AVE0991, a nonpeptide analogue of Ang-(1-7), attenuates aging-related neuroinflammation

Figures

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  Figure 1. Inflammatory markers are increased in the brains of SAMP8 mice during the aging process. (A) The dynamic changes of Il1b mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. (B) The dynamic changes of Il6 mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. (C) The dynamic changes of Tnf mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. Gapdh was used as an internal control. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and were expressed as a fold change relative to 4-month-old SAMR1 control mice. Columns represent mean ± SD (n=8 per group). *P<0.05. #P<0.05 versus age-matched SAMR1 control mice.

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  Figure 2. Ang-(1-7) levels are decreased in the brains of SAMP8 mice during the aging process. (A) The activity of ACE2 in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were assessed using a specific detection kit. (B) The Ang-(1-7) levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were detected by ELISA. (C) The expression of Mas1 mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were assessed by qRT-PCR. Gapdh was used as an internal control. Data from panel B and C were expressed as a fold change relative to 4-month-old SAMR1 control mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). *P<0.05. #P<0.05 versus age-matched SAMR1 control mice.

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  Figure 3. AVE0991 attenuates neuroinflammation in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. (A) The mRNA levels of Il1b in the brains were investigated by qRT-PCR. (B) The mRNA levels of Il6 in the brains were investigated by qRT-PCR. (C) The mRNA levels of Tnf in the brains were investigated by qRT-PCR. (D) The protein levels of IL-1β in the brains were investigated by ELISA. (E) The protein levels of IL-6 in the brains were investigated by ELISA. (F) The protein levels of TNF-α in the brains were investigated by ELISA. In panel A-C, Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). #P<0.05 versus vehicle-treated SAMP8 mice.

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  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).

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  Figure 5. AVE0991 elevates microglial M2 activation makers in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. (A) The mRNA levels of Arg1 in the brains were investigated by qRT-PCR. (B) The mRNA levels of Il10 in the brains were investigated by qRT-PCR. (C) The mRNA levels of Retnla in the brains were investigated by qRT-PCR. Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). #P<0.05 versus vehicle-treated SAMP8 mice.

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  Figure 6. AVE0991 elevates M2 activation makers in primary microglia. Primary microglia were directly isolated from the brain of 8-month-old SAMP8 mice. They 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. (A) The mRNA levels of Arg1 were investigated by qRT-PCR. (B) The mRNA levels of Il10 were investigated by qRT-PCR. (C) The mRNA levels of Retnla were investigated by qRT-PCR. 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).

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  Figure 7. A model illustrating the effects of AVE0991 on aging-related neuroinflammation and its potential mechanisms.