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Objective To investigate the role and possible mechanisms of transactive response DNA binding protein 43 (TDP-43) in mediating neuronal injury induced by oxidative stress in mouse neuro-2a (N2a) cells and mouse pain sensitization. Methods ① To evaluate the optimal induction concentration, N2a cells were treated with different concentrations of H2O2, and the cells were divided into control group, 200, 400 and 800 μmol/L H2O2 groups. ②To assess the optimal induction duration, N2a cells were treated with 400 μmol/L H2O2, and the cells were divided into control group, and the cell groups treated for 6, 12 and 24 h, respectively. ③To validate the mitochondrial DNA (mtDNA) release pathway, cyclosporin A (CsA) was used to inhibit the mitochondrial permeability transition pore (mPTP), and the cells were divided into control group, 24 h H2O2 group and 24 h H2O2+CsA group. ④To validate TDP-43-mediated cellular damage, the cells were divided into control group, 24 h H2O2 group and 24 h H2O2+siTDP-43 group. ⑤ Cell viability was assessed using CCK-8 assay, while cell proliferation was determined using EdU assay. Western blot analysis was employed to examine the expression levels of TDP-43, neuronal nuclei (NeuN), cyclic GMP-AMP synthase (cGAS), and stimulator of interferon genes (STING). qPCR was utilized to measure the release of mtDNA. Immunostaining was conducted to observe intracellular expression of TDP-43, and Calcein AM staining was employed to evaluate mPTP opening status. ⑥To elucidate the role of TDP-43 in neuropathic pain (NP), 24 healthy SPF male C57BL/6J mice (6~8 weeks old, 25~30 g) were randomly divided into control group, chronic constriction injury (CCI) group, and CCI+siTDP-43 group. In 1 d before and 7, 14 and 21 d after surgery, intrathecal injections of siTDP-43 were administered. Mechanical and thermal pain thresholds of the mice were assessed using von Frey filaments and radiant heat, respectively, on 1 preoperatively and 1, 3, 5, 7, 14 and 21 d postoperatively. Immunofluorescence assay was conducted on 21 d postoperatively to examine the changes in TDP-43 and NeuN in the lumbar spinal dorsal horn (L5-L6). Results Oxidative stress induced a significant increase in TDP-43 protein level in N2a cells, prompted mtDNA release through mPTP, markedly up-regulated the expression of cGAS and STING, and consequently impacted the viability of N2a cells (P |
