Micrographs illustrate results from at least three distinct animals in experiments conducted on separate days.

Figure 4

Increased MDA levels in spinal cord represents the presence of oxidative stress during morphine tolerance in mice. Mice that received morphine for 4 days showed an amount of MDA level. Co-administration of morphine and BPF (25 mg/kg) or MnTBAP (10 mg/kg) resulted in a substantial decrease in MDA. The results are shown as the mean ± SEM for 6 mice. * p < 0.0001 versus vehicle (Veh); † p < 0.01 versus vehicle + morphine.

Figure 5

(A,B) Nitration of GS and GLT1 proteins in spinal cord tissues as assessed by immunoprecipitation. Administering morphine for 4 days in combination with BPF (25 mg/kg) or MnTBAP (10 mg/kg) prevented the nitration of GS and GLT1. In these conditions, actin expression appeared statistically similar across the lanes. The reported data include densitometric analyses for all animals per group. GS, nitrated GS, GLT1 and nitrated GLT1 were first normalized to actin and then these values were used to obtain GS nitrated/GS and GLT1 nitrated/GLT1 ratio. The data are presented as the mean ± SEM for 6 mice; * p < 0.001 versus Veh; † p < 0.001 versus morphine.

Figure 6

Chronic morphine administration in mice induced nitration on mitochondrial proteins as shown by WES methodology. Co-administration of BPF (25 mg/kg) or MnTBAP (10 mg/kg) attenuated mitochondrial proteins nitration. No statistically significant difference for the TOM20 value was identified in the lanes under these conditions. (A,B) Lanes and (D) electropherogram are representative of the results from six animals; (blue lanes: morphine groups; pink lanes: BPF group; green lanes: MnTBAP group; and light grey: vehicle group). (C) The reported data include densitometric analyses for all animals per group. The results are presented as the mean ± SEM for six mice. * p < 0.05 versus Veh; † p < 0.05 versus vehicle + morphine.

Figure 7

(A) Nitration of MnSOD protein in spinal cord tissues as assessed by immunoprecipitation. Combined treatment with morphine and BPF (25 mg/kg) or MnTBAP (10 mg/kg), for four consecutive days, prevented MnSOD nitration. In these conditions, prohibitin expression appeared statistically similar across the lanes. Densitometric analyses for all animals in each group are reported. MnSOD and nitrated MnSOD were first normalized with prohibitin and then these values were used to obtain MnSOD nitrated/MnSOD ratio. (B) Nitration on MnSOD is linked to inactivation of its biological function, which is restored following the administration of BPF (25 mg/kg) or MnTBAP (10 mg/kg). The results are presented as the mean ± SEM for six mice; * p < 0.001 compared to Veh; † p < 0.001 compared to morphine.

Figure 8

(A) Nitration of SIRT3 protein in spinal cord tissues is identified by immunoprecipitation. Combined treatment with morphine and BPF (25 mg/kg) or MnTBAP (10 mg/kg), for 4 consecutive days, blocked SIRT3 nitration. Prohibitin levels appeared statistically similar across the lanes. Densitometric analyses for all animals in each group are reported. MnSOD and nitrated MnSOD were initially normalized using prohibitin, and these values were then utilized to calculate the MnSOD nitrated/MnSOD ratio. (B) SIRT3 activation, expressed in arbitrary fluorescence units (AFU), is restored following the administration of BPF (25 mg/kg) or MnTBAP (10 mg/kg). The results are presented as the mean ± SEM for six mice; * p < 0.001 compared to Veh; † p < 0.001 compared to morphine.

Figure 9

(A) SIRT3 inhibition induces acetylation on mitochondrial proteins during morphine tolerance in mice as shown by WES methodology. Co-administration of BPF (25 mg/kg) or MnTBAP (10 mg/kg) attenuated mitochondrial proteins acetylation. No statistically significant difference in TOM20 value was observed between the lanes under these conditions. (A,B) Lanes and (D) electropherogram are representative of results from six animals (green lanes: morphine groups; blue lanes: BPF group; dark grey lanes: MnTBAP group; and light grey lanes: vehicle group). (C) Densitometric analyses for all animals in each group are reported. Values are presented as the mean ± SEM for six mice. * p < 0.05 vs Veh; † p < 0.05 vs. vehicle + morphine.