Fig. 4

NLRP3 inhibition neutralizes the protective effect of rebamipide following α-synuclein + MPP⁺ intoxication. Overview of the experimental design. A BV2 microglia cells were treated with NLRP3 inflammasome inhibitor (MCC950) for 6 h, treated with rebamipide for 1 h, and stimulated with α-synuclein + MPP⁺ for an additional 11 h. IL-1ß (B) and IL-18 (C) levels were evaluated using enzyme-linked immunosorbent assay kits (ELISA). Overview of the experimental design (D). Effects of NLRP3 on IL-1ß and IL-18 regulation (E) in NLRP3 siRNA-transfected BV2 microglia cells. NLRP3 inhibition neutralized the protective effect of rebamipide following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. An MCC950 or saline was injected once daily in the MPTP intoxication model before rebamipide treatment. Overview of the experimental design. F Microglial activations were visualized 3 d after the last MPTP treatment using Iba-1-specific immunostaining. Iba-1-immunopositive microglia in the substantia nigra pars compacta (SNpc) were counted 3 d after the last MPTP treatment (G and N). IL-1ß (H) and IL-18 (I) levels were evaluated using enzyme-linked immunosorbent assays (ELISA). Moreover, dopaminergic neurons were visualized 7 d after MPTP injection, using tyrosine hydroxylase (TH)-specific immunostaining. TH-immunopositive neurons in the SNpc (J and O) were counted, and the relative TH fluorescence intensity in the striatum (ST) (K and O) was measured. Dopamine levels in the ST were measured using HPLC (L). Latency time on the rotarod was recorded 7 d post-MPTP injection, with a 300 s cutoff limit (M). Data are expressed as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, ***p < 0.001, compared with the control group; # p < 0.05, ## p < 0.01, ###p < 0.001, compared with the α-synuclein + MPP + or MPTP-treated group