Hydroxysafflor Yellow A Shows Protection against PPARγ Inactivation in Nitrosative Neurons

Figure 5

The inhibitory effects of HSYA on PPARγ nitration and inactivation in the SIN-1-injected rat hippocampus. (a) Inhibition of SIN-1-induced PPARγ nitration by HSYA in the hippocampus of rats. Rats were treated with increasing doses of HSYA (1, 5, or 10 mg/kg), 30 min before SIN-1 (25 mM) injection. The effects of HSYA were evaluated at 24 h after SIN-1 injection. The protein levels of nitro-PPARγ and PPARγ were characterized by immunoprecipitation or Western blotting. The bar graphs illustrate the densitometric analysis of the related bands. Data are expressed as mean ± SEM (). compared to control (untreated) and compared to SIN-1 alone. (b) Prevention of PPARγ inactivation by HSYA in the SIN-1-injected hippocampus. Rats were injected with HSYA (10 mg/kg), SIN-1 (25 mM), or their combination as described in Materials and Methods. At 24 h postinjection, rats were treated intracerebroventricularly (ICV) with either vehicle (filled bars) or 10 μg 15d-PGJ2 (open bars). Hippocampal nuclear extracts were prepared for the detection of PPARγ DNA-binding activity 6 h after ICV administration. Data are expressed as mean ± SEM (). compared to control (untreated) and compared to SIN-1 plus 15d-PGJ2. (c, d) Localization of nitrated PPARγ in the SIN-1-injected hippocampus. The rat hippocampus was injected with SIN-1 (25 mM). The neuronal distribution of nitro-PPARγ in the hippocampus was detected by immunofluorescent double labeling at 24 h after SIN-1 injection. (c) Photomicrographs show the colocalization of 3-NT (red) with NeuN (green) in the CA1 and dentate gyrus (DG) areas of the hippocampus. Scale bar, 20 μm. (d) Photomicrographs with increased magnification show the colocalization of PPARγ (green) with 3-NT (red) in the cytoplasm of most 3-NT-positive cells. (e) Rats were treated with HSYA (10 mg/kg), 30 min before SIN-1 (25 mM) injection. At 24 h postinjection, the cell loss was measured by counting the numbers of Hoechst 33258-stained nuclei. Summary bar graph illustrates cell counts in the hippocampus. Data are expressed as mean ± SEM ( fields counted in 6 animals). (f) Inhibition of SIN-1-induced Bcl2 expression by HSYA in the hippocampus of rats. Rats received HSYA treatment of 1, 5, or 10 mg/kg 30 min before SIN-1 (25 mM) injection. The effects of HSYA on the protein levels of Bcl-2, a downstream target of PPARγ signaling, were evaluated by Western blotting at 24 h after SIN-1 injection. The bar graphs illustrate the densitometric analysis of the related bands. Data are expressed as mean ± SEM (). compared to control (untreated) and compared to SIN-1 alone.

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