Comprehensive profiling and molecular characterization of alternative splicing regulation in synaptic remodelling associated with neuropathic pain induced by chronic constrictive injury in a rat model

Neuropathic pain (NP) is a chronic pain condition caused by injury to the nervous system. Although alternative splicing dysregulation has been implicated in NP, the contribution of RNA-binding proteins (RBPs) to this process remains unclear. We analysed 12 RNA-seq datasets (GSE217932) using the SUVA pipeline to identify splicing alterations in Sham, chronic constriction injury (CCI) and CCI treated with L-tetrahydropalmatine (CCI_THP) groups. Co-expression and co-disturbance analyses were performed to define splicing-associated RBP networks. Key findings were further validated in a CCI rat model by behavioural testing, junction-specific RT-qPCR, immunohistochemistry and immunofluorescence. Distinct splicing profiles were observed among the three groups. Differentially spliced genes were enriched in pathways related to axonogenesis, cell junction assembly and synapse assembly. Several genes, including Lrrc4c, Shank2, Nefh, and Grid2, showed aberrant splicing in CCI, which was partially reversed by THP treatment. K-means clustering supported a role for THP in restoring synaptic remodelling-related splicing alterations. In addition, Nefh splicing was associated with Rbm47 and Grn expression, and multiple RBPs, including Rbm47, Grn, Lcp2, Plek, Ptpn6 and Hcls1, were significantly upregulated in CCI. These results were supported by in vivo validation of selected splicing events and protein changes. This study provides a transcriptome-wide view of alternative splicing and RBP dysregulation in NP, and suggests that THP may alleviate neuropathic pain partly by modulating aberrant splicing programs. These findings offer insight into NP mechanisms and identify potential therapeutic targets.