Tryptase Delta 1 Gene Induces Neuropathic Pain through Activation of Microglia by NF-κB Signaling Pathway

Abstract

Background: The pathogenesis of neuropathic pain (NP) remains a mystery, and no ideal treatment methods or prevention measures exist. Our goal was to identify potential therapeutic targets for NP using the RNA sequencing (RNA-Seq) dataset GSE126611 from the Gene Expression Omnibus (GEO) database and experimentally validate the potential target. Methods: Dataset GSE126611 was used for screening differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment analyses were performed on the DEGs and key modules. Tryptase delta 1 (TPSD1) was screened out for experimental validation. A chronic constriction injury (CCI) model of a rat was used to induce NP. The levels of mechanical nociceptive threshold (MNT) and thermal pain threshold (TPT) of rats were measured on days 1, 7 and 14 after modeling. On day 14, levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and transforming growth factor (TGF)-β in serum were measured by enzyme-linked immunosorbent assay (ELISA). Expression levels of TPSD1 in the meningeal tissue, p65 and NF-κB (nuclear factor kappa-B) inhibitor alpha (IκBα) in the spinal dorsal horn tissue were measured by quantitative PCR (qPCR) and Western blot. Immunofluorescence measured the expression of ionized calcium-binding adapter protein 1 (Iba1) in the spinal dorsal horn tissue. TPSD1 and NF-κB inhibitor BAY 11-7082 were used to induce microglia. Cell supernatant was collected for IL-1β, TNF-α, IL-6, IL-10, and TGF-β detection. Cells were collected for p65, phosphorylated p65 (p-p65), IκBα, phosphorylated IκBα (p-IκBα), Iba1, CD86, and CD206 expression levels detection. Results: In vivo results showed that compared with the sham group, the levels of MNT, TPT, TGF-β, IL-10, IκBα, and TPSD1 in the CCI group were notably down-regulated, whereas the levels of IL-1β, TNF-α, IL-6, p-IκBα, and Iba1 were obviously up-regulated. Besides, we cultured microglia with TPSD1, and results showed that compared to the conventional culture microglia, the levels of TGF-β, IL-10, p-p65, p-IκBα, and M1 marker CD86 were significantly decreased, while the levels of IL-1β, TNF-α, IL-6, IκBα, and M2 marker CD206 were significantly increased. Conclusions: The TPSD1 gene induces the activation of the NF-κB signaling pathway, which activates microglia and polarizes them toward the M1 phenotype, leading to the secretion of pro-inflammatory factors and causing pain. The findings suggest that the management of neuropathic pain could be improved by targeting TPSD1.