Oregano Essential Oil Induces SOD1 and GSH Expression through Nrf2 Activation and Alleviates Hydrogen Peroxide-Induced Oxidative Damage in IPEC-J2 Cells

Figure 4

Effects of oregano essential oil (OEO) on nuclear factor-erythroid 2-related factor-2 (Nrf2) activation in IPEC-J2 cells. (a) IPEC-J2 cells were incubated with OEO (2.5–10 μg/mL) for 12 h. Relative Nrf2 mRNA expression levels were measured quantitatively using real-time RT-PCR. (b) IPEC-J2 cells were incubated with OEO 10 μg/mL for 24 h and then Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) were estimated in IPEC-J2 cells. (c) IPEC-J2 cells were incubated with OEO 10 μg/mL for 24 h; then Nrf2 was estimated in the cytosolic and nuclear fractions. Western blot results show the effects of OEO on protein levels of Nrf2 in the cytosolic and nuclear fractions. (d) Localization of endogenous Nrf2. IPEC-J2 cells were incubated with OEO 10 μg/mL for 24 h and then subjected to immunohistochemical staining with antibodies specific for Nrf2 followed by incubation with Alexa Fluor 568-conjugated secondary antibodies. For each condition, an image of the cell nucleus stained with DAPI specific for nuclear proteins is also presented. The immunohistochemical staining was observed using fluorescence microscopy (200x magnification). (e) IPEC-J2 cells were incubated with OEO 10 μg/mL for 24 h; then levels of Nrf2 and Keap1 were estimated in IPEC-J2 cells. (e) IPEC-J2 cells were cotransfected with a reporter plasmid (pGL6) and a control plasmid (pRL-TK). After transfection, IPEC-J2 cells were incubated with OEO (2.5–10 μg/mL) for 12 h; then luciferase activity was measured. Values represent means ± SEM, . and , compared with untreated control cells. Means without a common letter differ, .

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