Figure 4. AVE0991 suppresses microglial-mediated inflammatory response through a MAS1 receptor-dependent manner. Primary microglia were directly isolated from the brain of 8-month-old SAMP8 mice. (A) The localization of MAS1 receptor on microglia was confirmed by double immunofluorescence staining. Scale bar=15 μm. Next, microglia were treated for 4 h with 100 ng/ml LPS with or without 4 h pre-incubation with AVE0991 (1×10-8, 1×10-7 or 1×10-6 M) in the presence or absence of A-779 (1×10-6 M) and were harvested and lysed for analysis. (B) The mRNA levels of Il6 were investigated by qRT-PCR. (C) The mRNA levels of Ilb were investigated by qRT-PCR. (D) The mRNA levels of Tnf were investigated by qRT-PCR. In panel B-D,Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated microglia. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=4-6). *P<0.05 versus non-treated microglia. #P<0.05 versus microglia incubated with LPS alone. ⸹P<0.05 versus microglia treated with LPS+AVE0991 (1×10-7M).