The effect of clavulanic acid on the development of tolerance and dependence induced by morphine in mice: role of DRD2 and NR1 genes, oxidative stress, and GDNF protein.

Morphine tolerance and dependence are major challenges in opioid addiction treatment. Clavulanic acid (CA), a non-antibiotic β-lactam, has recently garnered attention for its potential neuromodulatory properties. This research was conducted to assess the impact of CA on the induction of tolerance and dependence following morphine administration, as well as to examine its modulatory effects on the gene expression levels of DRD2 and NR1, nitric oxide (NO) and malondialdehyde (MDA) concentrations, and the expression of GDNF protein in the brain of mice. Forty-nine adult male outbred mice (25-30 g) were randomly allocated to seven groups (n = 7). Morphine tolerance/dependence was induced using an escalating subcutaneous regimen for 3 consecutive days (50 mg/kg at 08:00, 50 mg/kg at 12:00, and 75 mg/kg at 16:00). Clavulanic acid (50, 100, or 200 mg/kg), clonidine (0.1 mg/kg; positive control), or normal saline was administered 30 min before each morphine injection. Antinociceptive tolerance was assessed by the tail-flick test following an acute morphine challenge (10 mg/kg, sc) on days 1 and 4. On day 4, locomotor activity was evaluated using the open-field test, and physical dependence was assessed by naloxone-precipitated jumping (5 mg/kg, ip). Brain DRD2 and NR1 mRNA levels were quantified by qRT-PCR; DRD2 and GDNF protein expression was measured by Western blotting, and oxidative stress indices (MDA and NO metabolites) were determined using TBARS and Griess assays, respectively. Repeated morphine administration induced tolerance compared with the control (P < 0.05). Treatment with CA significantly inhibited the development of morphine tolerance at doses of 50 (P < 0.05), 100 (P < 0.01), and 200 mg/kg (P < 0.05) and also suppressed morphine dependence notably at the dose of 200 mg/kg (P < 0.05). Furthermore, administration of CA markedly decreased the relative expression of DRD2 (P < 0.001) and NR1 (P < 0.05) genes and lowered the nitric oxide (NO) levels (P < 0.01) compared to the morphine-treated group. In addition, co-administration of CA significantly increased the expression level of GDNF protein (P < 0.01) in the brain of morphine-treated mice but had no significant effect on the expression level of DRD2 protein compared to the morphine group (P > 0.05). The findings of this study demonstrated that CA markedly attenuates the development of morphine tolerance and dependence. The observed behavioral effects were associated with changes in oxidative stress (NO levels), altered expression of DRD2 and NR1, and increased GDNF expression, suggesting the involvement of multiple interacting pathways.