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. 2017 Nov 27:8:868.
doi: 10.3389/fphar.2017.00868. eCollection 2017.

Pelargonidin Modulates Keap1/Nrf2 Pathway Gene Expression and Ameliorates Citrinin-Induced Oxidative Stress in HepG2 Cells

G R Sharath Babu  1 , Tamatam Anand  1 , N Ilaiyaraja  1 , Farhath Khanum  1 , N Gopalan  2
Affiliations

Pelargonidin Modulates Keap1/Nrf2 Pathway Gene Expression and Ameliorates Citrinin-Induced Oxidative Stress in HepG2 Cells

G R Sharath Babu et al. Front Pharmacol. .

Abstract

Pelargonidin chloride (PC) is one of the major anthocyanin found in berries, radish and other natural foods. Many natural chemopreventive compounds have been shown to be potent inducers of phase II detoxification genes and its up-regulation is important for oxidative stress related disorders. In the present study, we investigated the effect of PC in ameliorating citrinin (CTN) induced cytotoxicity and oxidative stress. The cytotoxicity of CTN was evaluated by treating HepG2 (Human hepatocellular carcinoma) cells with CTN (0-150 μM) in a dose dependent manner for 24 h, and the IC50 was determined to be 96.16 μM. CTN increased lactate dehydrogenase leakage (59%), elevated reactive oxygen species (2.5-fold), depolarized mitochondrial membrane potential as confirmed by JC-1 monomers and arrested cell cycle at G2/M phase. Further, apoptotic and necrotic analysis revealed significant changes followed by DNA damage. To overcome these toxicological effects, PC was pretreated for 2 h followed by CTN exposure for 24 h. Pretreatment with PC resulted in significant increase in cell viability (84.5%), restored membrane integrity, reactive oxygen species level were maintained and cell cycle phases were normal. PC significantly up-regulated the activity of detoxification enzymes: heme oxygenase 1 (HO-1), glutathione transferase, glutathione peroxidase, superoxide dismutase and quinone reductase. Nrf2 translocation into the nucleus was also observed by immunocytochemistry analysis. These data demonstrate the protective effect of PC against CTN-induced oxidative stress in HepG2 cells and up-regulated the activity of detoxification enzyme levels through Keap1/Nrf2 signaling pathway.

Keywords: Keap1/Nrf2 signaling pathway; citrinin; cytotoxicity; mitochondrial membrane potential; oxidative stress; pelargonidin chloride.

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Figures

GRAPHICAL ABSTRACT
GRAPHICAL ABSTRACT
Schematic model of the Keap1/Nrf2 signaling pathway. Pelargonidin chloride dissociates Nrf2 from Keap1 resulting in translocation of Nrf2 into the nucleus, where it binds to ARE and activates cytoprotective genes.
FIGURE 1
FIGURE 1
Effect of CTN on cell viability as measured by MTT assay. (A) Dose-response curve of CTN. (B) HepG2 cells were pretreated with PC (50 and 100 μM) for 2 h followed by with or without CTN (96 μM) treatment for 24 h. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 2
FIGURE 2
Effect of CTN on LDH leakage into the medium. (A) HepG2 cells after treatment with different concentrations of CTN (0–100 μM) for 24 h. (B) Protective effect of PC (50 and 100 μM) when pretreated for 2 h followed by with or without CTN (96 μM) treatment for 24 h. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 3
FIGURE 3
Effect of PC on the morphology of HepG2 cells, (a) Control, (b–e) CTN 25, 50, 75, and 100 μM, (f) PC 100 μM, (g) PC 50 μM + CTN 96 μM, (h) PC 100 μM + CTN 96 μM. HepG2 cells were treated with PC for 2 h followed by with or without CTN (96 μM) treatment for 24 h and morphological changes observed under phase-contrast microscopy. The figures shown are representative of three independent experiments. Scale bar, 100 μm.
FIGURE 4
FIGURE 4
Representative photomicrographs of HepG2 cells stained with acridine orange (AO, green) and ethidium bromide (EtBr, red) fluorescent dyes, (A) Vital cells (green), early apoptotic (bright green to yellow), late apoptotic (orange) and necrotic cells (red). (a) Control, (b–e) CTN 25, 50, 75, and 100 μM, (f) Control, (g) CTN 96 μM, (h) PC 100 μM, (i) PC 50 μM + CTN 96 μM, (j) PC 100 μM + CTN 96 μM. (B) The percentage of apoptotic and necrotic cells after staining with AO and EtBr. Data are expressed as the mean ± SD. ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells. Scale bar, 20 μm.
FIGURE 5
FIGURE 5
Effect of PC on CTN-induced ROS in HepG2 cells. (A) Cells were treated with CTN for 24 h and then intracellular ROS levels were detected. ROS levels were found to be increased compared to control cells. (B) PC pretreatment for 2 h followed by with or without CTN (96 μM) treatment for 24 h markedly decreased intracellular ROS. (C) Fluorescence images of HepG2 cells stained with H2DCF-DA, ROS accumulation leads to oxidation of the dye resulting in increased DCF fluorescence (green color) (a) Control, (b–e) CTN 25, 50, 75, and 100 μM, (f) Control, (g) CTN 96 μM, (h) PC 100 μM, (i) PC 50 μM + CTN 96 μM, (j) PC 100 μM + CTN 96 μM. Scale bar, 50 μm. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 6
FIGURE 6
The mitochondrial membrane potential is expressed as JC-1 fluorescence ratio in terms of red fluorescence to green fluorescence. (A) Effect of CTN on loss of MMP in HepG2 cells. (B) PC pretreatment for 2 h prevented loss of MMP by CTN (96 μM) treatment. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 7
FIGURE 7
Fluorescent microscopic images of HepG2 cells stained with JC-1 dye after CTN treatment. Red color indicates healthy cells with high MMP and green color indicates low MMP. (A) Effect of CTN on loss of MMP in HepG2 cells (a) Control, (b–e) CTN 25, 50, 75, and 100 μM. (B) PC pretreatment for 2 h prevented loss of MMP by CTN (96 μM) treatment (f) Control, (g) CTN 96 μM, (h) PC 100 μM, (i) PC 50 μM + CTN 96 μM, (j) PC 100 μM + CTN 96 μM. Scale bar, 50 μm.
FIGURE 8
FIGURE 8
Cell cycle analysis using propidium iodide (PI) staining and flow cytometry. (A) Flow cytometry histogram of HepG2 cells after CTN treatment and compared with pretreatment with PC. (B) Cell cycle phase distribution (%) of 24 h CTN treated HepG2 cells showing G2/M phase arrest and sub-G1 phase. (C) Cell cycle phase distribution (%) of PC pretreated (2 h) cells showing protection after 24 h CTN exposure.
FIGURE 9
FIGURE 9
Effect of PC on DNA damage induced by CTN. (A) Comet images in different treatment groups (a) Control, (b–e) CTN 25, 50, 75, and 100 μM, (f) Control, (g) CTN 96 μM, (h) PC 100 μM, (i) PC 50 μM + CTN 96 μM, (j) PC 100 μM + CTN 96 μM. (B) Effect of CTN on DNA damage in HepG2 cells. (C) Protective effect of PC on olive tail moment when pretreated for 2 h followed by with or without CTN (96 μM) treatment for 24 h. Scale bar, 50 μm.
FIGURE 10
FIGURE 10
Measurement of NQO1 activity in HepG2 cells. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 11
FIGURE 11
Effect of PC on mRNA expression levels of Nrf2, NQO1, Keap1, GST and HO-1 in HepG2 cells. mRNA was analyzed by quantitative real-time PCR; normalized gene expression levels are given as the mean fold change. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 12
FIGURE 12
Effect of PC on Nrf2-mediated mRNA expression levels of antioxidant enzymes, CAT, SOD1, and GPx1. mRNA was analyzed by quantitative real-time PCR; normalized gene expression levels are given as the mean fold change. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 13
FIGURE 13
Effect of PC on the expression of apoptotic markers, (A) Expression of p53, Bax, Bcl2 and Cyt-C analyzed by western blotting. (B) Densitometry analysis of the intensity of the protein bands. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 14
FIGURE 14
Western blot analysis of phase II detoxification proteins and antioxidant protein markers, (A) Expression of Nrf2, Keap1, NQO1, HO-1, CAT, SOD1, and GPx1 upon pretreatment with PC for 2 h followed by with or without CTN (96 μM) treatment for 24 h. (B) Densitometry analysis of the intensity of the protein bands. Data are expressed as the mean ± SD (n = 3). ∗p < 0.05 vs. control cells, #p < 0.05 vs. CTN treated cells.
FIGURE 15
FIGURE 15
Nrf2 nuclear localization in HepG2 cells was examined by confocal microscopy after pretreatment with PC for 2 h followed by with or without CTN (96 μM) treatment for 24 h (a) Control, (b) CTN 96 μM, (c) PC 100 μM, (d) PC 50 μM + CTN 96 μM, (e) PC 100 μM + CTN 96 μM. Green signals represent nuclear distribution of Nrf2 while nuclei were stained with Hoechst 33342 (blue signal) as assessed by secondary antibody conjugated with FITC staining in laser scanning confocal microscope. Scale bar, 10 μm.
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