Recombinant C8G protein inhibits LPS-induced microglial activation. (A) C8G inhibited LPS-induced NO production in BV-2 microglial cell lines and primary microglia. BV-2 cells were stimulated with LPS (100 ng/ml) for 24 h after a 2 h pretreatment with different concentrations of C8G (n = 3 each). (B) Recombinant C8G protein inhibits the levels of pro-inflammatory cytokines and NO synthase 2 (Nos2), which were upregulated by LPS in microglial cells. After a 2 h C8G pretreatment (1 μg/ml), BV-2 microglia were exposed to LPS (100 ng/ml) for 6 h (n = 3 each). (C) Recombinant C8G protein inhibits TNF-α production in BV-2 cells. After a 2 h C8G pretreatment (1 μg/ml), BV-2 microglia were exposed to LPS (100 ng/ml) for 24 h. The TNF-α protein level in cultured medium of BV-2 cells was assessed using sandwich ELISA (n = 3 each). The assay (n = 3) was repeated at least twice (inter-assay coefficients of variation were < 20%). (D) A schematic of the experiment with an intracerebroventricular LPS-induced neuroinflammation model. C57BL/6 mice (10 weeks old) were administered vehicle (PBS), C8G (1 μg/ml, 3 μl), and/or LPS (10 ng per brain, 3 μl) using a stereotaxic device and microinjector. (E) Immunohistochemistry of hippocampal gliosis. Reactive astrocytes and microglia in the hippocampus were immunostained with anti-GFAP and anti-Iba-1 at 3 days after LPS injection. Scale bar = 400 μm (inset = 50 μm). Quantified graphs of gliosis are shown in the right panels (n = 5 each). (F) Quantification of activated microglia morphology (n = 5 each). (G) Expression of pro-inflammatory cytokines (TNF-α and IL-1β) in the brains of mice (n = 5 per treatment). (H) Sucrose preference test (n = 5 per treatment). Data are shown as mean ± SEM. *P < 0.05 versus control (Con); ^#P < 0.05 versus LPS, unpaired non-parametric test (Mann-Whitney) or Welch’s t-test. White bars = control; grey = C8G; red = LPS; dark green = C8G + LPS.