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Figure 4 | Journal of Neuroinflammation

Figure 4

From: Inhibitory effect of a tyrosine-fructose Maillard reaction product, 2,4-bis(p-hydroxyphenyl)-2-butenal on amyloid-β generation and inflammatory reactions via inhibition of NF-κB and STAT3 activation in cultured astrocytes and microglial BV-2 cells

Figure 4

Effects of 2,4-bis( p -hydroxyphenyl)-2-butenal on protein expressions of iNOS and COX-2, and on NF-κB-dependent luciferase activity and NF-κB DNA binding activity in astrocytes and microglial BV-2 cells. Astrocytes (A) and microglial BV-2 cells (E) were treated with 1 μg/ml of LPS alone, or with LPS plus different concentrations (1, 5, 10 μM) of 2,4-bis(p-hydroxyphenyl)-2-butenal, at 37°C for 24 h. Equal amounts of total protein (40 μg/lane) were subjected to 10% SDS-PAGE, and the expression of iNOS and COX-2 were detected by western blotting using specific antibodies. β-Actin protein was used here as an internal control. Similar results were obtained from at least three different sets of experiment. Astrocytes were transfected with a p-NF-κB-Luc plasmid (5 × NF-κB), and then treated with LPS (1 μg/ml) either alone or with 2,4-bis(p-hydroxyphenyl)-2-butenal (1, 2, 5 μM) for 37°C for 8 h. Luciferase activity was then determined as described in Methods (B). Astrocytes (C) and microglial BV-2 cells (F) were treated with 1 μg/ml of LPS alone, or with LPS plus different concentrations (1, 5, 10 μM) of 2,4-bis(p-hydroxyphenyl)-2-butenal at 37°C for 1 h. Activation of NF-κB was investigated using EMSA as described in Methods. Nuclear extracts from astrocytes treated with LPS alone (1 μg/ml) or with 2,4-bis(p-hydroxyphenyl)-2-butenal (1, 2, 5 μM) and LPS were subjected to DNA binding reaction with 32P end-labeled oligonucleotide specific to NF-κB. Specific DNA binding of the NF-κB complex is indicated by an arrow. Similar results were obtained from at least three different sets of experiments. Equal amounts of nuclear extract (40 μg) were subjected to 10% SDS-PAGE. Nuclear translocation of p50 and p65, and degradation of IκB were detected by western blotting using specific antibodies. β-Actin protein was used here as an internal control (D). Values represent the mean ± S.E. for three independent experiments performed in triplicate, and each luciferase activity was calibrated using the amount of protein. * indicates significantly different from the LPS-treated group (P < 0.05).

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