Ferroptosis, recently discovered, is a distinctive type of regulated cell death characterized by the buildup of lipid peroxides. This accumulation is triggered by the disruption of antioxidant mechanisms reliant on glutathione. It distinguishes itself from apoptosis, autophagy, and necrosis through genetic, morphological, and biological distinctions (Mongin et al. 2020). Ferroptosis initiates lipid peroxidation through the production of reactive oxygen species (ROS) via the Fenton reaction (Lee et al. 2018). New research reveals that ferroptosis has a role in drug resistance (Alvarez et al. 2017); for example, the lipid-peroxidase pathway is required for anticancer drug resistance. Other features of ferroptosis include oxidative burst development, antioxidant depletion, and stimulation of lipid peroxidation (Doll and Conrad 2017). Previous research has demonstrated that ferroptosis is crucial in neurological diseases (Lei et al. 2018). It was shown that ferroptosis characterized by free-iron overload contributes to morphine tolerance (Chen et al. 2019). The use of ferrostatin, which is an inhibitor of ferroptosis, has been associated with positive outcomes in the central nervous system. Li et al. demonstrated that ferrostatin-1 attenuated mechanical hypersensitivity by suppressing spinal ferroptosis in rats with streptozotocin-induced diabetes (Li et al. 2021). An et al. indicated that ferrostatin-1 reversed acrylamide-induced biological activities and facilitated the repair of damaged DRG neurons by inhibiting ferroptosis (An et al. 2022). Li et al. revealed that striatum injection and cerebral ventricular injection of ferrostatin-1 produced neuroprotective effects after intracerebral hemorrhage induced by collagenase (Li et al. 2017). In the autologous blood infusion model of intracerebral hemorrhage, intraperitoneal injection of ferrostatin-1 was demonstrated to enhance long-term neurological processes (Alim et al. 2019).
These positive effects of ferrostatin-1 on the central nervous system and the hypothesis that inhibition of ferroptosis may reduce morphine tolerance prompted us to investigate the impact of ferrostatin-1, a ferroptosis inhibitor,on morphine tolerance which has not been studied so far. So this study was designed to investigate the effects of ferrostatin-1 on acute pain, morphine analgesia, and morphine tolerance devlopment, as well as the mechanisms that mediate these effects. In the our work, ferrostatin-1 decreased the development of morphine tolerance. However, this compound did produce any effect on acute pain and did not modify the analgesic effect of morphine when administered in a single dose. These outcomes are consistent with the earlier investigation conducted by Chen, reporting that ferroptosis suppression by liproxstatin-1 suppressed tolerance development, but ferroptosis activation by erastin accelerated it (Chen et al. 2019). Chen et al. also discovered that chronic morphine treatment caused neuronal loss, iron accumulation, inflammation, lipid peroxidation, and mitochondrial shrinkage in the spinal cord, but liproxstatin-1, a ferroptosis inhibitor, reversed all of these events (Chen et al. 2019).
Cysteine, glycine, and glutamic acid make up the tripeptide glutathione, which is present in most cells in remarkably high amounts (5 millimolar). It plays a vital role in protecting cellular macromolecules against exogenous and endogenous reactive oxygen and nitrogen species (Demirkol and Ercal 2014). Ferroptosis can be induced by molecules or situations that prevent the formation of glutathione or glutathione peroxidase (GPX4), a glutathione-dependent antioxidant enzyme (Cao and Dixon 2016). It was discovered that morphine injection reduced the intracellular GSH level in the rat brain (Abdel-Zaher et al. 2013). Glutathione peroxidase 4 (GPX4) is a specific type of selenoprotein that possesses one selenocysteine residue at its active site and seven cysteine residues. GPX4 plays an important role in the regulation of ferroptosis, and inhibiting its activity promotes the occurrence of ferroptosis (Mongin et al. 2020). It is believed that a deficiency in GPx4 activity causes ferroptosis in neurodegenerative disorders (Chen et al. 2019). Abdel-Zaher et al. showed that prolonged injection of morphine into mice reduced the amount of intracellular glutathione (GSH), a non-enzymatic antioxidant, and the activity of glutathione peroxidase GPX, an enzymatic antioxidant (Abdel-Zaher et al. 2013). The nuclear factor erythroid 2-related factor 2 (Nrf2) is an increasingly recognized regulator that influences the vulnerability of cells to oxidative stress. Nrf2 plays a crucial role in maintaining the expression of various genes containing antioxidant response elements, both under normal conditions and when stimulated, to mitigate the detrimental effects of exposure to oxidants (Ma 2013). It has been established that Nrf2 is essential for controlling ferroptosis and treating neurological diseases. Nrf2 can regulate the ferroptosis process by controlling the levels of GPX4 protein, mitochondrial activity, and intracellular free iron (Pozo et al. 2020).
It was shown that morphine treatment significantly influenced several antioxidant proteins involved in the Nrf2 pathway in HBMECs (human brain microvascular endothelial cells) (Reymond et al. 2022). In line with the above, our findings show that repeated injections of morphine to rats led to the production of oxidative stress and ferroptosis in the dorsal root ganglion tissues. This effect was confirmed by a decrease in glutathione, GPX4, and Nrf2 levels. Otherwise, Ferrostatin-1 suppressed the ferroptosis induced by repeated injection of morphine through increasing glutathione, GPX4, and Nrf2 levels in DRG. In line with our results, Chu, et al. demonstrated that ferrostatin-1 suppressed glutamate-induced downregulation of Gpx4 and Nrf2 (Chu et al. 2020). In addition, An et al. showed that ferrostatin-1 significantly enhanced glutathione levels in the dorsal root ganglion neurons injury induced by acrylamide (An et al. 2022). Ferrostatin-1 treatment significantly diminished the STZ-induced decrease in GPX4 levels in the spinal cord (Li et al. 2021). Morphine exposure not only declined the activities of antioxidants in target cells but also facilitated the production of free radicals, including reactive oxygen species (ROS) or reactive nitrogen species (RNS) (Skrabalova et al. 2013). Taskiran et al. reported that morphine administration in single-dose and repeated doses decreased TAS levels and increased TOS levels in DRG. It could indicate that morphine use inhibited the antioxidant system, which could be involved in tolerance development (Avci and Taşkiran 2020). In line with the above, our findings show that single and repeated injections of morphine to rats led to the development of oxidative stress in the dorsal root ganglion tissues. This effect was confirmed by a decrease in TAS levels and an increase in TOS levels.
On the other hand, Ferrostatin-1 suppressed the oxidative stress induced by morphine administration through increasing TAS levels and decreasing TOS levels in DRG with morphine tolerance. In line with our results, Chu, et al. showed that ferrostatin-1 can significantly reduce the levels of reactive oxygen species in glutamate-injured HT-22 cells (Chu et al. 2020). Additionally, Chen et al. reported that liproxstatin-, a ferroptosis inhibitor, massively diminished the elevation of MDA and ROS levels that morphine causes in the serum and spinal cord of mice (Chen et al. 2019).
All in all, our results suggest that Ferrostatin-1 could reverse the morphine tolerance development by suppressing ferroptosis and oxidative stress in DRG neurons, which proposes the potential therapeutic application of Ferrostatin-1 to prevent or reduce morphine tolerance formation.