S-Allyl Cysteine Alleviates Hydrogen Peroxide Induced Oxidative Injury and Apoptosis through Upregulation of Akt/Nrf-2/HO-1 Signaling Pathway in HepG2 Cells

Figure 5

SAC inhibits H₂O₂ induced apoptosis in HepG2 cells via upregulation of HO-1. Cells were transfected with HO-1 specific siRNA or control siRNA and incubated for 72 h followed by treatment with H₂O₂ for 24 h in the presence or absence of SAC. (a) Western blot with whole cell lysate was used to determine HO-1 protein levels. α-Tubulin was used as a loading control. The HO-1/α-Tubulin ratio was determined using densitometry and the fold protein expression is represented as a bar graph. Densitometry data represents mean ± SD of three independent experiments and a representative western blot gel image is shown below. (b) Cells were stained with Annexin V/PI followed by FACS analysis. Fold difference of apoptosis compared to control was plotted. (c) Mitochondrial membrane potential (∆Ψm) was estimated by JC-1 staining followed by FACS analysis. The ratio of aggregate and monomeric form of JC-1 was plotted. (d) HO-1 specific siRNA or control siRNA transfected cells were treated with H₂O₂ for 2 h with or without treatment with SAC. ROS level was measured by DCFH₂-DA staining followed by FACS analysis and represented as DCF fluorescence. Fluorescence intensities were plotted as a bar graph. P<0.01 and P<0.001 compared to the control group. ##P<0.01 and ###P<0.001 compared to the H₂O₂ treated group. ‡P<0.05, ‡‡P<0.01, and ‡‡‡P<0.001 compared to the control siRNA treated group. All data are represented by mean ± SD of three independent experiments.

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