Identification of key regulatory genes involved in myelination after spinal cord injury by GSEA analysis.

Multilayer dense myelin tissue provides insulating space and nutritional support for axons in healthy spinal cord tissue. Oligodendrocyte precursor cells (OPCs) are the main glial cells that complement myelin loss in the central nervous system and play an important role in the repair of spinal cord injury (SCI). However, the regulation of axonal remyelination after SCI is still insufficient. In this study, we focused on the changes in genes related to myelin repair after rat hemisection SCI by gene set enrichment analysis (GSEA). Key genes proteolipid protein 1 (Plp1), hexosaminidase subunit alpha (Hexa), and hexosaminidase subunit beta (Hexb) during remyelination after SCI were found. Through quantitative real-time polymerase chain reaction (qPCR) experiments, we confirmed that within 28 days after rat hemisection SCI, the mRNA expression of gene Plp1 gradually decreased, while the expressions of gene Hexa and Hexb gradually increased, which was consistent with RNA sequencing results. In vitro, we performed EdU proliferation assays on OPC cell line OLN-93 and primary rat OPCs. We found that interference of Plp1 promoted OPC proliferation, while interference of Hexa and Hexb inhibited OPC proliferation. In addition, we performed in vitro differentiation experiments on primary rat OPCs. By measuring myelin sheath branch outgrowth and the fluorescence intensity of the mature myelin sheath marker myelin basic protein (MBP), we found that interference of Hexa or Hexb promoted OPC differentiation and maturation, but interference of Plp1 inhibited this process. Finally, we injected Hexb siRNA in vivo and found that interfering Hexb could improve motor movements and myelin regeneration after SCI in rats. Our results provide new target genes that can selectively regulate the proliferation and differentiation of endogenous OPCs, providing new ideas for promoting remyelination and functional recovery after SCI.