Fig 2: CRAE induced to the M1-type macrophage polarization and the M1-type polarization markers were inhibited by the TLR4 protein inhibitors TAK-242 and NF-κB inhibitor Bay 11–7085 (Bay). (A) Protein expression levels of M1 macrophages markers and M2 macrophages markers were analyzed and semi-quantified through WB. (B) The corresponding expression of CD163, iNOS, CD86 and IL-1β levels are shown as histograms (one-way ANOVA or two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (C) The expression level and localization of p65 protein in macrophages were detected by immunofluorescence assay. Magnification, ×400.

Fig 3: CRAE induced phenotypic switch of macrophages from M2 polarization to M1 polarization in vivo and in vitro. (A) Detection of protein expression and localization of CD86 and F4/80 in tumor tissues were determined using an immunofluorescence assay. Magnification, ×100. (B) Immunohistochemistry results showed that CRAE decreased the expression level of CD163 and Arg1, increased the expression level of CD86 and iNOS compared with the Vehicle group. Magnification, ×200. (C) The expression levels of CD86 and iNOS (M1-like macrophage markers), and the expression levels of CD163 and Arg1 (M2-like macrophage markers) were detected by WB in vivo. (D) The expression of CD163, Arg1, CD86 and iNOS levels of macrophages in tumor tissue are displayed as histograms (one-way ANOVA or two-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001). (E and F) Flow cytometry were used to detect the percentages of CD86+ cells or CD163 cells of BMDM-M2 in vitro (****P < 0.0001).

Fig 4: NC009-1 down-regulated neuroinflammation and up-regulated cellular redox signaling in MPTP-treated mice. (A) Expression levels of striatal IL-1β, IL-6, and TNF-α analyzed by ELISA (n = 6). Expression levels of (B) NLRP3, CASP1, iNOS, and (C) SOD2, NRF2, and NQO1 in striatum analyzed by Western blot (n = 6). GAPDH was included as a loading control. To normalize, the relative NLRP3, CASP1, iNOS, SOD2, NRF2, and NQO1 of control mice was set as 100%. p values: comparisons between MPTP and control (# p < 0.05, ## p < 0.01, ### p < 0.001), or NC009-1-treated and untreated MPTP mice (* p < 0.05, ** p < 0.01).

Fig 5: MPP+-induced inflammation of HMC3 microglia and anti-inflammatory potentials of NC009 compounds. (A) Cytotoxicity of MPP+ against HMC3 cells by MTT and NO release assays. Cells were treated with MPP+ (0–10 mM) and cell viability and NO release in culture medium was measured the following day (n = 3). To normalize, the relative cell viability in MPP+-untreated (inactive) cells was set at 100%. p values: comparisons between inactive and activated cells (* p < 0.05, *** p < 0.001). (B) Experimental flow chart. HMC3 cells were plated on day 1. After 20 h, cells were pre-treated with test compounds (1–10 μM) for 8 h, followed by MPP+ (3 mM) treatment for 20 h. On day 3, the HMC3 cells were examined for cell viability (MTT assay) and NO release (Griess assay) in culture medium. In addition, cells with 10 μM compound treatment were examined for CD68 and CD11B expression (ICC staining), IL-1β, IL-6, TNF-α release (ELISA), and NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α expression (Western). (C) Cell viability and NO release assays (n = 3). The relative cell viability in MPP+-untreated cells was normalized as 100%. (D) CD68 and CD11B expression (n = 3). Cells were analyzed by immunofluorescence using antibodies against CD68 (yellow) and CD11B (green). Cell nuclei were counterstained with DAPI (blue). (E) IL-1β, IL-6, and TNF-α release assays (n = 3). (F) NLRP3, CASP1, iNOS, CREB, IL-1β, IL-6, and TNF-α levels analyzed by immunoblotting using GAPDH as a loading control (n = 3). To normalize, protein expression level in MPP+-untreated cells was set at 100%. (C–F), p values: comparisons between MPP+-untreated and treated cells (# p < 0.05, ## p < 0.01, ### p < 0.001), or with and without compound addition (* p < 0.05, ** p < 0.01, *** p < 0.001).