Fig 2: Effects of RBE treatment on protein expressions of (a) iNOS, (b) TNF-a, (c) IL-1a, and (d) IL-6 in LPS-stimulated J774A.1 macrophage-like cells. Expression levels of targeted proteins were normalized against reference control (ß-actin). Data are presented as means of fold changes of expressed proteins relative to LPS-stimulated control (0 µg/mL) ± SEM (n = 3). Significant differences are represented by: *p < 0.05; **p < 0.01 (two-tailed unpaired t-test). Corresponding images of developed gel blots were provided as supplementary data in ‘Supplementary A’.

Fig 3: Pregabalin inhibited inflammatory response by directly acting on neurons, rather than microglia or astrocyte in vitro. A Q-PCR analysis of Il-6 and Tnf-a mRNA levels in microglia with pregabalin treatment after radiation. B Q-PCR analysis the effects of pregabalin on Il-1ß, Tnf-a, and Icam-1 mRNA levels in BV2 cells after radiation. C Schematic diagram of microglia incubated with neuron supernatant. D Q-PCR analysis of Il-1ß, Il-6, and Tnf-a mRNA levels in BV2 cells after incubated with the supernatant from pregabalin-treated neurons. E–F Representative immunofluorescent images of IL-6 (E) and TNF-a (F) in BV2 cells among the different groups. Staining with ß-tubulin was used to visualize cytoskeleton. G-H The fluorescence intensity data of IL-6 (G) and TNF-a (H) were imaged by confocal microscopy and analyzed. I ELISA analysis of IL-1ß protein level in the BV2 cells culture supernatant. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 3–4 for each group. Data were presented as mean ± SEM, ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001

Fig 4: Pregabalin inhibited microglial inflammatory response through neuronal HMGB1 signaling pathway. A, B Immunofluorescence staining was used to detect microglial IL-6 and TNF-a expressions under different conditions in the co-culture system of neurons and microglia. C, D Statistical analysis of IL-6 and TNF-a expressions among the different groups. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 4 per group for immunofluorescence staining in vitro. Data were presented as mean ± SEM, ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001

Fig 5: Pregabalin protected neuron from radiative microglia-mediated damage. A Schematic diagram of the in vitro experiments. B Representative images of MAP-2 and ß-tubulin co-labeling in neurons. C Statistics of the neuronal dendric numbers among the different groups. D Q-PCR analysis of Il-1ß, Il-6, Tnf-a, Cox-2, iNos, and Icam-1 mRNA levels in BV2 cells incubated with supernatant from injured or non-injured neuron. E Schematic diagram of radiation-induced secondary damage of neurons. F Schematic diagram of the co-culture system between microglia and neurons. G Q-PCR analysis of Il-1ß, Il-6, and Tnf-a mRNA levels in microglia after co-culture with neurons. H-I ELISA analysis of IL-1ß and TNF-a protein levels in microglia after co-culture with neurons. J Representative images of MAP-2 and ß-tubulin co-labeling in neurons after co-culture with microglia. K Statistics of the neuronal dendric numbers. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 3–6 for each group. Data were presented as mean ± SEM, ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. Four groups were established for in vitro study: control cells (Con), control cells treated with pregabalin (PGB), irradiated cells (Rad), and irradiated cells treated with pregabalin (Rad + PGB)