Electroacupuncture Inhibits Neural Ferroptosis in Rat Model of Traumatic Brain Injury via Activating System Xc-/GSH/GPX4 Axis

Abstract

Background: Ferroptosis is an iron-dependent regulating programmed cell death discovered recently that has been receiving much attention in traumatic brain injury (TBI). xCT, a major functional subunit of Cystine/glutamic acid reverse transporter (System Xc^-), promotes cystine intake and glutathione biosynthesis, thereby protecting against oxidative stress and ferroptosis.

Objective: The intention of this research was to verify the hypothesis that electroacupuncture (EA) exerted an anti-ferroptosis effect via an increase in the expression of xCT and activation of the System Xc^-/GSH/GPX4 axis in cortical neurons of TBI rats.

Methods: After the TBI rat model was prepared, animals received EA treatment at GV20, GV26, ST36 and PC6, for 15 min. The xCT inhibitor Sulfasalazine (SSZ) was administered 2h prior to model being prepared. The degree of neurological impairment was evaluated by means of TUNEL staining and the modified neurological severity score (mNSS). Specific indicators of ferroptosis (Ultrastructure of mitochondria, Iron and ROS) were detected by transmission electron microscopy (TEM), Prussian blue staining (Perls stain) and flow cytometry (FCM), respectively. GSH synthesis and metabolism-related factors in the content of the cerebral cortex were detected by an assay kit. Real-time quantitative PCR (RT-QPCR), Western blot (WB), and immunofluorescence (IF) were used for detecting the expression of System Xc^-/GSH/GPX4 axisrelated proteins in injured cerebral cortex tissues.

Results: EA successfully relieved nerve damage within 7 days after TBI, significantly inhibited neuronal ferroptosis, upregulated the expression of xCT and System Xc^-/GSH/GPX4 axis forward protein and promoted glutathione (GSH) synthesis and metabolism in the injured area of the cerebral cortex. However, aggravation of nerve damage and increased ferroptosis effect were found in TBI rats injected with xCT inhibitors.

Conclusions: EA inhibits neuronal ferroptosis by up-regulated xCT expression and by activating System Xc^-/GSH/GPX4 axis after TBI, confirming the relevant theories regarding the EA effect in treating TBI and providing theoretical support for clinical practice.

Keywords: Traumatic brain injury; cell death.; electroacupuncture; ferroptosis; system Xc-/GSH/GPX4 axis; xCT expression.