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

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)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, IL-10, and transforming growth factor (TGF)-beta in serum were measured by enzyme-linked immunosorbent assay (ELISA). Expression levels of TPSD1 in the meningeal tissue, p65 and NF-kappa B (nuclear factor kappa-B) inhibitor alpha (I kappa B alpha) 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-kappa B inhibitor BAY 11-7082 were used to induce microglia. Cell supernatant was collected for IL-1 beta, TNF-alpha, IL-6, IL-10, and TGF-beta detection. Cells were collected for p65, phosphorylated p65 (p-p65), I kappa B alpha, phosphorylated I kappa B alpha (p-I kappa B alpha), Iba1, CD86, and CD206 expression levels detection.Results: In vivo results showed that compared with the sham group, the levels of MNT, TPT, TGF-beta, IL-10, I kappa B alpha, and TPSD1 in the CCI group were notably down-regulated, whereas the levels of IL-1 beta, TNF-alpha, IL-6, p-I kappa B alpha, and Iba1 were obviously upregulated. Besides, we cultured microglia with TPSD1, and results showed that compared to the conventional culture microglia, the levels of TGF-beta, IL-10, p-p65, p-I kappa B alpha, and M1 marker CD86 were significantly decreased, while the levels of IL-1 beta, TNF-alpha,Conclusions: The TPSD1 gene induces the activation of the NF-kappa B 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.

Automated summary

By using RNA sequencing and experimental validation, this study identified that TPSD1 gene induces the activation of the NF-kappa B signaling pathway, leading to the activation and M1 polarization of microglia, secretion of pro-inflammatory factors, and ultimately resulting in neuropathic pain. Targeting TPSD1 could improve the management of neuropathic pain.