Figure 5.

The protective effects of piracetam against the effects of psychostimulants and opioids on the mitochondrial protein expression of SIRT-3 and SIRT-5. Human primary astrocytes were exposed to cocaine (1 µM), METH (10 µM) and morphine (5 µM) alone or in combination with piracetam (10 µM) for 24 h to isolate the mitochondrial fraction. Controls were maintained in drug-free medium (without drug exposure). Representative blots showing the expression of mitochondrial proteins (A) SIRT-3 and (C) SIRT-5. (B) and (D) Densitometric analyses of each protein level to its corresponding COX-IV loading control (fold-change from control). Data are expressed as the means ± SD of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, NS—Nonsignificant. (E) Effects of psychostimulants and opioids on SIRT-3 expression and colocalization. SIRT-3 expression (green), mitochondrial membrane protein COX-IV (red) and nuclear stain DAPI (blue) were observed using confocal microscopy (magnification 100x, scale bar 100 μm). (F) Quantification of SIRT-3 colocalization with the mitochondrial membrane protein COX-IV was analyzed using Pearson’s coefficient r value in three different regions of interest (ROIs) for the same cell. (G) Effects of psychostimulants and opioids on intracellular ATP content. Data are expressed as the means ± SD of luminescent intensity in triplicate. ***P < 0.001, **P < 0.01, *P < 0.05, NS—Nonsignificant. Coexposure of psychostimulant or opioid with piracetam compared to psychostimulant or opioid alone. The supplementary information includes all full-length blots.