RNA-seq Analysis Reveals Neuroimmune Remodeling and NTSR1-Related Neuroactive Signatures After Combined rTMS and Press-Tack Needle Stimulation in TBI Rats.

Traumatic brain injury (TBI) is associated with complex transcriptional disturbances involving cortical suppression, neuroimmune responses, and altered neuroactive signaling. Although repetitive transcranial magnetic stimulation (rTMS) and acupuncture-related stimulation have been investigated as neuromodulatory strategies after brain injury, the transcriptomic features associated with combined rTMS and press-tack needle (PTN) stimulation remain incompletely understood. In this study, RNA sequencing was performed on prefrontal cortex tissue from Sham rats, TBI rats, and TBI rats treated with combined rTMS + PTN. Differentially expressed genes (DEGs), functional enrichment, pathway-gene interaction networks, protein-protein interaction networks, and gene set enrichment analysis (GSEA) were used to explore transcriptional alterations associated with TBI and combined intervention. TBI induced marked transcriptional changes in the prefrontal cortex, including genes enriched in neuroactive ligand-receptor interaction, calcium signaling, and injury-related pathways. Combined rTMS + PTN stimulation was associated with additional transcriptional remodeling, particularly involving immune-inflammatory pathways such as NF-κB signaling, IL-17 signaling, cytokine-cytokine receptor interaction, and Toll-like receptor signaling. Intersection analysis identified 55 shared TBI-associated and intervention-responsive genes as a candidate signature. Further PPI and GSEA analyses suggested that Nts/Ntsr1, Egr1, Drd1, and related neuroactive signaling molecules may participate in the transcriptional response to combined stimulation after TBI. These findings provide an exploratory RNA-seq-based framework for understanding NTSR1-associated neuroimmune and neuroactive molecular signatures after TBI. Targeted qRT-PCR assessment of selected neuroactive candidate genes, including Ntsrl, Egrl, and Drd1, provided complementary evidence of group-dependent expression differences in an independent animal cohort. Further protein-level and functional validation will be required to determine the causal relationships among these candidate molecules.