Article Figures & Data
Figures
- Fig. 1.
Chemical structure of FCPR03 and BV-2 microglial cells’ viability after pretreatment with FCPR03. Chemical structure of FCPR03 (A). BV-2 microglial cells were pretreated with various concentrations of FCPR03 (5, 10, and 20 μM) or rolipram (20 μM) for 1 hour before stimulation with LPS (1 μg/ml) for 24 hours. Cell viability was detected by the MTT assay (B). Values are mean ± S.E.M. (n = 6/group).
- Fig. 2.
The inhibition effects of FCPR03 on TNF-α, IL-1β, and IL-6 production in LPS-induced BV-2 microglial cells. BV-2 microglial cells were pretreated with various concentrations of FCPR03 (5, 10, and 20 μM) or rolipram (20 μM) for 1 hour in the presence of the selective PKA inhibitor H89 (10 μM) for 40 minutes before stimulation with LPS (1 μg/ml) for 24 hours. Then, the levels of TNF-α, IL-1β, and IL-6 in cell-culture supernatants were measured with commercial ELISA kits. Data are expressed as mean ±S.E.M. (n = 6/group). ##P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the LPS group; +P < 0.05 compared with the LPS + FCPR03 (20 μM) group.
- Fig. 3.
FCPR03 increased the level of intracellular cAMP in BV-2 microglial cells. BV-2 microglial cells were treated with various concentrations of FCPR03 for 1 hour (A), cells were pretreated with various concentrations of FCPR03 for 1 hour and then incubated with or without LPS (1 μg/ml) for 24 hours (B), and intracellular cAMP concentrations were measured by ELISA. Results are shown as the mean ± S.E.M. (n = 6/group). #P < 0.05 compared with the control group; *P < 0.05 compared with the LPS group.
- Fig. 4.
FCPR03 promoted the phosphorylation of CREB in BV-2 microglial cells. BV-2 microglial cells were pretreated with 20 μM of FCPR03 for the indicated times (A) or various concentrations of FCPR03 (0, 5, 10, and 20 μM) for 30 minutes (B). BV-2 microglial cells were pretreated with LPS (1 μg/ml) for the indicated times (C). After treatment of FCPR03 (20 μM) for 30 minutes, BV-2 microglial cells were incubated with LPS (1 μg/ml) for another 90 minutes (D). Levels of phosphorylated CREB (pCREB) and total CREB were determined by Western blot. The corresponding quantification data are shown in each panel. Data are expressed as mean ± S.E.M. (n = 3/group). #P < 0.05 compared with the FCPR03 (20 μM) group; *P < 0.05 compared with the control group.
- Fig. 5.
Effect of FCPR03 on NF-κB p65 activation in LPS-stimulated BV-2 microglial cells. NF-κB p65 protein levels in whole cells (A), cytosolic (B), and nuclear (C) were analyzed by Western blot. NF-κB p65 subunit translocation was assessed by immunofluorescence (D). Scale bars = 20 μm. The corresponding quantification data are shown in each panel. glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and histone H3 were used as internal controls. Data are expressed as mean ±S.E.M. (n = 3/group). #P < 0.05, ##P < 0.01 compared with the control group; *P < 0.05 compared with the LPS-treatment group; +P < 0.05 compared with the LPS + FCPR03 (20 μM) group.
- Fig. 6.
Effects of FCPR03 on production of LPS-induced proinflammatory cytokines in the hippocampus and cortex. After 7 consecutive days pretreatment with FCPR03 or rolipram, mice were injected i.p. with saline or 1.2 mg/kg LPS. Brains were removed 24 hours later, with the hippocampus, and the cortex was dissected and homogenized. Levels of the proinflammatory cytokines TNF-α (A), IL-1β (B), and IL-6 (C) were quantified by ELISA kits. Data are expressed as mean ± S.E.M. (n = 6–8/group). ##P < 0.01 compared with the control group; *P < 0.05; **P < 0.01 compared with the LPS group.
- Fig. 7.
Effects of FCPR03 on the cAMP/PKA/CREB signaling pathway in LPS-induced mice. 24 hour after LPS administration, the entire hippocampal and cortical extracts were homogenized, and cAMP levels were determined by ELISA (A). Western blot was used to assess pCREB and CREB protein levels in the cortex (B) and hippocampus (C). The corresponding quantification data are shown in each panel. Data are expressed as mean ± S.E.M. (n = 3/group). ##P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the LPS group.
- Fig. 8.
Effects of FCPR03 on LPS-induced NF-κB activation in the hippocampus and cortex. 24 hours after LPS administration, the total NF-κB p65 protein levels in cortex and hippocampus were detected (A, B), and then the cytosolic and nuclear proteins in the mouse hippocampus and cortex were separated. Cytosolic NF-κB p65 protein levels in the cortex and hippocampus (C, D), as well as nuclear NF-κB p65 protein amounts in the cortex and hippocampus (E, F), were analyzed by Western blot. The corresponding quantification data are shown in each panel. Data are expressed as mean ±S.E.M. (n = 3/group). #P < 0.05; ##P < 0.01 compared with the control group; *P < 0.05, compared with the LPS group.
- Fig. 9.
FCPR03 alleviated the sickness behaviors and cognitive deficits in female mice treated with LPS. After 7 consecutive days of pretreatment with FCPR03 or rolipram, mice were injected i.p. with saline or 1.2 mg/kg LPS; 24 hours after LPS injection, food intake (A) and body weight change (B) were measured. The novel recognition test (C) was carried out, and then the hippocampus and cortex were dissected and homogenized. Levels of the proinflammatory cytokines TNF-α (D), IL-1β (E), and IL-6 (F) were quantified by ELISA assay. Data are expressed as mean ± S.E.M. (n = 6–8/group). ##P < 0.01 compared with the control group; *P < 0.05 compared with the LPS group.
In this issue
FCPR03 Inhibits Neuroinflammation
