Electroacupuncture suppresses neuronal ferroptosis to relieve chronic neuropathic pain

Abstract

Growing evidence supports the analgesic efficacy of electroacupuncture (EA) in managing chronic neuropathic pain (NP) in both patients and NP models induced by peripheral nerve injury. However, the underlying mechanisms remain incompletely understood. Ferroptosis, a novel form of programmed cell death, has been found to be activated during NP development, while EA has shown potential in promoting neurological recovery following acute cerebral injury by targeting ferroptosis. In this study, to investigate the detailed mechanism underlying EA intervention on NP, male Sprague-Dawley rats with chronic constriction injury (CCI)-induced NP model received EA treatment at acupoints ST36 and GV20 for 14 days. Results demonstrated that EA effectively attenuated CCI-induced pain hypersensitivity and mitigated neuron damage and loss in the spinal cord of NP rats. Moreover, EA reversed the oxidative stress-mediated spinal ferroptosis phenotype by upregulating reduced expression of xCT, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH1) and superoxide dismutase (SOD) levels, and downregulating increased expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), malondialdehyde levels and iron overload. Furthermore, EA increased the immunofluorescence co-staining of GPX4 in neurons cells of the spinal cord of CCI rats. Mechanistic analysis unveiled that the inhibition of antioxidant pathway of Nrf2 signalling via its specific inhibitor, ML385, significantly countered EA's protective effect against neuronal ferroptosis in NP rats while marginally diminishing its analgesic effect. These findings suggest that EA treatment at acupoints ST36 and GV20 may protect against NP by inhibiting neuronal ferroptosis in the spinal cord, partially through the activation of Nrf2 signalling.

Keywords: electroacupuncture; ferroptosis; glutathione peroxidase 4; neuronal protection; neuropathic pain; oxidative stress.

FIGURE 1

EA improved CCI‐induced NP phenotype. (A) The study protocol of EA treatment in CCI rats. (B) Effects of EA on MWT induced by CCI surgery. (C) Effects of EA on PWL in CCI‐induced NP models. n = 8–10. *p < 0.05, ***p < 0.001. vs. Sham group. ^# p < 0.05. vs. CCI group.

FIGURE 2

EA protected the neuron damage and loss in the spinal dorsal horn caused by NP rats. (A) H&E staining (spinal cord dorsal horn) of each group. Scale bar, 100 μm. Green arrows pointed out the neurons. (B) Nissel staining in the spinal cord dorsal horn among groups. Red arrows pointed out the Nissel bodies. Scale bar, 100 μm. (C) IF staining of NeuN (red arrows) in the spinal cord dorsal horn among groups. Scale bar, 100 μm. (D) The representation of the number of Nissl‐stained cells in the spinal dorsal horn among groups. (E) Quantification of NeuN‐positive cells among groups. Data presented as means ± s.d. **p < 0.01. n = 3.

FIGURE 3

EA attenuated CCI‐induced ferroptosis phenotype in the spinal cord. (A) The protein expression of xCT, GPX4 and FTH1 in the spinal cord of each group. GAPDH was used as an internal control. n = 3. (B) IF staining of ACSL4 in the dorsal horn of the spinal cord of each group. Scale bar, 100 μm. n = 3. (C) Changes of the mitochondrial ultrastructure (red arrows) in the spinal cord dorsal horn among groups by TEM. Scale bar, 500 nm. n = 3. (D) Quantitative analysis of ACSL4‐positive cells in each group. n = 3. (E) Changes in iron levels in each group. n = 5–6. (F) The level of MDA in the spinal cord of each group. n = 4–7. (G) The level of SOD in the spinal cord of each group. n = 3–4. Data presented as means ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

EA restored the GPX4‐mediated ferroptosis in neuron cells but not in microglia or astrocytes following CCI operation. (A) Representative images of immunofluorescent analysis of NeuN⁺ (green), GPX4⁺(red) maker and DAPI (blue) staining of nuclei in the spinal cord dorsal horn among groups. Scale bars, 20 μm (top), 10 μm (bottom). (B) Representative images of immunofluorescent analysis of GFAP⁺(green), GPX4⁺(red) maker and DAPI (blue) staining of nuclei in the spinal cord dorsal horn among groups. Scale bars, 20 μm (top), 10 μm (bottom). (C) Representative images of immunofluorescent analysis of Iba‐1⁺ (green), GPX4⁺(red) maker and DAPI (blue) staining of nuclei in the spinal cord dorsal horn among groups. Scale bars, 20 μm (top), 10 μm (bottom). Quantification analysis the number of GPX4/NeuN⁺ (D), GPX4/GFAP⁺ (E) and GPX4/ Iba‐1⁺(F) in the spinal cord dorsal horn. Data presented as means ± s.d. ***p < 0.001, n = 3.

FIGURE 5

EA increased the activity of Nrf2 and a concurrent reduction in Keap1 expression in the spinal cord of CCI rats. (A) IHC staining of Keap1 in the spinal cord dorsal horn of each group. Scale bar, 100 μm. (B) IHC staining of Nrf2 in the spinal cord dorsal horn of each group. Scale bar, 100  μm. (C) Quantitative analysis of Keap1‐positive cells in the spinal cord dorsal horn. (D) Quantification analysis of the number of Nrf2‐positive cells in the spinal cord dorsal horn. Data presented as means ± s.d. **p < 0.01, ***p < 0.001. n = 3.

FIGURE 6

EA inhibited ferroptosis by partial activation of Nrf2 signalling, but inhibition of Nrf2 did not abolish the analgesic effect of EA therapy. (A) Schematic representation of the Nrf2 inhibitor ML385 upon intrathecal injection into the spinal cord. The study protocol of EA treatment in CCI rats following the use of ML385. (B) The protein expression of Nrf2, GPX4 and FTH1 in the spinal cord of each group. GAPDH was used as an internal control. (C–E) The relative expression of Nrf2, GPX4 and FTH1 protein in each group. Data presented as means ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. The effects of EA on MWT (F) and PWL (G) in CCI‐induced NP models. n = 5. Data presented as means ± s.d. *p < 0.05, ***p < 0.001. vs. Sham group. ^# p < 0.05, ^### p < 0.001. vs. CCI group.

FIGURE 7

A schematic diagram illustrating the mechanism of EA treatment in peripheral nerve injury‐induced NP.