Figure 3.

Effects of hPH treatment on D-GalN-induced apoptosis in HepG2 cells. (A) HepG2 cells were treated with 25, 50, or 100 mM D-GalN for 24 h. Treatment with 50 mM D-GalN yielded a cell viability of almost 50% compared with the vehicle control. The concentration of 50 mM D-GalN was used to determine the 50% inhibitory concentration. (B) HepG2 cells were treated with increasing concentrations of hPH or vehicle control for 24 h. HepG2 cells were treated with hPH (5%) for 2 h prior to D-GalN stimulation (50 mM). After 24 h, the hepatoprotective effects of hPH were assessed with the (C) Cell Counting Kit-8 assay and (D) LDH release test for cell viability, and with an (E) inverted phase-contrast microscope for morphological changes (scale bar=50 µm). HepG2 cells were stimulated with D-GalN (50 mM, 24 h) in the presence or absence of 5% hPH. (F) Cells were subjected to fluorescence microscopy (PI stain only) and (G) Annexin V/PI staining analyzed by a microplate (scale bar=50 µm). (H) DNA fragmentation (red arrow) and nuclear condensation (white arrow) were detected by DAPI staining under each condition (scale bar=5 µm). For western blot analysis, cell lysates were collected and subjected to sodium dodecyl sulfate-PAGE, followed by immunoblot analysis using anti-BCL2, anti-BAX and anti-PARP antibodies. Anti-GAPDH was used as a loading control. (I) Representative images and (J) densitometry. All data are presented as the mean ± standard error of the mean. ^**P<0.01, vs. D-GalN group. hPH, human placental hydrolysate; D-GalN, D-galactosamine; Cont, control; LDH, lactate dehydrogenase; BCL2, B-cell lymphoma 2; BAX, Bcl-2-associated X protein; PARP, poly (ADP) ribose polymerase; PI, propidium iodide.