Stimulation of Angiotensin II Type 2 Receptor Modulates Pro-Inflammatory Response in Microglia and Macrophages: Therapeutic Implications for the Treatment of Stroke

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  Figure 1. Dose-dependent effect of Compound 21 on C8-B4 microglia and RAW 264.7 macrophages demonstrate acceptable cytotoxicity at lower concentrations. The effect of different concentrations of C21 on cell viability was assessed via MTS (a tetrazolium salt that is reduced to purple formazan crystals) viability assay and trypan blue staining. C8-B4 microglia and RAW 264.7 macrophages were seeded into 96-well plates and treated with different concentrations of C21 (1 µM, 10 µM, 20 µM, 50 µM, and 100 µM) for a total of 24 h. Then, cells were incubated with MTS reagent for ~3 h at 37 °C. Absorbance was measured at a wavelength of 490 nm and the percentage of cell survival was calculated accordingly. C8-B4 cell proliferation was assessed using MTS assay (A), and trypan blue staining (B). Cell viability was assessed in RAW 264.7 macrophages via MTS viability assay (C). Values are the mean ± SD of 3–4 independent experiments analyzed by one-way ANOVA. ns p > 0.05, * p < 0.05, ** p < 0.01.

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  Figure 2. Compound 21 exhibits a profound anti-inflammatory response in microglia via decreasing the LPS-induced expression of pro-inflammatory cytokines/chemokines in a dose-dependent manner. C8-B4 mouse microglia cells were activated by LPS treatment (100 ng/mL) and co-treated with either a different concentration of C21 as indicated or the vehicle for 24 h. An amount of 500 ng of total RNA was reverse transcribed into complementary cDNA, and then gene expression was detected using SYBR Green-based RT-qPCR. PPIA was used as an endogenous control. As for protein expression, supernatants of cell culture medium were collected from the same experiment and centrifuged at 2000× g × 10 min for complete removal of cell debris and then the secreted cytokines/chemokines were assessed using ELISA kits. The mRNA expression level of pro-inflammatory cytokines and chemokines IL-1β (A), IL-12b (B), and IL-6 (C). The protein expression level of the pro-inflammatory cytokine, IL-1β (D). Values are the mean ± SD of 3–5 independent experiments analyzed by one-way ANOVA. ns p > 0.05, * p < 0.05, ** p < 0.01.

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  Figure 3. Compound 21 significantly ameliorates LPS-induced pro-inflammatory response (cytokine production) in macrophages in a dose-dependent manner. RAW 264.7 macrophage cells were cultured with LPS (100 ng/mL) and co-treated with different concentrations of C21, as indicated, or vehicle for 24 h. An amount of 500 ng of total RNA was reverse transcribed into complementary DNA, and then gene expression was detected using SYBR Green-based RT-qPCR. PPIA was used as an endogenous control. Protein expression was evaluated via ELISA. Cell culture supernatants were collected following a similar experimental design and centrifuged at 2000× g × 10 min for complete removal of cells’ debris, and then the secreted cytokines/chemokines were assessed. The mRNA expression level of pro-inflammatory cytokines and chemokines IL-1β (A), TNF-α (B), and CXCL1 (C). The protein expression level of pro-inflammatory cytokine/chemokines IL-1β (D), TNF-α (E), and CXCL1 (F). Values are the mean ± SD of 3–4 independent experiments analyzed by one-way ANOVA. ns p > 0.05, * p < 0.05, ** p < 0.01.

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  Figure 4. Compound 21 effectively regulates the production of NO and the activity of pro-inflammatory enzymes, iNOS and COX-2, in microglia. C8-B4 cells were incubated with LPS (100 ng/mL) and co-cultured with different concentrations of C21 as indicated for 24 h. The mRNA expression level of pro-inflammatory mediators, COX-2 (A) and iNOS (B). The Griess assay kit was used to quantify NO production. Cell culture supernatant was collected and centrifuged to eliminate cell debris, and then mixed with Griess reagent for 30 min at room temperature. The 548 nm absorbance was measured. Sodium nitrate was used as a reference. The level of nitrate (µM) was recorded using a microplate reader (C). Results are the mean ± SD of 3–5 independent experiments analyzed by one-way ANOVA. ns p > 0.05, * p < 0.05, ** p < 0.01.

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  Figure 5. Compound 21 regulates the generation of ROS and NO, and the activity of pro-inflammatory enzymes, iNOS and COX-,2 in macrophages. RAW 264.7 macrophage cells were incubated with LPS (100 ng/mL) and/or different concentrations of C21, as indicated, for 24 h. An amount of 500 ng of total RNA was reverse transcribed into complementary DNA, and then gene expression was detected using SYBR Green-based RT-qPCR. PPIA was used as an endogenous control. The mRNA expression level of pro-inflammatory mediators COX-2 (A) and iNOS (B). Nitrate level (μM) was evaluated by Griess assay kit (C). After 24 h of treatment with LPS (100 ng/mL) and/or C21, the cell supernatants were centrifuged and incubated with Griess reagent. Absorbance was measured at 548 nm and sodium nitrate was used as a reference. For ROS production, cells were cultured with LPS (100 ng/mL) and co-treated with either different concentrations of C21 or vehicle for 24 h. Then, cells were washed with PBS and treated with 5 µM CellROXGreen Reagent for 30 min (D). Fluorescence images were obtained using a Zeiss Observer Z1 microscope (Carl Zeiss). Values are the mean ± SD of 3–5 independent experiments analyzed by one-way ANOVA.…

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  Figure 6. Compound 21 promotes the expression of neurotrophic growth factors (BDNF and GDNF) in a dose-dependent manner. RAW 264.7 macrophage and C8-B4 microglial cells were incubated with LPS (100 ng/mL) +/− C21 for a total of 24 h. mRNA expression levels of neurotrophic factors were analyzed using SYBR Green-based RT-qPCR. PPIA was used as an endogenous control. The mRNA expression level of neurotrophic factors GDNF (A) and BDNF (B) in RAW 264.7 macrophages (A,B), and of GDNF in C8-B4 microglia (C). Values are the mean ± SD of 5 independent experiments analyzed by one-way ANOVA. ns p > 0.05, ** p < 0.01.

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  Figure 7. Schematic representation of the working hypothesis on Compound 21-mediated effects on microglia and macrophages on oxidative stress and cytokine release as determined by ELISA and quantitative real-time rt-PCR analysis. ↑ upregulation.