PMC

Effect of ZER on intracellular ROS levels and SA-β-gal activity in UVA-irradiated HSF cells, (a) Cells were pretreated with ZER (0, 2, 4, or 8 μM for 24 h) followed by irradiated with UVA in the absence or presence of 3 J/cm² UVA. The accumulation of UVA-induced ROS and SA-β-gal-positive cells was measured using fluorescence microscopy (200x magnification) as described. (b) The data was represented as fold change over control intracellular ROS levels. (c) The data was reported as fold increase over control SA-β-gal-positive cells. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗∗p < 0.001 compared to untreated control cells; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to UVA-irradiated cells.

ZER-mediated protective effect against UVA radiation was attenuated in Nrf2 knockdown HSF cells. (a, b) Cells were transfected with specific siRNA against Nrf2 or a nonsilencing control, followed by treatment with ZER (8 μM, 2 h), and the expression patterns of total Nrf2 and HO-1 proteins in both Nrf2-transfected and nonsilencing control were measured using western blot method. (c, d) In ZER-pretreated (8 μM, 24 h) and UVA-exposed HSF cells, the effect of Nrf2 silencing on intracellular ROS accumulation was measured using DCFH₂-DA (10 μM) fluorescence staining method. Data were presented as fold change of ROS in nonsilencing control and Nrf2 knockdown cells. Results were presented as mean ± SD of three or more assays. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control cells. ^#p < 0.05, ^##p < 0.01, compared to ZER-treated cells.

Effect of ZER on activator protein- (AP-) 1-associated protein expression in UVA-irradiated HSF cells. Cells were pretreated with ZER (0, 2, 4, or 8 μM for 24 h) followed by irradiation in the absence or presence of 3 J/cm² UVA for the indicated time. After irradiation, cells were allowed to incubate for 2 h and the subsequent experiments were performed. (a, b) The expression of p-c-Fos and p-c-Jun proteins in the absence or presence of UVA at various concentrations of ZER was measured using western blot method. Also, an immunofluorescence staining was also performed to measure the alterations in p-c-Fos (c) and p-c-Jun (d) expressions as described in the methodology. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control cells; ^#p < 0.05, ^##p < 0.01 compared to UVA-irradiated cells.

Role of signal transduction pathways mediating the activation of nuclear Nrf2 in ZER-pretreated HSF cells. Cells were pretreated with pharmacological inhibitors for MAPK p38 (SB203580, 20 μM), ERK (PD98059, 30 μM), JNK (SP600125, 25 μM), PI3K/AKT (LY294002, 30 μM), PKC (GF109203X, 2.5 μM), AMPK inhibitor (compound C, 10 μM), casein kinase II inhibitor (CKII inhibitor, 20 μM), or ROS inhibitor (NAC, 1 mM) for 30 min followed by ZER treatment (8 μM) for 2 h. Cells were harvested and nuclear protein fraction was analyzed using western blot method. Histone was used as an internal protein control. Results were presented as mean ± SD of three assays. ∗∗∗p < 0.001 compared to untreated control cells; ^##p < 0.01, ^###p < 0.001 compared to ZER alone-treated cells.

Effect of ZER on cell viability and altered MMP-1 and collagen III expressions in UVA-irradiated HSF cells. (a) Chemical structure of zerumbone (ZER). (b) Cells were pretreated with ZER (0, 2, 4, or 8 μM for 24 h) followed by UVA irradiation (3 J/cm² for 27 min). 24 h after UVA exposure, the percentage of cell viability was measured by MTT colorimetric assay as described. The formula used to calculate the percentage of viable cells was (A₅₇₀ of treated cells/A₅₇₀ of untreated cells) × 100. (c, d) HSF cells were pretreated with ZER (0, 2, 4, or 8 μM for 24 h), and the expression of MMP-1 and collagen III proteins in the absence (c) or presence (d) of UVA radiation was measured using the western blot method. Results were presented as mean ± SD of three or more assays. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control cells; ^#p < 0.05, ^##p < 0.01 compared to UVA-irradiated cells.

Effect of ZER on the nuclear translocation, activation of Nrf2, and its associated proteins in UVA-irradiated HSF cells: ZER pretreated (2-8 μM for 2 h) HSF cells were irradiated in the presence (a, b) or presence (c, d) of 3 J/cm² UVA (for the indicated time) were subjected to western blot for the measurement of total Nrf2 and Keap-1 protein expressions. The effect of ZER on this pattern was represented as fold change of Nrf2/Keap-1 ratio over the control values. This ratio determines the dissociation pattern of Nrf2 from Keap-1 in the cytoplasm. (e, f) HSF cells were treated with 8 μM of ZER at different time points (0, 0.5, 1, 2, or 4 h), and the expression of cytosolic, nuclear Nrf2 protein fractions was determined using western blot method. β-Actin and histone proteins were used as internal controls for cytosolic and nuclear Nrf2, respectively. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control cells.

Effect of ZER on ARE promoter activation and subsequent expression of HO-1 and γ-GCLC proteins in HSF cells. (a) Cells were treated with ZER (8 μM for 2 h) and subcellular localization of Nrf2 was determined using immunostaining method. (b) HSF cells were cotransfected with pGL3-ARE and treated with various concentrations of ZER (2-8 μM for 2 h) to measure the percentage of ARE promoter activity. Data was presented as fold over increase in the percentage of ARE promoter activity. (c, d) The effect of ZER treatment (8 μM) on the expression of total Nrf2 and antioxidant proteins (HO-1 and γ-GCLC) at different time points (0, 1, 2, 4, 8, 12, 16, or 24 h) was measured using western blot method against β-actin as an internal control. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control cells.