Figure 3

Fuzheng Huayu recipe (FZHY) inhibits Act D/TNF-α induced hepatocyte apoptosis. Z-VAD-FMK (FMK) is a synthetic peptide that irreversibly inhibits activity of caspase family proteases and blocks apoptosis, and was used as a positive control in this work. Twenty four hours after plating, primary hepatocytes were pretreated with 10% FZHY medicated serum or 10% rats’ serum plus 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. (A) Confocal microscopy observation of hepatocyte apoptosis stained by FITC labeled Annexin V (green) and propidium iodide (PI) (red) (×400). (B) Cells were trypsinized and stained with Annexin V and PI followed by analysis with flow cytometry. Early apoptotic cells (Annexin V positive and PI negative) were in the right lower quadrant. Late apoptotic or necrotic cells were in the right upper quadrant. (C) The apoptotic index (AI) of five high-power fields at × 400 magnification of confocal microscopic figures in Figure 3A. ^** P < 0.01 vs. control group; ^## P < 0.01 vs. Act D/TNF-α treated group. (D) Primary hepatocytes were pretreated with 10% FZHY medicated resum or 10% rats’ serum + 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. Cells were harvested, and DNA was purified by the DNeasy Kit. DNA was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light. Lane 1, marker; lane 2, phosphate-buffered saline (PBS) treated cells; lane 3, PBS and Act D/TNF-α treated cells; lane 4, FZHY and Act D/TNF-α treated cells; and lane 5, Z-VAD-FMK and Act D/TNF-α treated cells. Agarose gel electrophoresis showed that FZHY and Z-VAD-FMK inhibited apoptosis of hepatocytes induced by Act D and TNF-α.