Differential roles of 3-Hydroxyflavone and 7-Hydroxyflavone against nicotine-induced oxidative stress in rat renal proximal tubule cells

<div><p>Plant flavonoids are well known as antioxidants against oxidative stress induced by exposure to external pollutants. Nicotine (NIC) is one of those agents which increases renal oxidative stress, an important factor in the pathogenesis of renal epithelial injury in smokers. Although several studies had been conducted on flavonoids and oxidative stress, the mechanism of the protective pathways are not fully understood. Here, we present studies on antioxidant properties of two mono-hydroxyflavone isomers, 3-hydroxyflanove (3HF)- and 7-hydroxyflavone (7HF), against nicotine-associated oxidative stress and injury in cultured renal proximal tubule cells and correlate their antioxidant properties with their chemical structure. Our data clearly demonstrates, for the first time, that while both 3HF and 7HF protect renal cells from NIC-associated cytotoxicity, the mechanism of their action is different: 3HF elicits protective activity via the PKA/CREB/MnSOD pathway while 7HF does so via the ERK/Nrf2/HO-1 pathway. Molecular docking and dynamics simulations with two major signaling pathway proteins showed significant differences in the binding energies of 3HF (-5.67 and -7.39 kcal.mol^-1) compared to 7HF (-5.41 and -8.55 kcal.mol^-1) in the matrices of CREB and Keap1-Nrf2 proteins respectively, which corroborate with the observed differences in their protective properties in the renal cells. The implications of this novel explorative study is likely to promote the understanding of the mechanisms of the antioxidative functions of different flavones.</p></div

Molecular dynamics simulations.

<p>(A) Time dependence of RMS deviation of distance (RMSD) between alpha-carbon atoms from the crystal structure as a function of simulation time for free 1DH3 and conjugated 1DH3. (B) Time evolution of the solvent accessible surface area (SASA) during 18 ns of MD simulation of 1DH3, 1DH3 with 3HF and 1DH3 with 7HF. (C) Time dependence of RMS deviation of distance (RMSD) between alpha-carbon atoms from the crystal structure as a function of simulation time for free 2DYH and conjugated 2DYH. (D) Time evolution of the solvent accessible surface area (SASA) during 18 ns of MD simulation of 2DYH, 2DYH with 3HF and 2DYH with 7HF. Color codes are given on the figures.</p

Average values of RMSD, SASA and no. of H-bonds values of 1DH3 and 2DYH proteins in free and 3HF / 7HF bound systems.

<p>Average values of RMSD, SASA and no. of H-bonds values of 1DH3 and 2DYH proteins in free and 3HF / 7HF bound systems.</p

Docking summary of 7HF and 3HF with 2DYH and 1DH3 proteins.

<p>Docking summary of 7HF and 3HF with 2DYH and 1DH3 proteins.</p

3HF and 7HF rescues renal proximal tubule cells from nicotine exposure-associated cytotoxicity by inhibiting ROS production.

<p>(A) Chemical structure of 3-hydroxyflavone (3HF) and 7-hydroxyflavone (7HF). (B) NRK52E cells were pretreated with 20 μM 3HF or 7HF overnight prior to treatment with 200 μM NIC and cell viability was determined 24 hours later. Control cells were left untreated, or treated with 200 μM NIC, 20 μM 3HF or 7HF. Values were expressed as % of untreated control. n = 3; *p<0.05 compared to untreated control. Dotted line represents untreated control value. (C) NRK52E cells were pretreated with 20 μM 3HF or 7HF overnight and 200 μM NIC-mediated ROS production was determined. Control cells were left untreated, or treated with 200 μM NIC, 20 μM 3HF or 7HF. Values are expressed as % of untreated control. n = 3; *p<0.05 compared to untreated control or as indicated. Dotted line represents untreated control value.</p

3HF and 7HF activates distinct antioxidant genes via distinct signaling pathways.

<p>(A) NRK52E cells were transfected with a MnSOD promoter luciferase plasmid as described in Materials and Methods. A set of cells were infected with the M1CREB adenovirus overnight or treated with 10 μM H89 1 hr prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value. (B) NRK52E cells were transfected with an HO-1 promoter luciferase plasmid as described in Materials and Methods. A set of cells were co-transfected with 20 nM Nrf2 siRNA or infected with the dnMEK adenovirus 24 hours prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value. (C) NRK52E cells were transfected with a CRE luciferase plasmid as described in Materials and Methods. A set of cells were infected with a M1CREB adenovirus 24 hours or treated with 10 μM H89 1 hr prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value. (D) NRK52E cells were transfected with an ARE luciferase plasmid as described in Materials and Methods. A set of cells were co-transfected with 20 nM Nrf2 siRNA or infected with the dnMEK adenovirus 24 hours prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value.</p

An intriguing difference in the pathways of the protective response.

<p>Flavonol 3-hydroxyflavone (3HF) activates the antioxidant MnSOD gene via the PKA/CREB pathway, which is potentially augmented by high affinity binding of 3HF to CREB. In contrast, 7-hydroxyflavone (7HF) upregulates the HO-1 gene via the ERK/Nrf2 axis, which may be facilitated by 7HF binding to the Nrf2-binding domain of Keap1 that releases Nrf2 from Keap1 resulting in activation of Nrf2.</p

CHOP is involved in response of HEp2 cells to CBP.

<p>(A) Parental HEp2 and CBP-resistant 7T cells were transfected with negative control siRNA (nc siRNA) or with CHOP-specific siRNA (CHOP siRNA) The expression of CHOP was determined by western blot 48 h after transfection. ERK2 was used as equal loading control. Representative blot of two independent experiments that yielded similar results is presented. (B) HEp2 and 7T cells were seeded for MTT assay 48 h after transfection with nc siRNA or CHOP siRNA and treated with CBP 24 h after. MTT assay data are representative of two independently performed experiments ±S.D. (C) HEp2 and 7T cells were transfected with nc siRNA or CHOP siRNA, plated and 24 h later treated with 40 µg/mL CBP for 48 h. Cells were stained with fluorescein diacetate and propidium iodide and examined by fluorescence microscopy. Representative data of three independent experiments are presented (mean ±SD). *p<0.05.</p

CBP induced Grp78 and CHOP expression in HEp2 but not in 7T cells.

<p>(A) Twenty-four hours after the seeding, HEp2 and 7T cells were treated with 40 µg/mL CBP. At indicated time points the cells were collected and the expression of ER stress markers CHOP and Grp78 was determined. Expression of ERK2 was used as an internal loading control. (B) HEp2 and 7T cells were treated with 40 µg/mL CBP for 3–24 h. At indicated time points the cells were collected and the mRNA expression of ER stress markers CHOP and Grp78 was determined. Expression of GAPDH was used as an internal control.</p

CBP induced ROS formation in HEp2 and 7T cells.

<p>(A) The logarithmically growing cell lines were treated with 40 µg/mL CBP during the indicated period of time. The cells were collected, stained with CM-H₂DCFDA and ROS was measured by flow cytometry. (B) The HEp2 and 7T cells were pretreated with 0.1 mM tempol. Two hours later different concentrations of CBP were added. The cell survival was determined 72 hours later by MTT assay. Representative data of three independent experiments are presented (mean ±SD). *p<0.05. C_ic-isotype control, C_c-untreated cells, CBP-carboplatin treated cells, H₂O₂-hydrogen peroxide treated cells.</p