Fig. 6

Pregabalin mitigated NF-κB-mediated microglial inflammatory response by inhibiting neuronal HMGB1 cytoplasmic translocation and secretion. A Schematic diagram of CRISPR/Cas9-mediated HMGB1 knockout in neurons. B Western blotting was used to detect the knockout efficiency among different HMGB1 gRNAs in neurons. n = 3 per group for Western blotting analysis. C Co-culture of microglia and neurons after HMGB1 knockout. D Immunofluorescence staining was used to detect CD68 expression, p65 NF-κB nuclear translocation, and the cell body size of primary microglia in the co-culture system. E–G Statistical analysis of CD68 expression, p65 NF-κB nuclear translocation, and the cell body size of microglia among the different groups. n = 5 per group for immunofluorescence staining in vitro. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. Data were presented as mean ± SEM, ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001