Fig. 6

Neuronal STAT3 overexpression evoked pain hypersensitivity and elevated synthesis of proinflammatory cytokines, which was reversed by ACY-1215 administration. (A) The diagram illustrates the experimental timeline for behavioral and molecular tests following the overexpression of STAT3 in neurons of male mice. Mice were intrathecally injected with AAV-hsyn-STAT3-EGFP (AAV-STAT3) or control AAV virus (Vacant), followed by daily intraperitoneal injections of ACY-1215 or vehicle (10% DMSO in saline) from Day 21 to Day 31. Baseline (BL) thresholds were measured the day preceding any treatment. (B) Mechanical threshold and withdrawal latency data for male mice. Two-way ANOVA followed by Tukey’s post hoc tests (n = 8 mice per group). Data are individual values with means ± SEM. Vacant + DMSO vs. AAV-STAT3 + DMSO ***p < 0.001; AAV-STAT3 + DMSO vs. AAV-STAT3 + ACY-1215 ^#p < 0.05, ^###p < 0.001. (C) Representative western blots of total STAT3, p-STAT3, Ac-STAT3 and tubulin. (D-I) Statistical results showing the relative expressional levels of total STAT3, p-STAT3, Ac-STAT3 and p-STAT3/Ac-STAT3 ratio. (J-L) Western blots (J) and statistical results for protein levels of TNF-α (K) and IL-1β (L). The blot densities were quantified relative to STAT3 or tubulin and normalized as fold change compared to the AAV-Vacant group receiving DMSO. Values represent mean ± SEM; n = 3. Vacant + DMSO vs. AAV-STAT3 + DMSO **p < 0.01, ****p < 0.0001; AAV-STAT3 + DMSO vs. AAV-STAT3 + ACY-1215 ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; Vacant + DMSO vs. Vacant + ACY-1215 ****p < 0.0001. (M-N) ELISA analysis of TNF-α (M) and IL-1β (N) expression levels in L4-L6 spinal cords. Values represent mean ± SEM; n = 5. Vacant + DMSO vs. AAV-STAT3 + DMSO ****p < 0.0001; AAV-STAT3 + DMSO vs. AAV-STAT3 + ACY-1215 ^###p < 0.001. Two-way ANOVA followed by Tukey’s post hoc tests