Electroacupuncture combined with rTMS promotes neuronal regeneration via DNMT1-mediated PI3K-AKT pathway in cerebral...

Cerebral palsy (CP), resulting from perinatal brain injury, is characterized by persistent motor dysfunction largely due to impaired regeneration of the corticospinal tract. Electroacupuncture (EA) and repetitive transcranial magnetic stimulation (rTMS) are promising non-invasive neuromodulation approaches, but their combined effects and epigenetic mechanisms remain unclear. This study examined the synergistic neurorestorative potential of EA and rTMS in a hypoxic-ischemic brain damage (HIBD) model of CP, focusing on DNA methyltransferase 1 (DNMT1) regulation of the PI3K-AKT pathway. In vivo, neonatal rats subjected to HIBD were assigned to control, HIBD, EA, rTMS, or combined EA+rTMS groups and treated for four weeks. Motor function was assessed using rotarod, grid-walking, and grip strength tests, while neuronal and molecular changes were analyzed via histological and biochemical methods. In vitro, hypoxia-exposed cortical neurons underwent DNMT1 and PI3K modulation to clarify mechanisms. Combined EA+rTMS produced superior functional recovery compared to monotherapies, including a 2.1-fold increase in rotarod latency, 53% reduction in foot faults, and 80% improvement in grip strength vs untreated HIBD animals. Improvements were also greater than EA or rTMS alone. Histological findings showed enhanced neuronal density in cortical and spinal regions. At the molecular level, combined treatment suppressed DNMT1 expression and increased PI3K, phosphorylated AKT, GAP-43, and myelin basic protein. In vitro, DNMT1 knockdown enhanced PI3K-AKT signaling, neuronal survival, and axonal growth, while DNMT1 overexpression or PI3K inhibition negated these effects. Overall, EA+rTMS promotes functional and structural recovery in CP by epigenetically inhibiting DNMT1 and activating the PI3K-AKT pathway, supporting its translational potential for perinatal brain injury.