Hydroxysafflor Yellow A Shows Protection against PPARγ Inactivation in Nitrosative Neurons

Figure 2

Protection against SIN-1-induced PPARγ nitration and inactivation by HSYA in primary neurons. (a–c) Inhibition of PPARγ nitration by HSYA in SIN-1-treated neurons. The neurons were incubated with increasing concentrations of HSYA (0.01, 0.1, and 1 mM), 10 min before the addition of SIN-1 (1 mM). After 24 h coincubation, neurons were harvested for analysis of PPARγ nitration and total PPARγ accumulation. GAPDH expression was shown as a loading control. (a) The cell extracts were immunoprecipitated (IP) with antibody specific to nitrotyrosine (Nitrotyr.). The nitrotyrosine immunoprecipitates were successively immunoblotted (WB) with PPARγ Ab. (b) The cell extracts were IP with anti-PPARγ antibody followed by WB with nitrotyrosine Ab. The bar graph illustrates the densitometric analysis of the related bands. Data are expressed as mean ± SEM (). compared to control (untreated) and compared to SIN-1 alone. (d) Restoration of agonist-dependent PPARγ activation by HSYA in SIN-1-treated neurons. Primary neuron cultures were incubated with 1 mM HSYA, 10 min before the addition of 1 mM SIN-1. After 24 h coincubation, cells were treated for an additional 6 h in the absence (filled bars) or presence (open bars) of PPARγ agonist 15d-PGJ2 (5 μM) A or rosiglitazone (Ros) (1 μM) B. Nuclear proteins were extracted, and activated PPARγ was quantified by PPARγ DNA-binding activity utilizing the PPARγ transcription factor assay kit. Data are expressed as mean ± SEM (). compared to control (untreated), compared to PPARγ agonist (15d-PGJ2 or Ros) alone, and compared to SIN-1 plus agonist (15d-PGJ2 or Ros).

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