The Circadian Hormone Melatonin Inhibits Morphine-Induced Tolerance and Inflammation via the Activation of Antioxidative Enzymes

Abstract

Opioids are commonly prescribed for clinical pain management; however, dose-escalation, tolerance, dependence, and addiction limit their usability for long-term chronic pain. The associated poor sleep pattern alters the circadian neurobiology, and further compromises the pain management. Here, we aim to determine the correlation between constant light exposure and morphine tolerance and explore the potential of melatonin as an adjuvant of morphine for neuropathic pain treatment.

Methods: Wistar rats were preconditioned under constant light (LL) or a regular light/dark (LD) cycle before neuropathic pain induction by chronic constriction injury. An intrathecal (i.t.) osmotic pump was used for continued drug delivery to induce morphine tolerance. Pain assessments, including the plantar test, static weight-bearing symmetry, and tail-flick latency, were used to determine the impact of the light disruption or exogenous melatonin on the morphine tolerance progression.

Results: constant light exposure significantly aggravates morphine tolerance in neuropathic rats. Continued infusion of low-dose melatonin (3 μg/h) attenuated morphine tolerance in both neuropathic and naïve rats. This protective effect was independent of melatonin receptors, as shown by the neutral effect of melatonin receptors inhibitors. The transcriptional profiling demonstrated a significant enhancement of proinflammatory and pain-related receptor genes in morphine-tolerant rats. In contrast, this transcriptional pattern was abolished by melatonin coinfusion along with the upregulation of the Kcnip3 gene. Moreover, melatonin increased the antioxidative enzymes SOD2, HO-1, and GPx1 in the spinal cord of morphine-tolerant rats.

Conclusion: Dysregulated circadian light exposure significantly compromises the efficacy of morphine's antinociceptive effect, while the cotreatment with melatonin attenuates morphine tolerance/hyperalgesia development. Our results suggest the potential of melatonin as an adjuvant of morphine in clinical pain management, particularly in patients who need long-term opioid treatment.

Keywords: DREAM; Kcnip3; antioxidants; antioxidative enzyme; chronic constriction injury; circadian rhythms; melatonin; morphine tolerance; neuropathic pain.

Figure 1

Circadian light disruption in rats with neuropathic pain. Rats were randomly preconditioned and housed in either a regular light/dark cycle (LD) or constant light (LL) room. The baseline paw withdrawal threshold (A) and weight-bearing test (B) were measured before chronic constriction injury of sciatic nerve (CCI) surgery and on day 3 post-CCI as the basal value before morphine treatment. Morphine (MOR) was infused (15 μg/h, i.t.) to CCI rats under both LD and LL cycles on day 4 (CCI-MOR(LD) and CCI-MOR(LL)) to induce tolerance. Pain behavior was assessed on days 5, 7, and 10 post-CCI surgery. The sham-operated rats under LD and LL circadian cycles served as non-neuropathic controls (Sham-LD and Sham-LL). * denotes a statistically significant difference between CCI + MOR(LD) vs CCI + MOR(LL); * p < 0.05.

Figure 2

Low-dose melatonin coinfusion with morphine reduces morphine tolerance progression. (A) Continuous intrathecal melatonin (Mela, 3 μg/h) or solvent-DMSO infused via an osmotic pump. Antinociceptive tail-flick latencies (52 ℃ water) were measured 1 day before (−1), 1 day after (+1), and 5 days after (+5) after osmotic pump implantation (day 0). (B) After 5 days of i.t. pump infusion, rats were further challenged with 15 μg of morphine (i.t.), and the tail-flick test was performed every 30 min for 120 min. (C) Continuous administration of 3 μg/h Mela, melatonin metabolite N-Acetyl-N-formyl-5-methoxykynurenamine (AFMK), or solvent-DMSO in morphine (MOR)-induced tolerant rats. Tail-flick test was measured before and 1 and 5 days after pump implantation (day 0) for morphine tolerance measurement. (D) After 5 days of i.t. infusion, rats were further challenged with morphine (15 μg, i.t.), and the tail-flick test was performed for 120 min to validate the tolerance. * denotes statistically significant differences between saline + DMSO and MOR + DMSO, # denotes statistically significant differences between MOR + Mela and MOR + DMSO, and “a” denotes statistically significant differences between MOR + AFMK and MOR + DMSO: # p < 0.05; ##/aa p < 0.01; ###/***/aaa p < 0.001.

Figure 3

Melatonin protects morphine tolerance via an MT receptor-independent pathway. After five days of continuous melatonin administration (3 μg/h, i.t.) with morphine (15 μg/h) (MOR + Mela) infusion, the basal tail-flick was measured before (A) 10 μg of luzindole or 5 μg of 4P-PDOT i.t. injection for 60 min. (B) After luzindole or 4P-PDOT i.t. injection to MOR + Mela rats was further challenged with morphine (15 μg, i.t.), the tail-flick test was performed at 0–120 min to validate the morphine responsiveness. * denotes statistically significant differences between MOR + Mela and MOR + DMSO. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Melatonin cotreatment upregulated antioxidative enzymes in morphine-tolerant rat spinal cord. (A) Spinal cord (dorsal horn) total protein was collected from rats treated with saline alone, morphine (15 μg/h, MOR) alone, MOR combined with melatonin (3 μg/h) (MOR + Mela), or AFMK (3 μg/h) (MOR + AFMK). Relative protein levels of NOX2, SOD2, HO-1, GPx1, and GAPDH were determined by Western blot analysis. (B) Densitometry of Western blot. Each probe signal was normalized to GAPDH, and fold change was calculated relative to the saline control. Statistics were analyzed from four independent samples of each group. * denotes statistically significant differences compared to saline + DMSO. * p < 0.05; ** p < 0.01; *** p < 0.001. # denotes statistically significant differences compared to MOR + DMSO. # p < 0.05; ## p < 0.01.

Figure 5

Normal saline + DMSO, MOR (15 μg/h) + DMSO, or MOR + Mela (3 μg/h) was administrated via i.t. infusion to CCI rats in a normal LD cycle room. The paw withdrawal threshold (A) and weight-bearing test (B) were measured before CCI surgery as a baseline and at day 3 post-CCI as the treatment baseline. After osmotic pump implantation at day 4 post-CCI, the behavior tests were measured at days 5, 7, and 10 post-CCI surgery. * denotes statistically significant differences between CCI-MOR + DMSO and CCI-saline + DMSO. # denotes statistically significant differences between CCI-MOR + Mela and CCI-saline + DMSO. “a” denotes statistically significant differences between CCI-MOR + Mela and CCI-MOR + DMSO. #/a p < 0.05; **/aa p < 0.05; ***/### p < 0.001.