ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3β in a mouse sciatic nerve injury model

The present study investigates the molecular mechanisms of peripheral nerve regeneration by examining the ROCK/PI3K/Akt/GSK3β pathway's role in promoting morphological and functional recovery after peripheral nerve injury (PNI). Using a mouse sciatic nerve crush (SNC) injury model and a dorsal root ganglion (DRG) explant axotomy model, mice and DRG were divided the experimental (treated with DMSO) group, Y27632 group (treated with ROCK inhibitor Y27632), Y + LY group (treated with Y27632 + PI3K inhibitor LY294002), and Y + LY + SB group (treated with Y27632 + LY294002 + GSK3β inhibitor SB216763). Immunofluorescence was used to assess axon density, diameter, myelin thickness and Schwann cell proliferation, while retrograde tracing with cholera toxin subunit B evaluated peripheral‑to‑central reconnection. Behavioral tests measured functional recovery, and in DRG explants, axon regeneration length and growth cone size were quantified. Protein expression analysis of RhoA, ROCK, PI3K, Akt, GSK3β, and their phosphorylated forms was conducted on day 3 post‑axotomy, both <i>in vivo</i> and <i>in vitro</i>. Additionally, RSC96 Schwann cell migration and proliferation were evaluated using scratch assays and EdU staining. Results showed that ROCK inhibition with Y27632 significantly enhanced axonal regeneration, growth cone expansion, retrograde transport, and reinnervation of acetylcholine receptors and Merkel cells, and promoted Schwann cell proliferation and RSC96 migration, leading to thicker myelin sheaths after SNC. These changes mitigated gastrocnemius muscle atrophy, improved muscle strength, gait, and thermal/tactile sensitivity. Co‑treatment with LY294002 blocked these effects, but adding SB216763 restored them. Protein analysis indicated that ROCK inhibition increased phosphorylated PI3K, Akt and GSK3β, whereas PI3K inhibition reduced GSK3β phosphorylation. These findings suggested that ROCK inhibition promotes axon regeneration and remyelination after PNI by enhancing PI3K/Akt phosphorylation and suppressing GSK3β activity, highlighting the therapeutic potential of targeting the ROCK/PI3K/Akt/GSK3β pathway for peripheral nerve repair.