Luteolin, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), xanthine, xanthine oxidase, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), MTT, thiobarbituric acid (TBA), Hoechst 33342, N-acetyl cysteine (NAC), and propidium iodide (PI) were obtained from Sigma-Aldrich (Merck KGaA). Additionally, 7-amino-4-chloromethylcoumarin (CMAC) and diphenyl-1-pyrenylphosphine (DPPP) were purchased from Molecular Probes (Thermo Fisher Scientific, Inc.) The primary Bax, Bcl-2, GPx, CAT and HO-1 antibodies were obtained from Santa Cruz Biotechnology, Inc.; primary β-actin, phosphorylated (phospho)-H2A histone family member X (H2A.X), H2A.X, caspase-3 and caspase-9 antibodies were obtained from Cell Signaling Technology, Inc.; primary γ-glutamylcysteine ligase (γ-GCL) antibody was obtained from Thermo Fisher Scientific, Inc.; primary Cu/Zn SOD was obtained from Enzo Life Science.

Chinese hamster lung fibroblasts (V79-4) were purchased from American Type Culture Collection and cultured in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% heat-inactivated fetal calf serum, at 37°C in a humidified incubator with 5% CO₂.

Cells (1.5×10⁵ cells/ml) were treated with 0.625, 1.250, 2.500, 5.000 or 10.000 µg/ml luteolin for 24 h at 37°C. To investigate the cytoprotective effect of luteolin against H₂O₂ exposure, cells were pretreated with 2.5 µg/ml luteolin for 1 h before exposure to 1 mM H₂O₂ for 24 h, all at 37°C. The MTT assay was performed and formazan crystals were dissolved in dimethyl sulfoxide, then absorbance was measured using a scanning multi-well spectrophotometer at 540 nm, as previously described (22).

Cells were exposed to luteolin (0.625, 1.250, 2.500, 5.000 or 10.000 µg/ml, respectively) and 2 mM NAC for 30 min, followed by 1 mM H₂O₂ treatment for 1 h, all at 37°C. Following staining with 25 µM H₂DCFDA at 37°C for 10 min, the fluorescence was monitored and quantified using a spectrofluorometer (PerkinElmer FL 6500 Fluorescence Spectrometer with Spectrum FL Software 1.1 version, PerkinElmer Inc.) or a confocal microscope (Zeiss LSM 510 confocal microscope with Zen 2.5 version, Carl Zeiss Inc.), as previously described (23) using 40× magnification.

The xanthine/xanthine oxidase system was used to produce O₂⁻, which was captured by the nitrone spin trap, DMPO. The resulting DMPO/·OOH adducts were identified using electron spin resonance (ESR) spectrometer (JEOL, Ltd.) as previously described (23).

Hydroxyl radical was generated by the Fenton reaction (H₂O₂ + FeSO₄) and detected by capturing with DMPO to form DMPO/·OH adducts, measured using ESR spectrometer as previously described (23).

Following cell treatment with 5 µM DPPP at 37°C for 30 min, a fluorescence microscope (Zeiss LSM 510 confocal microscope with Zen 2.5 version; 20× magnification) was used to assess images of DPPP fluorescence. The cells were rinsed with cold PBS, scraped and homogenized in ice-cold 1.15% KCl, resulted cell lysate was subjected to further assessment. For the detection of TBA reactive substances (TBARS), 100 µl cell lysates were mixed with 0.2 ml sodium dodecyl sulfate (SDS, 8.1%), 1.5 ml 20% acetic acid (pH 3.5) and 1.5 ml TBA (0.8%) and combined with 5 ml 15:1 (v/v) n-butanol and pyridine solutions. The resulting supernatant absorbance was measured using a spectrophotometer at 532 nm.

Following treatment with luteolin and H₂O₂, cells were collected and centrifuged at 15,000 × g for 5 min, following washing with PBS to obtain cell pellets. Cell pellets on 1% agarose-coated slides were subjected to gel electrophoresis at 300 mA and 25 V for 20 min in darkness at 20°C. Ethidium bromide (20 µg/ml) stained slides at 20°C for 5 min were examined under a fluorescence microscope (Komet 7 with Zyla 5.5 USB 3.0 sCMOS camera, Andor Technology; 20× magnification). Each slide contained 50 cells and data on tail length and total fluorescence percentages were analyzed using Komet version 5.5 image analyzer software (Andor Technology).

Total proteins from the cells was extract by using PRO-PREP™ protein extraction solution (iNtRON Biotechnology). Thereafter total protein levels were estimated by protein assay reagent kit (Bio-Rad). A total of 30 µg/lane cell lysates were subjected to electrophoresis on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, subjected for blocking with 3% bovine serum albumin (Bovogen Biologicals Pty Ltd.) for 1 h at 20°C, and then incubated with the corresponding primary antibodies whereas phospho-H2A.X (cat. #9718), H2A.X (cat. #2595), β-actin (cat. #4967), Bax (cat. sc-7480), Bcl-2 (cat. sc-7382), caspase-9 (cat. #9508), caspase-3 (cat. #9662), γ-GCL (cat. #RB-1697-P0, -P1), Cu/Zn SOD (cat. ADI-SOD-100), CAT (cat. sc-34285), GPx (cat. sc-22145), HO-1 (cat. sc-10789) (all 1:1,000 ratio, respectively) and for overnight at 4°C subsequently treated with the relevant secondary (diluted 1:10,000, respectively) goat anti-rabbit IgG (H+L) Secondary antibody, HRP (cat. #31460) and goat anti-mouse IgG (H+L) secondary antibody, HRP (cat. #31430; Thermo Fisher Scientific, Inc.) at 20°C for 1 h. Each corresponding protein band was observed on an X-ray film following treat with enhanced chemiluminescence western blotting detection kit (Amersham) as previously described (23).

Cells were stained with Hoechst 33342, a DNA-specific probe, at 37°C for 10 min. The degree of nuclear condensation was evaluated using a fluorescence microscope (BH2-RFL-T3; Olympus; 20× magnification) fitted with a CoolSNAP-Pro color digital camera (Media Cybernetics) to observe stained cells.

Cells were fixed with 70% ethanol at 4°C for 30 min, subjected for washing with PBS, mixed in 1 ml PBS containing 100 µg PI solution and 100 µg RNase A and incubated in dark conditions at 37°C for 30 min. The percentage of apoptotic sub-G₁ cells was determined using FACSCalibur flow cytometer with CellQuest pro software 4.02 (Becton Dickinson).

Cells were incubated with 5 µM CMAC at 37°C, a GSH-sensitive fluorescent dye, for 30 min. Images of CMAC fluorescence in response to GSH were analyzed using a fluorescence microscope (BH2-RFL-T3; Olympus; 10× magnification) (23).

Collected cells were sonicated twice for 15 sec in 10 mM phosphate buffer (pH 7.5) on ice to lyse. 1% Triton X-100 was added to the lysates and incubated on ice for 10 min. After centrifugation at 5,000 × g for 10 min at 4°C, the lysates were cleared of debris and the protein concentration of the supernatant was measured utilizing using the Bradford method. Cell lysates were mixed with 500 mM phosphate buffer (pH 10.2) and 1 mM epinephrine at 20°C, which auto-oxidizes to form adrenochrome, and the reactants were measured at 480 nm using a ultraviolet/visible spectrophotometer in kinetic mode. SOD activity was calculated as unit/mg protein as previously described (23).

Harvested cells were suspended in 10 mM phosphate buffer (pH 7.5) and sonicated twice for 15 sec on ice. After adding 1% TritonX-100 to the lysates, they were incubated on ice for 10 min. Protein content was measured after centrifuging lysates at 5,000 × g for 30 min at 4°C to eliminate cellular debris. Then cell lysates were reacted with 50 mM phosphate buffer (pH 7) and 100 mM H₂O₂, at 37°C for 2 min. Absorbance changes at 240 nm over 5 min were measured by spectrophotometer (X-ma 1000; Human Corporation) to determine the rate of H₂O₂ decomposition.

Cells were lysed by sonicating twice for 15 sec in 10 mM phosphate buffer (pH 7.5) on ice. 1% Triton X-100 was added to lysates and incubated on ice for 10 min. After centrifugation at 5,000 × g for 10 min at 4°C, debris was removed and protein concentration was assessed using the Bradford technique. Then cell lysates were mixed with 25 mM phosphate buffer (pH 7.5), 1 mM EDTA, NaN₃, GSH, 0.25 units of glutathione reductase, and 0.1 mM NADPH. Following 10 min incubation at 37°C, 1 mM H₂O₂ was added for 1 min at 37°C and absorbance was measured using spectrophotometer (X-ma 1000, Human Co.) at 340 nm for 5 min.

Cells were washed with PBS, collected in PBS (pH 7.4), allowed to 15 min incubation on ice facilitating brief sonication and added sucrose solution obtaining 0.25 M sucrose as final concentration. Homogenates were centrifuged 10 min at 1,000 × g at 4°C. The supernatants were centrifuged at 12,000 × g for 15 min at 4°C and afterward at 105,000 × g for 60 min at 4°C. Resulted pellet was resuspended in 50 mM PBS (pH 7.4) and protein concentration was measured using the Bradford technique. Cell lysates were suspended in a reaction mixture containing 0.2 mM hemin, 0.5 mg/ml rat liver cytosol (supplying biliverdin reductase; Creative Bioarray; cat. no. DDM-M063), 2 mM glucose-6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase, 1 mM NADPH and 50 mM PBS (pH 7.4) for 2 h at 37°C. The chloroform-extracted layer was assessed using a spectrophotometer at 464 and 530 nm.

All experiments were performed in triplicate, with results presented as the mean ± standard error. Data were analyzed using one way analysis of variance, followed by Tukey's post hoc test to determine the differences using SigmaStat 3.5 version software (Systat Software Inc.). P<0.05 was considered to indicate a statistically significant difference.

Luteolin exhibited no cytotoxicity toward V79-4 cells at concentrations of 0.625, 1.25 and 2.5 µg/ml. Cytotoxic effects were observed at concentrations of 5 and 10 µg/ml (Fig. 1A). Fluorescence spectrometry indicated that luteolin scavenged intracellular ROS in a dose-dependent manner, decreasing ROS by 31% at 0.625, 51% at 1.250, 58% at 2.500, 68% at 5.000 and 75% at 10.000 µg/ml (Fig. 1B). The ROS scavenger NAC, serving as a positive control, eliminated 72% of ROS. Given its cell viability and ROS-scavenging ability, 2.5 µg/ml luteolin was selected as the optimal dose for further evaluation. ESR spectrometry was used to quantify the superoxide anion generated by the xanthine/xanthine oxidase system. ESR measurement revealed an elevation in the superoxide anion signal to 902 in this system. However, when the superoxide anion was treated with luteolin, the superoxide anion signal was reduced to 573, indicating the direct scavenging effect of luteolin on the superoxide anion produced via the xanthine/xanthine oxidase pathway (Fig. 2A). Furthermore, ESR spectrometry was used to detect the hydroxyl radical produced via the Fenton reaction. ESR measurement demonstrated that neither the control nor luteolin at 2.5 µg/ml exhibited a signal, whereas the signal of the hydroxyl radical increased to 2,621 in the Fenton reaction system. Treatment with luteolin markedly decreased the hydroxyl radical signal to 237, indicating the capacity of luteolin to directly mitigate the hydroxyl radical generated through the Fenton reaction system (Fig. 2B). Additionally, confocal microscopy images from the H₂DCFDA assay showed that luteolin suppressed red fluorescence intensity, which increased in response to H₂O₂ treatment in association with elevated ROS levels (Fig. 2C).

The capacity of luteolin to counteract membrane lipid peroxidation in cells exposed to H₂O₂ was assessed. Lipid peroxidation, evidenced by formation of the highly fluorescent compound DPPP oxide upon DPPP stoichiometric reaction with lipid hydroperoxides, was assessed (24). H₂O₂ treatment enhanced DPPP fluorescence intensity, indicating the elevation of lipid peroxidation levels (Fig. 3A). However, this increase was notably attenuated by treatment with 2.5 µg/ml luteolin. Additionally, protective effect of luteolin against lipid peroxidation was demonstrated by TBARS formation in H₂O₂-treated V79-4 cells whereas luteolin significantly decreased H₂O₂-induced TBARS formation (Fig. 3B).

The comet assay, a sensitive technique for measuring DNA damage (25), demonstrated that H₂O₂ resulted in a 77% increase in the length and quantity of DNA in the comet tail, signifying considerable DNA damage; however, pretreatment with luteolin reduced this increase to 51% (Fig. 4A). Furthermore, evaluation of phospho-H2A.X, a marker for DNA double-strand breaks (25), showed that luteolin pretreatment diminished expression levels of phospho-H2A.X in H₂O₂-exposed cells (Fig. 4B). There was no change in total H2A.X expression.

Pretreatment with 2.5 µg/ml luteolin in conjunction with H₂O₂ significantly increased cell viability to 77% compared with 54% in cells treated solely with H₂O₂ (Fig. 5A). The cytoprotective effect of luteolin against H₂O₂-induced apoptosis was determined by staining nuclei of cells with Hoechst 33342 and observing them under a microscope. H₂O₂-treated cells showed considerable nuclear fragmentation and apoptotic morphology; however, luteolin pretreatment alleviated H₂O₂-induced cellular apoptosis (Fig. 5B). The proportion of sub-G₁ in H₂O₂-treated cells was 33% (a 31% increase relative to the control; Fig. 5C); however, pretreatment with luteolin decreased the sub-G₁ apoptotic cells to 12%. Pretreatment with luteolin led to an alleviation of H₂O₂-induced pro-apoptotic protein Bax expression and increased the anti-apoptotic Bcl-2 protein expression, which was decreased by H₂O₂ (Fig. 5D). Furthermore, luteolin alleviated H₂O₂-mediated activation of caspase-9 and caspase-3 (Fig. 5D).

Confocal microscopy showed a notable reduction in GSH level in H₂O₂-treated cells, showing lower fluorescence intensity than that of the control. However, an increase in GSH levels was observed in the luteolin-pretreated group (Fig. 6A). As GSH synthesis involves γ-GCL and GSH synthase (26), γ-GCL expression was evaluated via western blotting. The expression of γ-GCL was notably downregulated by H₂O₂-treated cells but upregulated by luteolin pretreatment (Fig. 6B). Moreover, enzymatic assays demonstrated that luteolin pretreatment enhanced activities of SOD, CAT, GPx, and HO-1 to 29, 17 and 17 unit/mg protein and 7,162 pmol bilirubin/mg protein, respectively, compared with diminished activities in cells treated with H₂O₂-alone (23, 9, and 9 unit/mg protein and 4,987 pmol bilirubin/mg protein, respectively; Fig. 6C and D). Also, the levels of SOD, CAT, GPx and HO-1 were reduced in H₂O₂-treated cells; however, luteolin partially restored expression of these proteins (Fig. 6E).