Conditioned Medium of Epidermal Neural Crest Stem Cells Improved Functional Recovery and Tissue Repairing after Spinal Cord Injury by Suppressing Neuronal Apoptosis Through the PI3K/AKT Signaling Pathway

Abstract Aims: This study evaluated the effects of conditioned medium from epidermal neural crest stem cells (EPI-NCSCs-CM) on functional recovery after spinal cord injury (SCI) and investigated the role of PI3K-AKT signaling pathway in regulating the neuronal apoptosis. Methods: EPI-NCSCs were isolated from 10-day-old rats and cultured for 48 hours and EPI-NCSCs-CM was extracted. H2O2 was used to establish apoptosis model in SHSY-5Y cells. Cell viability and survival rate were assessed using CCK-8 assay and Calcein-AM/PI staining. A SCI contusion model was established in adult Sprague-Dawley rats. Functional recovery was evaluated using the Basso-Bresnahan-Beattie (BBB) scoring system, inclined test, and footprint observation. Electrophysiological recording was used to analyze neurological restoration after SCI. Histological organization was assessed using Hematoxylin-eosin (H&E) staining and Nissl staining. TUNEL staining and ROS detection were applied to measure the apoptosis and oxidative stress levels. Western blot was conducted to detect the expression levels of apoptosis markers and PI3K/AKT signaling-related proteins. Results: EPI-NCSCs-CM significantly promoted functional and histological rehabilitation in SCI rats by suppressing neuronal apoptosis through regulating PI3K/AKT signaling pathway. In vitro study indicated that EPI-NCSCs-CM administration alleviated neurotoxicity caused by H2O2 in SHSY-5Y cells. The administration of LY294002 (a PI3K inhibitor) implied that the PI3K/AKT signaling pathway plays a vital role in regulating neuronal apoptosis. Conclusions: This study presents a new strategy for repairing SCI using EPI-NCSCs-CM, and provides evidence that EPI-NCSCs-CM can inhibit neuronal apoptosis by regulating the PI3K/AKT signaling pathway in SCI rats.