Electroacupuncture Ameliorates Chronic Inflammatory Pain and Depression Comorbidity by Inhibiting Nrf2-Mediated Ferroptosis in Hippocampal Neurons

Abstract

Chronic inflammatory pain and depression are highly comorbid, with nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated ferroptosis in hippocampal neurons strongly associated with the onset and progression of the comorbidity. Electroacupuncture (EA), widely used to treat pain and mood disorders, may ameliorate chronic inflammatory pain and depression comorbidity (CIPDC) by inhibiting Nrf2-mediated ferroptosis in hippocampal neurons, though its mechanism of action remains partially understood. In this study, we established the CIPDC model by administering a subcutaneous injection of complete Freund's adjuvant (CFA) into the left hind paw. Evaluations of EA's effects on pain thresholds and depressive behaviors in CIPDC rats included paw withdrawal mechanical threshold, paw withdrawal thermal latency, sucrose preference test, open field test, and forced swim test assessments. HE staining was performed to assess the pathological and morphological alterations in hippocampal neurons. FJB staining was utilized to evaluate neuronal degeneration, while transmission electron microscopy (TEM) was employed to examine ultrastructural changes in hippocampal neuronal mitochondria. Prussian blue staining was conducted to visualize ferrous ion deposition in the hippocampus, and the contents of ferrous ion (Fe²⁺), malondialdehyde (MDA), and glutathione (GSH) were measured using colorimetric assay kits. Western blotting (WB) was performed to determine the relative protein expression of Nrf2, FTH1, FTL, xCT, GPX4, ACSL4, LPCAT3, and LOX in the hippocampus. Additionally, the relative mRNA expression of FTH1, FTL, xCT, GPX4, ACSL4, LPCAT3, and LOX was analyzed by PCR. Flow cytometry was used to quantify ROS levels in the hippocampus, and immunofluorescence staining was applied to detect nuclear expression of Nrf2 as well as co-localization of GPX4 with the neuronal marker NeuN. Our results demonstrate that EA upregulates nuclear Nrf2 expression in hippocampal tissue, thereby alleviating iron metabolism dysregulation, enhancing antioxidant system activity, and reducing lipid peroxidation. This process inhibits ferroptosis in hippocampal neurons, promoting their repair and remodeling, and effectively treating CIPDC.

Keywords: Chronic inflammatory pain; Depression; Electroacupuncture; Ferroptosis; Hippocampal neurons; Nrf2 signal pathway.