Primary antibodies against occludin (cat. no. 27260-1-AP), Sirt1 (cat. no. 13161-1-AP) and GAPDH (cat. no. 60004-1-Ig), as well as HRP-conjugated goat anti-mouse (cat. no. SA00001-1), goat anti-rabbit (cat. no. SA00001-2) and rabbit anti-goat (cat. no. SA00001-4) secondary antibodies were purchased from Wuhan Sanying Biotechnology. Primary antibodies against zonula occludens-1 (ZO-1) (cat. no. ab190085), Gpx4 (cat. no. ab125066), Nrf2 (cat. no. ab92946) and 4-hydroxynonenal (4-HNE; cat. no. ab46545) were purchased from Abcam. Quercetin, EX527 (Sirt1 inhibitor) and ferrostatin-1 were obtained from Selleck Chemicals. LPS (055: B5) was obtained from Beijing Solarbio Science & Technology Co., Ltd.

Primary mouse type II alveolar epithelial (AT2) cells (cat. no. CP-M003) were obtained from Procell Life Science & Technology Co., Ltd. Cells were cultured in DMEM/F-12 containing 10% FBS and maintained at 37°C in a humidified 5% CO₂ incubator. The Sirt1 small interfering (si)RNA and negative control (NC) sequences were designed and synthesized by Genomeditech (Shanghai) Co., Ltd. The siRNA sequences were as follows: siSirt1#1 sense, 5′-GCU GAG GUA UAU UCA GAC U-3′ and antisense, 5′-AGU CUG AAU AUA CCU CAG C-3′; siSirt1#2 sense, 5′-GAC UCA AGU UCA CCU GAA AGA-3′ and antisense, 5′-UCU UUC AGG UGA ACU UGA GUC-3′; siSirt1#3 sense, 5′-CCU CAA GCG AUG UUU GAU A-3′ and antisense, 5′-UAU CAA ACA UCG CUU GAG G-3′; and siNC sense, 5′-UUC UCC GAA CGU GUC ACG U-3′ and antisense, 5′-ACG UGA CAC GUU CGG A GAA-3′. Cell transfection was performed according to the manufacturer's instructions of Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The final concentration of siRNA) was 20 nM. After 6 h of culture at 37°C in a humidified 5% CO₂ incubator, the medium that transfection medium was replaced with fresh complete culture medium. Subsequent experiments were performed after 36 h.

To explore the protective effects of quercetin in vitro, an LPS-induced AT2 cell injury model was established. Briefly, AT2 cells were seeded in 96-well plates (5,000 cells/well or 6-well plates (1×10⁵ cells/well), pretreated with quercetin (0, 5, 10 and 20 μM) for 2 h, and then LPS (5, 10 or 20 μg/ml) was used to induce the acute cell injury model. The cells were cultured at 37°C in a 5% CO₂ incubator. Cells were collected at 6, 12 and 24 h for subsequent experiments.

CCK-8, cell death and LDH release assays were performed to measure cell injury as previously described (26). CCK-8 (cat. no. C0039), LDH (cat. no. C0016) and cell death reagents (7-AAD; cat. no. C1053S) were purchased from Beyotime Institute of Biotechnology. AT2 cells were seeded in 96-well plates (5,000 cells/well) for CCK-8 and LDH assays or 6-well plates (1×10⁵ cells/well) for cell death detection. For the CCK-8 assay, 10 μl CCK-8 reagent was added to each well, incubated at 37°C for 1 h, and the optical density value was measured at 450 nm. For cell death detection, after the AT2 cells were treated as aforementioned, the cells were digested with trypsin without EDTA, and then the cells were harvested and washed twice with PBS. A total of 500 μl 7-AAD working solution was added to each well and incubated at 37°C for 10 min. Cell death was detected using a flow cytometer (FACSCalibur; Becton, Dickinson and Company). CytExpert 2.4 software (Beckman Coulter, Inc.) was used to analyze the data.

RT-qPCR was performed as previously described (27). Total RNA was extracted from AT2 cells according to the instructions of the Total RNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd.). RNA samples were reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. The cDNAs were then added to the reaction with the indicated primers using SYBR Green Supermix (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions and amplified using qPCR, performed at 95°C for 2 min, followed by 40 cycles of amplification at 95°C for 10 sec, 62°C for 30 sec and 72°C for 30 sec, using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). Data were analyzed with 2^-ΔΔCq method (28), using β-actin for normalization. The following primers were used: Sirt1-forward, 5′-TCC TTC AGT GTC ATG GTT CCT TTG C-3′ and reverse, 5′-CTC CAC GAA CAG CTT CAC AAT CAA C-3′; and β-actin forward, 5′-GTG CTA TGT TGC TCT AGA CTT CG-3′ and reverse, 5′-ATG CCA CAG GAT TCC ATA CC-3′.

A total of 102 male C57BL/6 mice (age, 6-8 weeks; weight, 18-22 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd. (https://www.cd-dossy.cn/). All mice were housed in cages with free access to food and water at 25°C with a 12/12-h light/dark cycle and ~55% relative humidity, and were quarantined and acclimatized before the experiment. The mice were randomly divided into five groups (n=6 mice/group): Control (saline), LPS (5 mg/kg, dissolved in saline) and LPS + quercetin (20, 40 or 60 mg/kg, dissolved in DMSO) for lung injury detection. The ALI model was established based on previous studies (29). Briefly, quercetin (20, 40 or 60 mg/kg) was administered via oral gavage. For ferroptosis detection, the mice were randomly divided into four groups (n=6 mice/group): Control (saline), LPS (5 mg/kg, dissolved in saline), quercetin (40 mg/kg, dissolved in DMSO), LPS + quercetin. Inhibition of Sirt1 in vivo, mice were divided into four groups (n=6 mice/group): Control (saline), LPS (5 mg/kg), LPS + quercetin (40 mg/kg) and LPS + quercetin + EX527 (5 mg/kg). For alveolar epithelial barrier integrity detection, the mice were randomly divided into four groups (n=6 mice/group): Control (saline), LPS (5 mg/kg), LPS + quercetin (40 mg/kg), LPS + ferrostatin-1 (5 mg/kg). The use of ferrostatin-1 as a ferroptosis inhibitor was based on our previous study (27). After 1 h of treatment with quercetin, Sirt1 inhibitor (EX527) or ferrostatin-1, the mice were anesthetized with sodium pentobarbital (intraperitoneal injection, 50 mg/kg), and LPS was intratracheally injected to induce lung injury. A total of 12 h after LPS administration, the mice were euthanized by intraperitoneal injection of 120 mg/kg sodium pentobarbital, and the lung tissue and bronchoalveolar lavage fluid (BALF) were collected. Death was verified by observing the cardiac arrest and spontaneous respiratory arrest.

During the experimental process, the health and behavior of the animals were monitored hourly. Humane endpoints included indicators, such as huddled posture, immobility, ruffled fur, failure to eat, hypothermia (colonic temperature of <34°C), weight loss >20%, inability to stand, agonal breathing, severe muscular atrophy, severe ulceration or uncontrolled bleeding. There was no accidental death of mice during the present study, and all the mice were euthanized at the end of the experiment.

IHC, IF and western blot assays were used to evaluate the protein levels of Sirt1, Nrf2, ZO-1, occludin, 4-HNE and Gpx4. For IHC, mouse lung tissues were fixed overnight with 4% paraformaldehyde at room temperature, followed by paraffin embedding and slicing (5 μm). The lung tissue sections were waxed at 65°C for 2 h, dewaxed twice in xylene (5 min each time), immersed in 100, 95, 85 and 75 % ethanol for 2 min each, and then immersed in PBS for 2 min. Subsequently, the tissue sections were immersed in EDTA Antigen Retrieval solution (1:50, diluted in deionized water; cat. no. P0085; Beyotime Institute of Biotechnology) in a pressure cooker for 10 min, cooled naturally at room temperature, and washed twice with PBS. After incubation in 3% H₂O₂ for 10 min, the sections were washed three times with PBS. Blocking with 5% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) was performed at room temperature for 30 min, following by primary antibody addition at 4°C overnight. On the next day, the sections were washed twice with PBS, followed by incubation with the secondary antibody (HRP-labelled; ready-to-use; cat. no. PV-9000; OriGene Technologies, Inc.) was added to cover the lung tissue and incubated at 37°C for 20 min, and the sections were washed twice with PBS (5 min each). DAB (cat. no. ZLI-9017; OriGene Technologies, Inc.) was used to stain the sections. Finally, the sections were incubated with hematoxylin staining solution for 1.5 min at room temperature.

For IF, AT2 cells were fixed with 4% paraformaldehyde at room temperature for 15 min, incubated with 0.1% Triton X-100 for 5 min to permeate the cell membrane, and blocked with 5% goat serum at room temperature for 30 min, followed by primary antibody incubation (1:100) overnight at 4°C. The secondary antibody, Cy3-labeled goat anti-rabbit (cat. no. A0516; Beyotime Institute of Biotechnology) or Cy3-labeled donkey anti-goat (cat. no. A0502; Beyotime Institute of Biotechnology) was diluted 1:300 and incubated at 37°C in the dark for 1 h. After washing with PBS three times, DAPI (1:5,000; diluted in PBS; cat. no. C0060; Beijing Solarbio Science & Technology Co., Ltd.) was added at 37°C in the dark for 5 min.

For WB, AT2 cells or lung tissue proteins were extracted using RIPA lysis Buffer (cat. no. P0013C; Beyotime Institute of Biotechnology,) and protein determination was performed with BCA. Protein samples (30 μg) were separated on 10% gels using SDS-PAGE and transferred to 0.2-μm polyvinylidene fluoride membranes. The membranes were blocked using Tris-buffered saline containing 0.1% Tween-20 and 5% skimmed milk for 1 h at 25°C. The membranes were incubated with the primary antibodies overnight at 4°C. After washing, the membranes were incubated with corresponding HRP-conjugated secondary antibodies (1:5,000) at 25°C for 1.5 h. The bands were visualized in conjunction with ECL Detection Reagents (EMD Millipore) using the Quantity One 5.2 software (Bio-Rad Laboratories, Inc.). All primary antibodies were used at a 1:100 dilution for IHC and IF and 1:1,000 dilution for western blotting.

H&E staining was performed as previously described (30). The hematoxylin staining solution was incubated for 1.5 min and the eosin dye solution for 30 sec at room temperature. Random images for IHC, IF, and H&E were captured at a magnification of ×200 or ×400 using a fluorescence microscope (DM4000B; Leica Microsystems, Inc.) and analyzed using ImageJ v1.48 software (National Institutes of Health). The severity of lung injury was scored was evaluated based on the alveolar wall thickness, the amount of cellular infiltration and the level of hemorrhaging, as previously described with minor modifications (31). Lung injury scores were graded on a scale of 0-8 as follows: 0, no injury; 2, mild injury (up to 25% injury of the field of view); 4, moderate injury (up to 50% injury of the field of view); 6, severe injury (up to 75% injury of the field of view); and 8, extremely severe injury (diffused injury).

The levels of IL-6 (cat. no. ml098430), IL-1β (cat. no. ml098416) and TNF-α (cat. no. mIC50536-1) in mouse BALF were detected using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the manufacturer's protocols. A myeloperoxidase (MPO) activity assay kit (cat. no. RXSH0539; Quanzhou Ruixin Biotechnology Co., Ltd.) was used to measure MPO activity in lung tissues.

Dihydroethidium (DHE; Molecular Probes; Thermo Fisher Scientific, Inc.) staining was used to determine ROS levels in the lung tissues. Briefly, the DHE probe was diluted with PBS (1:5,000) and added to the lung tissue sections at room temperature for 10 min. ROS levels in AT2 cells were measured using a ROS Assay kit (cat. no. S0033S; Beyotime Institute of Biotechnology) by flow cytometry as previously described (23). Briefly, AT2 cells, seeded in 6-well plates (1×10⁵/well), were treated as aforementioned and incubated with RPMI-1640 medium containing dichlorodihydrofluorescein diacetate (10 μM; included in the ROS kit) at 37°C for 20 min. The cells were then washed three times with PBS, and fluorescence intensity was determined with a flow cytometer (FACSCalibur; Becton, Dickinson and Company). CytExpert 2.4 software (Beckman Coulter, Inc.) was to analyze the data. GSH levels in AT2 cells and lung tissues were measured using a GSH assay kit (cat. no. BC1170; Beijing Solarbio Science & Technology Co., Ltd.). Lipid peroxidation levels and relative iron concentrations in AT2 cells and lung tissues were evaluated by measuring malondialdehyde (MDA) concentrations using a lipid peroxidation assay kit (cat. no. S0131M; Beyotime Institute of Biotechnology) and an Iron Assay Kit (cat. no. E1042; Applygen Technologies, Inc.), respectively. All kits were used according to the manufacturers' instructions.

The W/D ratio measurement of lung samples were conducted as previously described (27). For W/D ratio measurement, after the mice were sacrificed the whole lung tissue was excised and quickly rinsed with pre-cooled normal saline. A filter paper was used to remove excess water, and the weight of the lung tissue of each group of mice was immediately weighed. At this point, the measurement data was recorded as W weight. Subsequently, the lung tissue was dried in a constant temperature oven at 65°C for 48 h, and then weighed. The measured data was recorded as D weight.

The EDU assay was conducted as previously described (27). Briefly, AT2 cells, seeded in 24-well plates (2×10⁴/well), were treated as aforementioned and incubated with EDU (cat. no. C0071S; Beyotime Institute of Biotechnology) working solution at 37°C for 30 min. After washing with PBS three times, DAPI (1:5,000, diluted in PBS) was added at 37°C in the dark for 5 min. Random images were captured at ×200 magnification using a fluorescence microscope (Leica Microsystems, Inc.).

The mitochondrial morphology of AT2 cells was analyzed via TEM (FEIG2; Thermo Fisher Scientific, Inc.) as previously described (27). Briefly, the cells were first fixed in 2.5% glutaraldehyde and then incubated in 0.1 M osmium tetroxide (prepared in 0.1 M PBS, pH 7.4) for 2 h or longer at room temperature. Following further dehydration, permeation and embedding (in araldite) steps, ultra-thin sections (65 nm) were obtained and viewed with the transmission electron microscope.

All in vitro experiments were performed independently at least three times, and all animals were randomly assigned to their experimental groups. Data are presented as the mean ± standard deviation or as the median + interquartile range and were analyzed using GraphPad Prism 7 software (GraphPad Software; Dotmatics). Statistical significance was determined using one-way ANOVA followed by Tukey's post hoc test or Kruskal-Wallis followed by Dunn's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Firstly, LPS was used to establish a mouse model of ALI and examine the potential protective effects of quercetin (Fig. 1A) against ALI. The results showed that quercetin reduced LPS-induced edema (Fig. 1B) and decreased the alveolar wall thickness and the amount of cellular infiltration induced by LPS (Fig. 1C and D), as measured by the lung injury score. Furthermore, the total cell count and protein concentrations in BALF were markedly increased after LPS treatment compared with the control group (Fig. 1E and F). However, pretreatment with quercetin markedly decreased the total number of cells and protein leakage in BALF. In addition, quercetin significantly decreased MPO activity compared with the LPS-only group (Fig. 1G). Moreover, quercetin pretreatment effectively reduced TNF-α, IL-6 and IL-1β levels in mouse BALF (Fig. 1H-J). Collectively, the present results indicate that quercetin significantly alleviated the pathological changes and inflammatory response in the lung tissues of LPS-induced mice.

To further evaluate the effects of quercetin on LPS-induced alveolar epithelial cell injury, an LPS-induced AT2 cell injury model was established. The CCK-8 and LDH release assays showed that LPS treatment markedly inhibited the proliferation of AT2 cells and increased the LDH release in a time- and dose-dependent manner (Fig. 2A and B). Next, to determine the most effective quercetin concentration, AT2 cells were separated into five groups: Control (saline), LPS (10 μM) and LPS + quercetin (5, 10 or 20 μM). CCK-8 and LDH release assays were subsequently performed (Fig. 2C and D). The results indicated that 10 and 20 μM quercetin had similar protective effects on the viability and LDH release of AT2 cells. Based on these data, a 10 μM concentration for both LPS and quercetin, as well as the 12-h time point were selected for subsequent cell experiments. The results demonstrated that quercetin treatment significantly reduced inflammatory cytokine secretion (Fig. 2E-G), promoted cell proliferation (Fig. 2H and I) and inhibited cell death (Fig. 2J and K) after LPS treatment. Collectively, these results suggest that quercetin exhibited protective effects against LPS-induced alveolar epithelial cell injury.

To investigate whether quercetin inhibits ferroptosis in ALI, ferroptosis parameters were evaluated in the LPS-induced ALI mouse model. Quercetin decreased the alveolar wall thickness and the amount of cellular infiltration in LPS-treated mice, as evaluated through H&E staining (Fig. 3A) and the lung injury score (Fig. 3B). In addition, quercetin significantly attenuated LPS-induced ferroptosis in the lungs of mice with ALI. Specifically, quercetin reversed the decrease in the levels of GSH (Fig. 3C) and Gpx4 (Fig. 3H and J) and reduced the levels of MDA (Fig. 3D), iron (Fig. 3E), ROS (Fig. 3F and G) and 4-HNE (Fig. 3I and J) in LPS-treated mice. To explore whether quercetin modulated the Sirt1/Nrf2/Gpx4 signaling pathway in LPS-induced ALI, the protein expression levels of Sirt1, Nrf2 and Gpx4 were detected. As shown in Fig. 3K-N, the protein expression of Sirt1, Nrf2 and Gpx4 was upregulated in the LPS + quercetin group compared with that in the LPS group. Collectively, these results indicate that quercetin inhibited LPS-induced ferroptosis via the Sirt1/Nrf2/Gpx4 pathway.

Consistent with the in vivo results, quercetin inhibited the decrease in the levels of GSH (Fig. 4A) and Gpx4 (Fig. 4E and F) and reduced the levels of MDA (Fig. 4B), iron (Fig. 4C), ROS (Fig. 4D) and 4-HNE (Fig. 4E and G) in the LPS-induced AT2 cell model. Moreover, quercetin inhibited the LPS-induced mitochondrial shrinkage and decreased the mitochondrial cristae (Fig. 4H). Similarly, the expression levels of Sirt1, Nrf2 and Gpx4 were significantly decreased after LPS treatment compared with the control group, and quercetin effectively reversed this decrease (Fig. 4I-L). These data suggest that quercetin inhibited LPS-induced ferroptosis in alveolar epithelial cells via the Sirt1/Nrf2/Gpx4 pathway.

To further confirm that quercetin alleviates ALI by inhibiting ferroptosis via the Sirt1 pathway, in vivo experiments were performed using the Sirt1 inhibitor EX527 (5 mg/kg) based on a previous study (32). EX527 abolished the protective effect of quercetin on LPS-induced ALI (Fig. 5A-D, F and G) and ferroptosis (Fig. 5E and H-L). The results indicated that quercetin markedly reduced LPS-induced edema (Fig. 5A), alveolar wall thickness and the amount of cellular infiltration (Fig. 5F and G) and MPO activity in lung tissue (Fig. 5D) and decreased the total number of cells (Fig. 5B) and protein leakage (Fig. 5C) in BALF. However, EX527 reversed these protective effects of quercetin. Furthermore, EX527 reversed the effect of quercetin on inhibiting Gpx4 degradation (Fig. 5J and K) and reducing the levels of iron (Fig. 5E), ROS (Fig. 5H and I) and 4-HNE (Fig. 5J and L) in LPS-treated mice.

To further verify the role of Sirt1 in the quercetin-induced inhibition of ferroptosis in vitro, we downregulated Sirt1 expression was knocked down in primary mouse AT2 cells using siRNA. The knockdown efficiency was confirmed by RT-qPCR and western blotting (Fig. 6A-C), and siSirt1#3 was selected for subsequent experiments. Sirt1 knockdown reduced the protective effect of quercetin against LPS-induced cell injury, including reducing the viability and increasing the death of AT2 cells (Fig. 6D-F). Moreover, Sirt1 knockdown abolished the protective effect of quercetin on LPS-induced ferroptosis in AT2 cells, including increasing the levels of ROS (Fig. 6I and J), MDA (Fig. 6G) and iron (Fig. 6H), and inducing mitochondrial shrinkage (Fig. 6K), confirming the previous findings.

Using LPS-induced ALI in vivo and in vitro models, the tight junction protein expression levels of ZO-1 and occludin were evaluated using IHC (Fig. 7A-C), western blotting (Fig. 7D-I) and IF (Fig. 7J-L). The expression of these proteins was significantly decreased following LPS treatment. To demonstrate the role of ferroptosis in LPS-induced epithelial barrier destruction, a ferroptosis inhibitor was used, the dose of which was selected based on our previous study (27). The results showed that both quercetin and ferrostatin-1, significantly enhanced the protein expression of ZO-1 and occludin in LPS-treated mice and cells. Together, these results indicate that quercetin attenuated the LPS-induced reduction of tight junction proteins both in vivo and in vitro by preventing ferroptosis.