Fig 1.
Effects of agmatine on cell viability, NO production and iNOS expression in LPS-induced RAW 264.7 cells.
(A) Cell viability; after treatment in 96-well plates with various doses of agmatine for 24 h, the MTT-based cytotoxicity assay was performed. Absorbance value was read at 570 nm. Results are expressed as the mean ± SEM from three independent experiments. Data were analyzed by one-way ANOVA. (B) NO production; RAW264.7 cells, seeded at a density of 2×105 cells/well in 24-well plates for 24 h, were treated with or without LPS (10 μg/mL) and co-incubated with agmatine (1 mM, 30 min following LPS treatment). The culture medium was removed to measure NO according to the Griess protocol. (C) iNOS detection; cells were seeded at a density of 5×106 cells/well into 10 cm plates, treated with LPS and agmatine, and assayed for iNOS expression at 24 h.
Fig 2.
Agmatine therapeutically reduces LPS-induced ROS generation.
ROS detection was performed using a fluorescence probe. (A) RAW264.7 cells were cultured in RPMI 1640 for 24 h followed by incubation with the DCFH-DA probe (20 μmol/L) at 37°C for 30 min. (B) RAW264.7 cells were stimulated with 10 μg/mL LPS for 24 h followed by incubation with the DCFH-DA probe for 30 min. (C) RAW264.7 cells were treated with 1 mM agmatine for 24 h. (D) RAW264.7 cells were incubated with 10 μg/mL LPS and 1 mM agmatine added 30 min later for 24 h, then the DCFH-DA probe was used for 30 min. (E) ROS production was analyzed by flow cytometry. A minimum of 10000 events/sample were acquired. Results were means ± SEM of three independent experiments and were assessed by one-way ANOVA. * P < 0.05 indicates significant differences compared with the control group.
Fig 3.
Agmatine induces an up-regulation of HO-1 and activates the transcription factor Nrf2.
RAW264.7 cells were incubated with 10 μg/mL LPS for 30min, and then 1 mM agmatine was added into the medium and incubated for additional (A)12h, (B)24h or (C)36h, respectively. Then the mRNA expressions of HO-1 and NQO-1 were analyzed by qPCR. (D and E) RAW264.7 cells were incubated with 10 μg/mL LPS and 1 mM agmatine added 30 min later for 24h(D) and 6 h(E), and the protein expression of Nrf2 was analyzed by western blotting. Results were expressed as mean ± S.E.M (n = 3) and were assessed by one-way ANOVA. * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group.
Fig 4.
RAW264.7 cells were incubated with 10 μg/mL LPS and 1 mM agmatine added 30 min later for 24 h. The DNA binding activity of Nrf2 was measured by EMSA as described in the Materials and Methods section.
Fig 5.
PI3K/Akt signal regulates agmatine-induced Nrf2 activation and HO-1 expression in RAW264.7 Cells.
(A) RAW264.7 cells were treated with 1 mM agmatine for 24 h, and total Akt and phospho-Akt (p-Akt) levels were analyzed by western blotting. (B, C) RAW264.7 cells were pretreated for 1 h with 10 μM LY294002, and total Akt, p-Akt and Nrf2 expression were analyzed by western blotting. (D) HO-1 mRNA expression after LY294002 pretreatment were analyzed by qPCR. Results are means ± SEM of three independent experiments and were assessed by one-way ANOVA. *P < 0.05 and **P < 0.01 indicate significant differences compared with the agmatine-treated group.
Fig 6.
Agmatine inhibits LPS-induced ROS generation via the PI3K/Akt pathway.
(A) RAW264.7 cells were cultured in RPMI 1640 for 24 h followed by incubation with the DCFH-DA probe (20 μmol/L) at 37°C for 30 min. (B) RAW264.7 cells were stimulated with 10 μg/mL LPS for 24 h followed by incubation with the DCFH-DA probe for 30 min. (C) RAW264.7 cells were treated with 1 mM agmatine for 24 h. (D) RAW264.7 cells were incubated with 10 μg/mL LPS and 1 mM agmatine added 30 min later for 24 h, and then ROS was detected with the DCFH-DA probe for 30 min. (E) RAW264.7 cells were pretreated for 1 h with 10 μM LY294002, and then incubated with 10 μg/mL LPS and 1 mM agmatine added 30 min later for 24 h. (F) ROS production was analyzed by flow cytometry. Results are means ± SEM of three independent experiments and were assessed by one-way ANOVA. * P < 0.05 and ** P < 0.01.
Fig 7.
agmatine exhibits its antioxidant effects on inflammatory macrophages mainly through a HO-1-dependent manner.
(A) Raw 264.7 cells were cultured as described in Fig 2 legend and were treated with ZnPP (1 μm) for 1 h before incubation with LPS (10 μg/mL) and agmatine (1 mM) added 30 min later for 24 h. HO-1 protein expression was analyzed by western blotting. (B and C) Cells were treated as described in A. ROS was labeled by DCFH-DA probe. ROS level was assessed by fluorescence microscopy (B) and flow cytometry (C). (D) Bilirubin accumulated over time was measured in the supernatant of Raw 264.7 cells 24h after exposure to increasing concentrations of agmatine (0.25, 0.5 and 1 mM) and is shown here as fold increase over the control.(E) Nitrite levels in the supernatant were evaluated using a colorimetric assay based on the Griess reaction. Data represent the mean ± SEM of 3 independent experiments per group and were assessed by one-way ANOVA. * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group.
Fig 8.
Membrane receptors contribute to the anti-oxidant effects of agmatine in RAW264.7 cells.
RAW264.7 cells were treated with 10 μg/mL LPS for 30 min and then co-incubated with agmatine (1 mM) for 24 h in the presence of 100 μM efaroxan (EFA), 100 μM idazoxan (IDA) or 100 μM yohimbine (YOH). (A, B and C) HO-1 mRNA expression after intervention was analyzed by qPCR. None of these inhibitors blocked the protective effects of agmatine. Each column represents the mean ± SEM of three independent experiments. Statistical analysis was performed by one-way ANOVA.