Fig. 7: Nerve ligation induces allodynia via TNF-α-impeded spinal Fbxo45-dependent Munc13-1 ubiquitination.

a Intrathecal application of TNF-α-neutralizing antibody (SNL 7D + TNF-α Ab; 100 ng, 10 μL) to SNL rats significantly increased the withdrawal threshold (von Frey test). **P < 0.01 compared with SNL 7D (n = 7). b Intrathecal application of TNF-α-neutralizing antibody (SNL 7D + TNF-α Ab; 100 ng, 10 μL) to SNL rats increased Munc13-1 ubiquitination in the synaptic plasma membrane (SPM) of the ipsilateral dorsal horn. In input control, SPM IP synaptic plasma membrane immunoprecipitation, IB immunoblotting. **P < 0.01 compared with SNL 7D (n = 5). c Representative Western blot and statistical analysis (normalized to N-Cad) demonstrating that intrathecal application of TNF-α-neutralizing antibody (SNL 7D + TNF-α Ab; 100 ng, 10 μL) to SNL rats decreased Munc13-1 expression but increased the Fbxo45 expression in the SPM of the ipsilateral dorsal horn. **P < 0.01 compared with SNL 7D (n = 6). d Intrathecal application of TNF-α (TNF-α, 1 pM, 10 μL) to naive rats significantly decreased the withdrawal threshold at 2 h post injection, which was markedly reversed by pretreating animals with TNF-α-neutralizing antibody (TNF-α + TNF-α Ab; 100 ng, 10 μL) and Munc13-1 siRNA (Munc13-1 siRNA + TNF-α; 3 μg, 10 μL; once daily for 4 days) (von Frey test). **P < 0.01 compared with naive. ^##P < 0.01 compared with TNF-α (n = 7). e Intrathecal injection of TNF-α (TNF-α, 1 pM, 10 μL) into naive animals decreased Munc13-1 ubiquitination in the SPM of the ipsilateral dorsal horn, which was markedly reversed by pretreatment with TNF-α-neutralizing antibody (TNF-α Ab + TNF-α, 100 ng, 10 μL). **P < 0.01 compared with naive. ^##P < 0.01 compared with TNF-α (n = 5). f Immunoprecipitation showing that spinal TNF-α (TNF-α, 1 pM, 10 μL) injection to naive rats significantly increased the abundance of Munc13-1-bound Munc13-1 but decreased the abundance of Munc13-1-bound Fbxo45 in the SPM of ipsilateral dorsal horn, which were markedly reversed by pretreating animals with TNF-α-neutralizing antibody (TNF-α Ab + TNF-α, 100 ng, 10 μL). In input control. **P < 0.01 compared with naive. ^#P < 0.05, ^##P < 0.01 compared with TNF-α. g Representative Western blot and statistical analysis (normalized to N-Cad) demonstrating that intrathecal injection of TNF-α (TNF-α, 1 pM, 10 μL) into naive rats increased Munc13-1 expression but decreased Fbxo45 expression in the SPM of the ipsilateral dorsal horn, which was markedly reversed by spinal pretreatment with TNF-α-neutralizing antibody (TNF-α Ab + TNF-α, 100 ng, 10 μL). **P < 0.01 compared with naive. ^#P < 0.05, ^##P < 0.01 compared with TNF-α (n = 6). Results were expressed as mean ± SEM