The prolonged use of opioid medications can lead to tolerance, ultimately reducing the analgesic effect and limiting its clinical application [1]. Therefore, a combination therapy that uses different analgesics can be a viable alternative for pain management. One typical instance is the co-administration of non-steroidal anti-inflammatory drugs (NSAIDs) with opioid agonists for the treatment of severe and persistent pain [2].

Studies have shown that inflammatory mediators like bradykinin, prostaglandins, tumor necrosis factor-α (TNF-α), and interleukin-1β induce pain and increase neuronal sensitivity [3]. Thus, an anti-inflammatory therapeutic strategy has the potential to improve pain symptoms. For example, acetaminophen and L-carnosine were found to alleviate neuropathic pain through the nuclear factor kappa B (NF-κB) pathway and antioxidant properties in a model of chronic constriction injury [4]. It's important to mention that the development of tolerance to analgesic effects is not limited to opioids. Non-opioid painkillers can also lead to tolerance, either individually or through cross-tolerance with opioids [5,6]. For instance, chronic use of NSAIDs such as lornoxicam, sodium salicylate (SS), ketorolac, diclofenac sodium, and dipyrone is associated with a gradual decrease in the analgesic effects [7–10]. SS, a metabolite of aspirin, is an effective treatment for inflammation-related illnesses. The pharmacological effects of salicylate include inhibiting cyclooxygenases and the NF-κB signaling pathway. These actions contribute to the anti-inflammatory and analgesic properties associated with salicylate [11]. To treat moderate to severe pain, this medication is frequently used either by itself or in conjunction with opioids. Nevertheless, long-term usage of these can lead to adverse effects such as tolerance to their analgesic effects. Biguanide-class drugs such as metformin (MET) are effective as a first-line treatment for type 2 diabetes, providing robust glucose-lowering effects, a well-established safety profile, and a cost-effective option for patients [12]. MET has been found to penetrate the blood-brain barrier in prior investigations, enabling it to exert numerous beneficial effects within the central nervous system, including anti-inflammatory and neuroprotective properties [13,14]. Several studies have demonstrated the anti-inflammatory effects of MET in various experimental models of inflammation. Additionally, several studies have demonstrated the neuroprotective effects of MET. These studies suggest that MET protects against neuronal damage by reducing oxidative stress, inflammation, and apoptosis [15]. These studies indicate that MET's anti-inflammatory effects are likely mediated by inhibition of NF-κB signaling [16]. Pan et al. [1] reported that MET has the potential to reduce morphine tolerance in mice by inhibiting microglial activation and suppressing central sensitization in the spinal cord. The findings suggest that MET could be useful for managing opioid tolerance in chronic pain treatment. However, further studies are required to understand its mechanisms and determine its clinical applicability. Thus, the present study aimed to investigate the possible effect of acute and chronic administration of MET on tolerance to anti-nociceptive effects of SS in male rats.

1. Animals

Fifty-six male Wistar rats were used in this research (weight 200–250 g). Rats were randomly divided into 8 groups (n = 7). All the rats were housed in special Plexiglas cages with dimensions of 30 cm × 40 cm × 15 cm and in a situation with controlled conditions in repeated periods of 12 hours of light and 12 hours of darkness (the light cycle starts at 7 a.m.). Food and water were freely available, and the temperature was kept at 23°C ± 2.0°C. The instructions for the care and use of laboratory animals were followed throughout all experimental procedures (National Academic Press, Washington D.C, 2010). All experimental protocols were approved by the ethics committee at Torbat Heydariyeh University of Medical Sciences (IR.THUMS.REC.14000.027). All animals were handled cautiously to minimize undesired stress.

2. Drugs and experimental groups

SS (Sigma-Aldrich), and MET (Merck) were each injected intraperitoneally after being completely dissolved in saline (0.9%). The rats were divided into the following 8 groups: (1) the control, which received saline (as a vehicle) daily for 7 days; (2) SS, which received salicylate (300 mg/kg) daily for 7 days; (3) Met50, which received MET (50 mg/kg) for 7 days; (4) Met75, which received MET (75 mg/kg) for 7 days; (5) Met100, which received MET (100 mg/kg) for 7 days; (6) SS + Met50, which received salicylate (300 mg/kg) and MET (50 mg/kg) for 7 days; (7) SS + Met75, which received salicylate (300 mg/kg) and MET (75 mg/kg) for 7 days; and (8) SS + Met100, received salicylate (300 mg/kg) and MET (100 mg/kg) for 7 days. The drug doses were chosen based on studies evaluating the analgesic effects of intraperitoneally injected SS and MET in rats [17–20].

3. Hot plate test

A study demonstrated that tolerance to NSAID's antinociceptive effects is mediated by the endogenous opioid system, potentially involving descending pain modulatory systems [21]. The hot plate test is used to assess supraspinal pain pathways in rats, as it was shown that hind limb withdrawal does not occur in rats with spinal transection [22]. Therefore, to assess the anti-nociceptive effects of the drugs the hot plate test (Borj Sanat Azma) was performed. For this procedure, the heat was adjusted to a constant temperature of 50°C ± 2°C. Individual rats were placed on a hot plate, and their response latency (the time taken to withdraw a hind paw, lick, or jump) was recorded in seconds. A cut-off duration of 50 seconds was specified to prevent tissue injury. To assess acute drug response, the mean reaction time was recorded at 15, 30, 45, and 60 minutes after drug administration.

4. Statistical analysis

All data were presented as mean ± standard error of the mean. One-way analysis of variance (ANOVA) test and Tukey's post hoc test were used for statistical analysis of data with a single independent variable. Two-way ANOVA was performed for data with numerous independent variables, followed by Tukey's post hoc test. The GraphPad Prism software (version 8.0) was used to perform statistical analyses and prepare figures. Statistical significance for differences between means was considered as P < 0.05.

1. Response time evaluation of acute treatments

To determine of best response time, after the first injection of the treatments, hot plate latencies were recorded each 15 minutes until 60 minutes. As shown in Fig. 1 most responses to the treatments were 45 minutes after injection.

Figure 1. Line chart of hot plate latencies after injection of the treatments. Data were presented as mean ± standard error of the mean, n = 7. Met50: received MET (50 mg/kg) for 7 days, Met75: received MET (75 mg/kg) for 7 days, Met100: received MET (100 mg/kg) for 7 days, SS: received salicylate (300 mg/kg) daily for 7 days, MET: metformin.

2. The analgesic effect of MET

Different doses of MET significantly increased hot plate latency on the first day after injection as compared to the control group (Met50, P = 0.048; Met75, P = 0.014). Moreover, the Met50 group still showed a significant effect compared with the control group on the second day after injection (P = 0.025; Fig. 2A). Analysis of the line chart area under the curve (AUC) was shown MET-treated groups have significantly higher hot plate latencies than the control group (Met50, Met75, P < 0.001; Met100, P = 0.019). Also, the Met100 group had a significantly lower AUC as compared to the Met50 group (P = 0.002; Fig. 2B).

Figure 2. Effect of different doses of metformin (MET) on hot plate latencies. (A) Line chart of hot plate latencies after MET injection at different doses over time. (B) Area under curve of the line chart. Data were presented as mean ± standard error of the mean, n = 7, and analyzed by ANOVA followed by Tukey’s multiple comparison tests. (a) Compared to the control group, (b) Compared to the Met50 group. Met50: received MET (50 mg/kg) for 7 days, Met75: received MET (75 mg/kg) for 7 days, Met100: received MET (100 mg/kg) for 7 days. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

3. The effect of MET co-treatment on salicylate tolerance

Salicylate injection significantly increased hot plate latency as compared to the control group (P < 0.001) but this latency decreased through the days after injection, and after day 4, salicylate injection did not significantly increase the latency, which indicated tolerance development to the salicylate. SS + Met co-treatment somehow prevented this tolerance development and the latency remained significant after day 4 (SS + Met50, P = 0.002; SS + Met75, P = 0.025). Moreover, after day 5 the SS + Met50 group latency became significant to the SS group (P = 0.018; Fig. 3A). Analysis of the line chart AUC showed that the salicylate salicylate-treated groups have significantly higher hot plate latency than the control group (P < 0.001). Co-treatment of SS + Met50 had a stronger analgesic effect as compared to the SS group (P = 0.015; Fig. 3B).

Figure 3. Effect of salicylate and metformin (MET) co-treatment on hot plate latencies. (A) Line chart of the groups hot plate latencies thorough the days. (B) Area under curve of the line chart. Data were presented as mean ± standard error of the mean, n = 7, and analyzed by ANOVA followed by Tukey’s multiple comparison tests. (a) Compared to the control group, (b) Compared to the SS group, (c) Compared to the Met50 group, (d) Compared to the Met75 group. Met50: received MET (50 mg/kg) for 7 days, Met75: received MET (75 mg/kg) for 7 days, Met100: received MET (100 mg/kg) for 7 days, SS: received salicylate (300 mg/kg) daily for 7 days. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

In the present study, the authors investigated the MET effect on salicylate tolerance development. MET is a well-known drug and widely used to treat patients with type 2 diabetes. It was shown that MET activates AMP-activated protein kinase (AMPK), which plays a role in nociceptive processing. Therefore, it is expected to have an effect on nociception [23,24]. Studies show that MET reduces neuropathic pain in rodents by suppressing aberrant translation pathways in primary afferent neurons and inhibiting neuronal excitability [25]. Moreover, MET has the potential to prevent the development of pain and thermal hyperalgesia in inflammatory pain induction models [26,27]. In agreement with previous studies, it was found that administration of the MET induced analgesic effects, as evidenced by the increased hotplate latency observed in the MET-treated groups (Fig. 2). A study reported that MET significantly increased nociceptive response latency in the hot-plate model [28]. A dose-response curve study of MET has revealed that the efficacy of the drug is biphasic and reduces at both very low and high doses [29]. Another study demonstrated that MET at 50 mg/kg attenuates IL-1β and TNF-α levels better than high doses of 100 and 200 mg/kg, and also this dose has stronger analgesic efficacy than higher doses in the spinal cord injury model [19]. The authors’ results, in accordance with the mentioned studies, indicate that MET at the dose of 50 mg/kg had the highest analgesic effect, and the Met100 group had significantly lower AUC than the Met50 group (Fig. 2B). NSAIDs are widely used for mild pain relief and produce their effects by inhibiting cyclo-oxygenase, a key enzyme in the production of prostaglandins which potentiate the pain caused by other mediators, e.g., histamine, serotonin, and bradykinin [30]. Studies have shown that NSAIDs could inhibit spinal cord nociceptive neurons [31]. It is confirmed that the usual analgesics, like lysine-acetylsalicylate, induce antinociception through the activation of neurons in opioid-related brain structures [32]. Also, another study showed that a morphine antagonist, naloxone can block the analgesic effects of NSAIDs [5]. This evidence suggests that the analgesic effect of NSAIDs may be related to the endogenous opioid system. In this study, after injecting salicylate, the hot plate latency increased. However, after repeated administration, this effect was reduced and on day 5, the latency reached the same level as the control group (Fig. 3A). Consistent with the authors’ observations, a study showed that administering diclofenac prolongs the response time of the hot plate in rats [33]. Another study reports that systemic injections of NSAIDs attenuate tail-flick and hot plate responses and repeated treatment leads to tolerance development. Moreover, this confirms that the tolerance was related to an opiate-mediated mechanism [34]. In the present study, it was found that co-treatment of MET with salicylate could prevent tolerance development as evidenced by hot plate response persistence in the co-treatment groups (Fig. 3A). Also, the SS + Met50 group had a significantly higher analgesic effect than the SS group (Fig. 3B). It was reported that MET reduced the development of analgesic tolerance resulting from repeated intraperitoneal injections of morphine in mice [35]. A study found that MET could enhance the analgesic effects of ibuprofen and aspirin in an inflammatory pain model, and this effect is not related to pharmacokinetic interactions [20]. MET activates AMPK, a kinase present in all cells that regulates various functions. In neural cells, it inhibits mitogen-activated protein kinase and mammalian target of rapamycin kinase pathways, which are important for pain plasticity and sensitization [36,37]. As tolerance to the analgesic effects of NSAIDs is related to the endogenous opioid system, and a recent study showed MET could prevent opioid-related tolerance, it is possible that MET, through AMPK activation, affects the opioid system and reduces salicylate tolerance development.

This study did not have sufficient tests to confirm this possibility, and further research is needed to explore the mechanism. Specifically, changes in the excitability of the ascending and descending pain pathways must be investigated by membrane potential recording. In addition, examining changes in gene expression and receptor density could help identify the mechanism behind this effect. Unfortunately, due to budget and device limitations, these changes were not studied. From the obtained results it could be concluded that MET has the potential to reduce analgesic tolerance induced by repeated intraperitoneal injections of SS in Wister rats. This suggests that MET could be a new approach to preventing NSAID analgesic tolerance.

The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.

No potential conflict of interest relevant to this article was reported.

No funding to declare.

Elham Akbari: Investigation; Dawood Hossaini: Investigation; Farimah Beheshti: Investigation; Mahdi Khorsand Ghaffari: Writing/manuscript preparation; Nastran Roshd Rashidi: Writing/manuscript preparation; Masoumeh Gholami: Project administration.

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