AAD-2004 through clearing H2O2 reduces astrocyte proliferation and promotes neural regeneration after spinal cord injury

To assess the effect of AAD-2004 on spinal cord injury (SCI) and to explore its mechanism, we employed an in vitro model using OGD/R-challenged astrocytes to investigate the effects of AAD-2004 against cell death (terminal deoxynucleotidyl transferase dUTP nick-end labeling, tunel), oxidative stress (H₂O₂ level), and the expression of the key neuroprotective factor MAP2.AAD-2004[2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] is a hydrogen peroxide(H₂O₂) scavenger primarily used for the treatment of amyotrophic lateral sclerosis and Alzheimer disease that has demonstrated certain neuroprotective properties. In parallel, modified allen's method was adopted, further exploring the potential molecular mechanism in vivo. Based on these conditions, histological and behavioral analysis were performed by Nissl staining, basso mouse scale and footprint analysis. The level of molecules associated with glial scar formation, nerve regeneration, axonal regeneration and H₂O₂ level were analyzed using western blot, immunofluorescence staining and H₂O₂ kit. AAD-2004 significantly improved the movement function after SCI and inhibited the proliferation of astrocytes, thus preventing the formation of glial scar by inhibiting of H₂O₂. At the same time, AAD-2004 promoted nerve regeneration, and the effect was due to neuronal regeneration and axonal regeneration pathways. The expression levels of GFAP and vimentin were significantly downregulated in AAD-2004-treated, and the expression level of Ki67 and PH3 were downregulated. The mean fluorescence intensity of neuronal regeneration (Neun⁺and MAP2⁺) and axonal regeneration-related (NF⁺ and GAP43⁺) were significantly upregulated after AAD-2004 treatment. Scavenging H₂O₂ level is a viable therapeutic strategy, and that AAD-2004 is prospective, and that scavenging H₂O₂ facilitated nerve regeneration and inhibited glial scar formation for SCI.