Figure 7

HCT1026 inhibits MPTP-induced increased Mac-1, PHOX, iNOX and 3-Nitrotyrosine expression in SNpc. A-B: Western blotting of phagocyte oxidase PHOX (A) and Mac-1 (B) within the VM at different time-intervals after saline or MPTP injection in mice fed with the control or HCT1026 diet. The data from experimental bands were normalized to β-actin, before statistical analysis of variance. Values are expressed as % of saline-injected controls. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. * p < 0.05 compared to saline; ° p < 0.05 vs MPTP fed with the control diet. C. Semi-quantitative RT-PCR for iNOS. The 250 ng of cDNA were used for PCR, by using Super Taq DNA polymerase with specific primer pairs for iNOS (500 bp) and Classic GADPH Standard (270 bp). Samples from PCR reactions were processed as described. Fluorescent bands of amplified gene products were analyzed, the values normalized against GADPH and ratios expressed as % of control, within each experimental group (C), see text for details. Differences were analyzed by ANOVA as above. ** p < 0.05 vs saline; ° p < 0.05 vs MPTP + control diet. D: Mean numbers of Mac1⁺iNOS⁺ cells within the SNpc in saline and MPTP mice fed with the control or HCT1026 diet. Cell counts obtained as described in Material section. E-F: Representative confocal images showing double staining with Mac-1 (green) and iNOS (red) in MPTP mice fed with the control (E) or HCT1026 (F) diets. G: Percent (%) of TH⁺ neurons colocalizing with 3-nitrotyrosine (3-NT), a peroxynitrite footprint. Dual stained TH⁺ 3-NT⁺ neurons were counted as described and values expressed as % of total TH⁺ neurons. H-I: Representative confocal images showing dual immunostaining with 3-NT (green) and TH (red) in MPTP mice fed with a ct diet (H) showing that a large proportion of DAergic neurons colocalize (orange-to-yellow) as opposed to TH neurons of mice fed with HCT1026 (E) where no colocalization was observed in the large part of SNpc neurons.