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 4

SAC activates the Nrf-2/HO-1 pathway via the induction of Akt phosphorylation in H₂O₂ induced HepG2 cells. (a) Cells were treated with 1 mM H₂O₂ for 24 h with or without treatment with SAC. PCR was performed to determine HO-1 mRNA levels. A representative PCR gel is shown. The HO-1/β-actin ratio was determined using densitometry and the fold mRNA expression is represented as a bar graph. Densitometry data represents mean ± SD of four independent experiments. (b) Cells were treated with 1 mM H₂O₂ for 24 h with or without treatment with SAC in the presence or absence of 10 μM wortmannin. Western blot with whole cell lysate was performed to determine HO-1 protein levels. α-Tubulin was used as an internal 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 blot is shown below. (c) Cells were treated with 1 mM H₂O₂ for the indicated time points. Upper panel: Nrf-2 protein levels in both nuclear and cytosolic extracts were determined using western blot analysis. A representative western blot gel is shown. The ratio of the band intensities of nuclear (normalized to that of nuclear Histone H3) and cytosolic (normalized to that of cytosolic α-tubulin) levels of Nrf-2 was determined using densitometry and the fold protein expression is represented as a bar graph. Densitometry data represents mean ± SD of three independent experiments. Lower panel: Western blot with whole cell lysate was used to determine Akt phosphorylation levels using p-Akt antibody and the blot was reblotted for Akt levels (loading control). The band intensities of p-Akt levels were normalized to that of Akt and the data represented as mean ± SD of three independent experiments above a representative western blot. (d) Cells were treated with 1 mM H₂O₂ for 6 h with or without treatment with SAC in the presence or absence of 10 μM wortmannin. Nrf-2 protein levels in both nuclear and cytosolic extracts were determined using western blot analysis. A representative western blot gel is shown. The ratio of the band intensities of nuclear (normalized to that of nuclear Histone H3) and cytosolic (normalized to that of cytosolic α-tubulin) levels of Nrf-2 was determined using densitometry and the fold protein expression is represented as a bar graph. Densitometry data represents mean ± SD of three independent experiments. (e) Cells were treated with 1 mM H₂O₂ for 6 h with or without treatment with SAC in the presence or absence of 10 μM wortmannin. Western blot with whole cell lysate was used to determine Akt phosphorylation levels using p-Akt antibody and the blot was reblotted for Akt levels (loading control). The band intensities of p-Akt levels were normalized to that of Akt and the data represented as mean ± SD of three independent experiments above a representative western blot. P<0.05 and P<0.01 P<0.001 compared to the control group. ##P<0.01, ###P<0.001 compared to the H₂O₂ treated group. ‡‡P<0.01 and ‡‡‡P<0.001 compared to H₂O₂ and SAC treated group.

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