Protective effect of luteolin against oxidative stress‑mediated cell injury via enhancing antioxidant systems

Abstract

Physiological stress such as excessive reactive oxygen species (ROS) production may contribute normal fibroblasts activation into cancer-associated fibroblasts, which serve a crucial role in certain types of cancer such as pancreatic, breast, liver and lung cancer. The present study aimed to examine the cytoprotective effects of luteolin (3′,4′,5,7-tetrahydroxyflavone) against hydrogen peroxide (H₂O₂)-generated oxidative stress in lung fibroblasts. To examine the effects of luteolin against H₂O₂-induced damages, cell viability, sub-G₁ cell population, nuclear staining with Hoechst 33342, lipid peroxidation and comet assays were performed. To evaluate the effects of luteolin on the protein expression level of apoptosis, western blot assay was performed. To assess the antioxidant effects of luteolin, detection of ROS using H₂DCFDA staining, O₂⁻ and ·OH using electron spin resonance spectrometer and antioxidant enzyme activity was performed. In a cell-free chemical system, luteolin scavenges superoxide anion and hydroxyl radical generated by xanthine/xanthine oxidase and the Fenton reaction (FeSO₄/H₂O₂). Furthermore, Chinese hamster lung fibroblasts (V79-4) treated with H₂O₂ showed a significant increase in cellular ROS. Intracellular ROS levels and damage to cellular components such as lipids and DNA in H₂O₂-treated cells were significantly decreased by luteolin pretreatment. Luteolin increased cell viability, which was impaired following H₂O₂ treatment and prevented H₂O₂-mediated apoptosis. Luteolin suppressed active caspase-9 and caspase-3 levels while increasing Bcl-2 expression and decreasing Bax protein levels. Additionally, luteolin restored levels of glutathione that was reduced in response to H₂O₂. Moreover, luteolin enhanced the activity and protein expressions of superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase-1. Overall, these results indicated that luteolin inhibits H₂O₂-mediated cellular damage by upregulating antioxidant enzymes.

Introduction

Reactive oxygen species (ROS), such as superoxide anion (O₂⁻), hydroxyl radical (·OH), and hydrogen peroxide (H₂O₂), are natural byproducts of oxygen metabolism that serve key roles in cell proliferation and immune responses (1,2). Excessive ROS can damage cellular molecules, leading to DNA damage, lipid peroxidation and protein oxidation. These processes are key in the development of various diseases, including cancer and lung fibrosis (3–5). Cells contain a range of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and non-enzymatic antioxidants, such as reduced glutathione (GSH), for defense against ROS (6,7). SOD, a metalloenzyme, catalyzes the conversion of O₂⁻ to molecular O₂ and H₂O₂ and functions as a key component of the cellular antioxidant defense mechanism (8). CAT breaks H₂O₂ into O₂ and H₂O and GPx uses GSH as an electron donor to convert H₂O₂ into its corresponding alcohol or water (9,10). In addition, heme oxygenase-1 (HO-1) serves as a catalyst for the oxidative transformation of heme to carbon monoxide, iron and biliverdin, which is then transformed into bilirubin by biliverdin reductase (11). Furthermore, ROS levels are notably boosted by exposure to environmental stressors, such as ultraviolet light, pollutants and heavy metals (12).

Lung fibroblasts serve a major role in lung development, such as alveolar unit development, aid production of extracellular matrix, and facilitate wound healing and tissue repair (13). Physiological stresses (ROS and disrupted metabolism) may turn normal fibroblasts into cancer-associated fibroblasts (CAFs) (14). CAFs are key factors in the tumor microenvironment and serve a crucial role in non-small cell lung cancer drug resistance (15). Therefore, the present study aimed to identify a potent antioxidant compound capable of ameliorating oxidative stress-mediated cellular defects in fibroblasts.

Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a flavonoid found abundantly in vegetables and fruits, including green pepper and chamomile tea (16). It exhibits antitumor effects against gastric, ovarian, and hepatocellular carcinomas (17–20). Luteolin also possesses numerous biological benefits, such as anti-inflammatory, anti-allergic, and antioxidant properties (21). Given its wide range of therapeutic potentials, the present study aimed to evaluate cytoprotective effects of luteolin on lung fibroblasts via the initiation of antioxidant enzyme activity.

Materials and methods

Reagents and antibodies

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.

Cell culture

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.

MTT assay

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).

Evaluation of ROS levels

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.

Detection of superoxide anion

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).

Detection of hydroxyl radical

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).

Assessment of lipid peroxidation

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.

Comet assay

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).

Western blot analysis

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).

Hoechst 33342 nuclear staining

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.

Detection of sub-G₁ 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).

GSH detection

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).

Assessment of SOD activity

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).

Assessment of CAT activity

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.

Assessment of GPx activity

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.

Assessment of HO-1 activity

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.

Statistical analysis

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.

Results

Effect of luteolin on ROS scavenging

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).

Figure 1.

Effect of luteolin on cytotoxicity and ROS scavenging following H₂O₂ treatment. (A) Cells were treated with luteolin for 24 h. Cell viability was determined using the MTT assay. *P<0.05 vs. 0. (B) Cells were pretreated with luteolin, followed by treatment with 1 mM H₂O₂ after 30 min. Following H₂DCFDA treatment, intracellular ROS levels were evaluated by spectrofluorometry. The antioxidant NAC was employed as the positive control. *P<0.05 vs. 0. ROS, reactive oxygen species; NAC, N-acetyl cysteine.

Figure 2.

Effect of luteolin on scavenging of superoxide anion, hydroxyl radical and intracellular ROS. (A) Superoxide anion produced through xanthine and xanthine oxidase interacted with DMPO. The resulting DMPO/·OOH adducts were identified via ESR spectrometry. (B) Hydroxyl radical interacted with DMPO and the resulting DMPO/·OH adducts were identified by ESR spectrometry. (C) 2′,7′-Dichlorodihydrofluorescein diacetate technique was utilized to estimate intracellular ROS levels. ROS, reactive oxygen species; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; ESR, electron spin resonance.

Effect of luteolin on H₂O₂-induced lipid peroxidation

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).

Figure 3.

Protective ability of luteolin on H₂O₂-induced cellular lipid peroxidation. Cellular lipid peroxidation was determined via (A) diphenyl-1-pyrenylphosphine staining and (B) TBARS assay. *P<0.05 vs. control; ^#P<0.05 vs. H₂O₂. TBARS, thiobarbituric acid reactive substances.

Effect of luteolin against DNA damage

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.

Figure 4.

Cytoprotective effect of luteolin on H₂O₂-induced DNA damage. (A) Alkaline comet assay was used for the identification of DNA damage. Representative pictures and cellular DNA damage (% fluorescence in the tail) are presented. *P<0.05 vs. control; ^#P<0.05 vs. H₂O₂. (B) Western blot analysis with antibody against total and phospho-H2A.X was performed. β-actin was used as the loading control. Phospho-H2A.X, phosphorylated-histone H2A histone family member X.

Effect of luteolin on cell survival following H₂O₂ treatment

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).

Figure 5.

Cytoprotective effect of luteolin on H₂O₂-induced apoptosis. (A) MTT assay was used to assess cell viability. *P<0.05 vs. control; ^#P<0.05 vs. H₂O₂. (B) Apoptotic bodies (arrow) were revealed by Hoechst 33342 staining. (C) Sub-G₁ cells, which is indicative of apoptosis, were identified by flow cytometry. (D) Western blot analysis with antibodies against Bax, Bcl-2, active caspase-9 and caspase-3 was performed. β-actin was employed as a loading control.

Effect of luteolin on antioxidant systems

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).

Figure 6.

Effect of luteolin on antioxidant systems. (A) 7-Amino-4-chloromethylcoumarin dye was used to detect intracellular GSH levels. (B) Western blot analysis with antibody against γ-GCL was assessed. (C) The activities of SOD, CAT, and GPx were presented as unit per mg of protein. (D) HO-1 enzyme activity was shown as pmol bilirubin/mg protein. (E) Western blotting analysis with antibodies for Cu/Zn SOD, CAT, GPx and HO-1. β-actin was employed as a loading control. *P<0.05 vs. control; ^#P<0.05 vs. H₂O₂. GSH, glutathione; γ-GCL, γ-glutamylcysteine ligase; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; HO-1, heme oxygenase-1.

Discussion

Flavonoids are categorized based on their molecular structures into flavan-3-ols, flavones, flavonols, flavanones, isoflavones, and anthocyanins (27). These substances are abundantly found in coffee, fruit, vegetables and cocoa-containing products (28). Flavonoids are known for broad therapeutic benefits, including anticancer, antioxidant, anti-inflammatory, anti-microbial and antiangiogenic effects (29). Extensive research, including our prior studies, has demonstrated that luteolin induces apoptosis in various types of cancer cells, such as human colon, melanoma, lung, cervical, monocytic leukemia and breast cancer cells (23,30–32). Notably, Liu et al (33) reported the protective effects of luteolin against angiotensin II-induced renal damage in apolipoprotein E-deficient mice. The present study evaluated the antioxidant potential of luteolin against H₂O₂-induced oxidative stress in lung fibroblasts and demonstrated that luteolin inhibited H₂O₂-mediated cellular damage by upregulating antioxidant enzymes.

ROS induce cellular damage and cause disease progression, including chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis (34,35). Here, luteolin showed potent scavenging capabilities for O₂⁻ and ·OH in a cell-free system. Additionally, pretreatment with luteolin resulted in a significant decrease in intracellular levels of ROS. Given its efficacy in neutralizing ROS, including O₂⁻ and ·OH, luteolin may serve as a promising therapeutic agent for management and treatment of conditions such as COPD and pulmonary fibrosis.

ROS-induced lipid peroxidation compromises cellular membranes (36). Polyunsaturated fatty acids, particularly those vulnerable to ROS, undergo peroxidation, leading to a cascade of free radical reactions (34,37,38). Here, H₂O₂ strongly promoted lipid peroxidation and TBARS production; however, luteolin inhibited lipid peroxidation in the cell membranes. GPx catalyzes conversion of H₂O₂ into water and lipid peroxides into their corresponding alcohols (6). Moreover, bilirubin, a product of HO-1 activity, offers protection against lipid peroxidation (39). The present study indicates that luteolin directly scavenges ROS and upregulates antioxidant enzymes such as GPx and HO-1, thereby mitigating lipid peroxidation. Additionally, luteolin decreased DNA damage and phospho-H2A.X protein levels that were elevated by H₂O₂ treatment. The present study investigated proteins associated with the mitochondrial cell death pathway to gain insight into the mechanisms of apoptosis as mitochondria-mediated apoptosis can be induced in response to H₂O₂ exposure (40,41). Luteolin suppressed active caspase-9 and caspase-3 while lowering the levels of the pro-apoptotic protein Bax and increasing the levels of anti-apoptotic protein Bcl-2. As luteolin attenuated cellular lipid peroxidation and DNA damage and inhibits H₂O₂-mediated cell apoptosis in the present study, we focused on the antioxidant capacity of luteolin to decrease the adverse effect of H₂O₂.

The cellular antioxidant system, comprising enzymes such as SOD, CAT, GPx, and HO-1, serves a crucial role in mitigating oxidative stress-induced cellular damage (42,43). As evidenced by CMAC staining and western blotting, luteolin pretreatment restored H₂O₂ mitigated GSH and γ-GCL levels. H₂O₂ treatment reduced levels and enzymatic activity of SOD, CAT, GPx, and HO-1 proteins, which were subsequently restored by luteolin pretreatment. Previous research has indicated that luteolin upregulates NRF2 and HO-1 expression, thereby decreasing H₂O₂-induced oxidative damage in intestinal epithelial cells (44). Additionally, luteolin is known to promote autophagy and antioxidant processes via activation of the p62/KEAP1/NRF2 pathway, which has been identified for its neuroprotective effect (45). Luteolin scavenges ROS by enhancing antioxidant enzymes by regulating the NRF2 signaling pathway (46,47). The NRF2 signaling pathway is integral to cellular antioxidant defenses and maintenance of redox homeostasis, regulating the expression of antioxidant and drug-metabolizing enzymes such as SOD, CAT, GPx, and HO-1 (48). The present study demonstrates that luteolin activates these antioxidant enzymes to counteract H₂O₂-induced oxidative stress, suggesting induction of these enzymes via the NRF2 signaling pathway. Taken together, the present results suggested that luteolin can prevent ROS generation by mitigating cellular damage and improving antioxidant enzyme activity.

In conclusion, luteolin inhibited H₂O₂-induced lipid peroxidation, DNA damage, and cell apoptosis while augmenting the activity of cellular antioxidant enzymes (CAT, SOD, GPx, and HO-1), thus safeguarding V79-4 cells from oxidative harm. These properties position luteolin as a potential agent for protecting lung fibroblasts against oxidative damage, warranting further clinical investigation to assess its efficacy in alleviating oxidative stress-associated pulmonary conditions.

Funding Statement

The present study was supported by the National Research Foundation of Korea, funded by the Ministry of Education (grant no. RS-2023-00270936) and the Ministry of Science and ICT (grant no. NRF-2023R1A2C1002770).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

PDSMF, DOK, and JWH conceived and designed the study and wrote the manuscript. DOK, HMULH, MJP, and KAK performed the experiments and data analysis and interpretation. PDSMF and JWH confirm the authenticity of all the raw data. PDSMF, DOK, HMULH, and JWH revised the manuscript for important intellectual content. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.