Fujian tablet regulates the Foxo3a/GPX4 axis to promote remyelination and improve motor function in ischemic stroke

Abstract

Ethnopharmacological relevance: Fujian Tablet (FJT), a traditional Chinese herbal compound formulation developed under the theoretical framework of "nourishing the liver and kidney, replenishing essence and marrow", has been clinically applied for over two decades to treat post-stroke neurological deficits. Preliminary studies demonstrated its efficacy in improving motor function and promoting cervical spinal cord neuroaxonal growth in a middle cerebral artery occlusion (MCAO) rat model. Building upon these findings, this study integrates metabolomic evidence of Foxo3a-GPX4 axis activation to systematically elucidate Fujian Tablet's neurorestorative mechanisms through three interconnected pathways: regulation of ferroptosis, promotion of oligodendrocyte proliferation, and remyelination. By bridging traditional TCM therapeutic principles with contemporary molecular neuroscience, this investigation aims to provide mechanistic insights for optimizing Fujian Tablet's clinical application and advancing its global recognition in neurorehabilitation.

Aim of the study: This study aims to verify the molecular mechanism by which FJT activates Forkhead box O3a (Foxo3a) to promote glutathione peroxidase 4 (GPX4) expression, thereby regulating oligodendrocyte survival and remyelination, based on the regulatory network of "traditional Chinese medicine compound - target - cellular function". This study provides experimental evidence for explaining the material basis and action targets of the compound's efficacy.

Materials and methods: The middle cerebral artery embolization method was employed to establish a rat model of MCAO. Then, FJT was used for intervention via intragastric administration at doses of 0.72 g/kg and 1.44 g/kg, twice daily for 14 consecutive days. Behavioral observations of the rats were carried out using the Catwalk system and beam walking test (BWT). remyelination was assessed via luxol fast blue (LFB) staining and transmission electron microscopy. In addition, in vitro experiments with oligodendrocytes were conducted in combination. The expressions of myelin basic protein (MBP) was detected by immunofluorescence. The expressions of Ferritin, and GPX4 were detected by RT-qPCR and Western blot. The levels of ferroptosis - related metabolites were measured using flow cytometry and enzyme - linked immunosorbent assay (ELISA). The transcriptional regulatory effect of Foxo3a on GPX4 was examined by ChIP-qPCR.

Results: Fujian Tablet dose-dependently improved the motor function of MCAO rats, and upregulated the expression of Foxo3a to enhance its binding activity to the GPX4 promoter, thereby increasing GPX4 expression. Knockdown of Foxo3a or treatment with the GPX4 inhibitor (RSL3) reversed the inhibitory effect of FJT on ferroptosis (decrease in lipid reactive oxygen species (ROS) and malondialdehyde (MDA), and increase in reduced glutathione (GSH)). This reversal led to a reduction in the expression of MBP and myelin oligodendrocyte glycoprotein (MOG), inhibited the level of remyelination (decrease in LFB optical density and increase in the electron microscopy G-ratio), and hindered the recovery of motor function. In vitro experiments confirmed that serum containing FJT inhibited ferroptosis through the Foxo3a-GPX4 axis, and promoted the proliferation of oligodendrocytes.

Conclusions: FJT exerts a neuroprotective effect by activating the Foxo3a - GPX4 axis to inhibit ferroptosis, promote the proliferation of oligodendrocytes and remyelination, and ultimately improve motor function. This study clarifies that Fujian Tablet exerts a neuroprotective effect through regulating the "ferroptosis - oligodendrocyte proliferation - remyelination" axis, providing a scientific basis for its clinical application in ethnic medicine.

Keywords: Ferroptosis; Foxo3a-GPX4 axis; Fujian tablet; Motor function; Myelin sheath.