Figure 3.

Dipyrone inhibits the release of apoptogenic factors from mitochondria, thereby rescuing cells from cell death. PCNs were pretreated for 2 hours with 1 μM dipyrone and challenged by 3-hour OGD. 16 hours after completion of OGD, cells were used for examinations. A, Cell morphology was studied by phase-contrast (200× magnification) and immunofluorescence microscopy (200× and 600× magnification). Apoptosis is evident from the change in the cells’ shape upon OGD. Pretreatment with dipyrone decreases the extent of cell death. Immunostaining with antibodies against MAP2 (red) and DAPI (blue) reveals the cells’ dendrites and nuclei, respectively. Scale bars, 40 μm. B, Dipyrone retains mitochondrial membrane potential (ΔΨ_m) in PCNs after OGD. The living cells were stained with 2 μmol/L rhodamine 123 to determine ΔΨ_m. Green fluorescence resulted from the accumulation of rhodamine 123 within negatively charged mitochondria, and diminished fluorescence, as was observed with apoptotic cells, indicated mitochondrial depolarization and the dissipation of ΔΨ_m. C, D, The release of mitochondrial factors and concomitant activation of caspase in response to OGD was analyzed by western blot. Cells were fractionated to obtain either cytosolic components or total cell lysates, and then used respectively for determination of cytochrome c and AIF (cytosolic fractions, C), or caspase-3 (total lysates, D). β-actin staining was used as an internal loading control. The blots are representative of three independent experiments. E, COX-2 activity was detected by the COX fluorescent assay in cell lysates. OGD caused a statistically significant increase in COX fluorescence in PCNs, and this increase there was not diminished by preincubation with dipyrone. Data are presented as mean ± S.E.M. of four independent experiments. #, P < 0.05 versus null-control group.