Ferroptosis inhibitor ferrostatin-1 attenuates morphine tolerance development in male rats by inhibiting dorsal root ganglion neuronal ferroptosis

Abstract

Background: Ferrostatin-1 and liproxstatin-1, both ferroptosis inhibitors, protect cells. Liproxstatin-1 decreases morphine tolerance. Yet, ferrostatin-1's effect on morphine tolerance remains unexplored. This study aimed to evaluate the influence of ferrostatin-1 on the advancement of morphine tolerance and understand the underlying mechanisms in male rats.

Methods: This experiment involved 36 adult male Wistar albino rats with an average weight ranging from 220 to 260 g. These rats were categorized into six groups: Control, single dose ferrostatin-1, single dose morphine, single dose ferrostatin-1 + morphine, morphine tolerance (twice daily for five days), and ferrostatin-1 + morphine tolerance (twice daily for five days). The antinociceptive action was evaluated using both the hot plate and tail-flick tests. After completing the analgesic tests, tissue samples were gathered from the dorsal root ganglia (DRG) for subsequent analysis. The levels of glutathione, glutathione peroxidase 4 (GPX4), and nuclear factor erythroid 2-related factor 2 (Nrf2), along with the measurements of total oxidant status (TOS) and total antioxidant status (TAS), were assessed in the tissues of the DRG.

Results: After tolerance development, the administration of ferrostatin-1 resulted in a significant decrease in morphine tolerance (P < 0.001). Additionally, ferrostatin-1 treatment led to elevated levels of glutathione, GPX4, Nrf2, and TOS (P < 0.001), while simultaneously causing a decrease in TAS levels (P < 0.001).

Conclusions: The study found that ferrostatin-1 can reduce morphine tolerance by suppressing ferroptosis and reducing oxidative stress in DRG neurons, suggesting it as a potential therapy for preventing morphine tolerance.

Keywords: Ferroptosis; Ganglia; Glutathione; Morphine; Oxidative Stress; Pain; Phospholipid Hydroperoxide Glutathione Peroxidase; Spinal.

Fig. 1

Experimental design of the study. DMSO: dimethyl sulfoxide (ferrostatin dissolved in dimethyl sulfoxide but morphine dissolved in saline), i.p.: intraperitoneally, s.c.: subcutaneousl, GPX4: glutathione peroxidase 4, Nrf2: nuclear factor erythroid 2-related factor 2, TAS: total antioxidant status, TOS: total oxidant status.

Fig. 2

This figure illustrates the impact of ferrostatin-1 on nociception and morphine analgesia in the tail flick (A) and hot plate (B) tests. The values are presented as the means ± standard error of the mean of % MPE (n = 6). **P < 0.01, indicating a significant difference compared to the control group.

Fig. 3

This figure depicts the impact of ferrostatin-1 on the development of morphine tolerance in the tail-flick (A) and hot plate (B) tests. The values are presented as the means ± standard error of the mean of % MPE (n = 6). **P < 0.01 indicate a significant difference compared to the control group. ++P < 0.01 indicate a significant difference compared to the morphine group. ##P < 0.01 indicate a significant difference compared to the morphine tolerance group.

Fig. 4

This figure illustrates the impact of ferrostatin-1 on the levels of glutathione (A), GPX4 (B), and Nrf2 (C) in the dorsal root ganglion during morphine analgesia and tolerance. Values are presented as the means ± standard error of the mean of six rats. GPX4: glutathione peroxidase 4, Nrf2: nuclear factor erythroid 2-related factor 2. ***P < 0.001, compared to the control group. +P < 0.001, compared to the morphine group. #P < 0.001, compared to the morphine tolerance group.

Fig. 5

This figure illustrates the impact of ferrostatin-1 on TAS (A) and TOS (B) in morphine analgesia and tolerance in dorsal root ganglion. Values are presented as the means ± standard error of the mean of six samples. TAS: total antioxidant status, TOS: total oxidant status. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the control group. +P < 0.01 and ++P < 0.001, compared to the morphine group. #P < 0.001, compared to the morphine tolerance group.