Baicalein (CAS no. 491-67-8) was purchased from Chengdu Must Bio-Technology Co., Ltd. Lipopolysaccharide (LPS) was obtained from MilliporeSigma and dexamethasone (DEX; CAS no. 50-02-2) from Shanghai Yuan Ye Bio-Technology Co., Ltd. The antibodies against hexokinase 2 (HK2; cat. no. 66974-1-Ig), phosphofructokinase-1 (PFK1; cat. no. 55028-1-AP), pyruvate kinase M2 (PKM2; cat. no. 10078-2-AP) and HIF1α (cat. no. 20960-1-AP) were purchased from Proteintech Group, Inc.

A total of 30 male C57BL/6 mice (weight, 18–22 g; age, 6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. In the present study, animal welfare considerations included all possible efforts to minimize suffering and distress, as well as the use of anesthetics and controlled housing conditions. For example, mice were housed in a temperature-controlled (23.0±1.0°C) and humidity-controlled (40–60%) cage under a 12-h light/dark cycle and with free access to food and water. The bedding material was composed of fine and softwood chips. To maintain a hygienic environment, the bedding material was changed at 2-day intervals. Animal health and behavior were monitored every day, and body weights were assessed weekly over the course of the study. Mice were randomly divided into the following five groups (n=6 mice/group): Normal, LPS, 10 mg/kg baicalein, 20 mg/kg baicalein and DEX groups. All groups received one-time tracheal instillation of LPS, with the exception of the normal group. A total of 7 days before modeling, the groups designated to receive medication were administered baicalein and DEX (2 mg/kg) by gavage. The ALI mouse model was established by a one-time tracheal instillation of LPS (5 mg/kg), which was a non-invasive procedure. Firstly, mice were anesthetized via intraperitoneal injection of pentobarbital sodium (50 mg/kg; MilliporeSigma). Subsequently, the mice were suspended on the experimental operating table, and their tongues were pulled to the side to expose the tracheal opening. A catheter was then inserted into the trachea along the opening. The needle core was immediately removed, and the corresponding LPS and saline solution was injected into the catheter using a 1-ml syringe. The normal group of mice was only injected with the corresponding volume of saline solution. Finally, the mice were placed under room temperature to recover from anesthesia before they were returned to their cages. At 24 h after modeling, the mice were euthanized by cervical dislocation, and bronchoalveolar lavage fluid (BALF), serum and lung tissue samples were collected. In addition, 0.3 ml blood was collected from the retro-orbital vein just before sacrifice and serum was obtained by centrifuging the blood at 1,500 × g for 15 min at 4°C. The death of the mice was verified by lack of respiration, heartbeat and corneal reflex. The duration of the experiment was 15 days, including 7 days of adaptation, 7 days of dosing and 1 day of modeling. Animal health and behavior were monitored every day and no spontaneous death occurred during the present study. The present study was approved by the Ethics Committee of the Zhumadian Hospital of Traditional Chinese Medicine (approval no. 2024104001; Zhumadian, China). Mice that reached the humane endpoints (including but not limited to >15% weight loss, inability to eat or drink, and signs of extreme distress or pain) or completed the experiments were euthanized. In the present study, no mice reached the predefined humane endpoints before the end of the experiments.

MH-S mouse alveolar macrophages (cat. no. GNM43; The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) were cultured in RPMI 1640 complete medium (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 10% fetal bovine serum (Lonsera; Shanghai Shuangru Biotechnology Co., Ltd.). The RPMI 1640 medium used in the experiment contains 1% penicillin and streptomycin. The cells were divided into the following five groups: Control group; model group (100 ng/ml LPS); and baicalein groups (40, 20 and 10 µM). With the exception of the control group, the other groups were stimulated with LPS. Cells were stimulated with LPS (100 ng/ml) and baicalein (40, 20 and 10 µM) in an incubator at 37°C and 5% CO₂. After 6 h of treatment, cells were harvested for testing.

The left lungs obtained from the mice were washed with saline and dried, and the wet weight of the lungs was then measured. Subsequently, the left lungs were dried overnight in an oven at 65°C and the dry weight was measured. The W/D ratio was calculated based on the wet and dry weights.

The left lungs were lavaged three times with 0.6 ml PBS to collect BALF, and the protein concentration in the BALF was then determined using a BCA kit. In addition, BALF was centrifuged at 1,000 × g for 10 min at 4°C, and the cell pellet and supernatant of BALF were collected separately. Subsequently, the cell pellet was resuspended in PBS and total cells were counted using a hemocytometer.

The upper lobes of the left lungs were fixed in 4% paraformaldehyde for 48 h at room temperature, embedded in paraffin, sectioned (4 µm) and stained with 1% hematoxylin for 10 min at room temperature and 1% eosin for 1 min at room temperature. Subsequently, pathological changes in the lung tissues were observed under a light microscope. The lung tissues stained with hematoxylin and eosin (H&E) were analyzed based on the Szapiel score (26).

The levels of lactic acid in lung tissues and cell supernatants were measured using a lactate colorimetric assay kit (cat. no. E-BC-K044-M; Elabscience^®; Elabscience Bionovation Inc.), according to the manufacturer's instructions. The absorbance was then measured at 530 nm and lactic acid levels in the intervention groups were normalized to those of the control group, as previously described (27).

The aforementioned paraffin-embedded tissue sections from the upper lobes of the left lungs were deparaffinized with xylene and rehydrated in a graded alcohol series (100, 90 and 70%). The sections were then heated in 10 mM sodium citrate buffer (pH 6.0), for 15 min in a 95°C water bath for antigen retrieval. The sections were then washed with PBS three times (3 min each). Endogenous peroxidase activity was blocked by incubation with 3% H₂O₂ for 15 min at room temperature, and nonspecific immunoreactions were blocked using 5% inactivated goat serum (Beijing Solarbio Science & Technology Co., Ltd.) in PBS for 30 min at room temperature. The sections were then incubated with HK2 (1:200), PFK1 (1:200), PKM2 (1:400) and HIF-1α (1:200) antibodies overnight at 4°C. After washing with PBS, the sections were incubated with goat anti-rabbit IgG-HRP secondary antibody (1:500; cat. no. sc-2030; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. After counterstaining with hematoxylin for 5 min at room temperature, images of the sections were captured using the light microscope (Carl Zeiss AG).

The paraffin-embedded lung tissue was cut in 4-µm sections. After dewaxing and rehydrating, the slides were subjected to antigen retrieval, by immersing in 10 mM sodium citrate (pH 6.0) and microwaving at 1,000 W for 30 min. The sections were then blocked with 5% inactivated goat serum at room temperature for 30 min. Subsequently, the sections were incubated with an antibody against HIF-1α (1:400) at 4°C overnight. Subsequently, Alexa Fluor^® 488-conjugated Goat Anti-Rabbit IgG H&L (1:1,000; cat. no. 4412; Cell Signaling Technology, Inc.) was added and incubated at 37°C for 1 h. The sections were incubated with DAPI (cat. no. 4083; Cell Signaling Technology, Inc.) in the dark at room temperature for 5 min for nuclear counterstaining. The collected images were captured under a laser confocal microscope (LSM700; Carl Zeiss AG).

The concentrations of TNF-α and IL-6 in mouse BALF, serum and cell supernatants were measured using the TNF-α ELISA kit (cat. no. E-EL-M3063; Elabscience^®; Elabscience Bionovation Inc.) and the IL-6 ELISA kit (cat. no. E-EL-M0044; Elabscience^®; Elabscience Bionovation Inc.), according to the manufacturer's instructions and previous studies (28).

Cell viability was detected using the Cell Counting Kit-8 (CCK-8) assay kit (Dojindo Molecular Technologies, Inc.). Briefly, cells (3×10³ cells/well) were seeded into 96-well plates and incubated at 37°C with 5% CO₂. After incubation with baicalein at varying concentrations (40, 20, 10, 5 and 2.5 µM) for 6 h, 10 µl CCK-8 solution was added to each well and incubated for an additional 4 h at 37°C. Absorbance was subsequently measured at a wavelength of 450 nm.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was reverse-transcribed into cDNA using a HiScript^® II 1st Strand cDNA Synthesis Kit (cat. no. R211-01; Vazyme Biotech Co., Ltd.), according to the manufacturer's instructions. The mRNA expression levels of HK2, PFK1, PKM2 and HIF-1α were detected using AceQ^® qPCR SYBR^® Green Master Mix (cat. no. Q111-02; Vazyme Biotech Co., Ltd.). mRNA expression levels were normalized to ACTB and calculated using the 2^−ΔΔCq method (29). The primer sequences used are listed in Table I. The following thermocycling conditions were used for qPCR: 95°C for 30 sec, followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec, 95°C for 15 sec, and final extension at 60°C for 60 sec and 95°C for 15 sec.

ALI-associated genes were identified using the GeneCards (https://www.genecards.org) database with ‘acute lung injury’ used as the key word. The targets of baicalein were obtained through screening in the SwissTargetPrediction database (http://old.swisstargetprediction.ch).

The overlapping targets between baicalein and ALI were imported into the STRING v11.5 database (https://string-db.org/) and a PPI network was constructed using Cytoscape software (version 3.7.1; http://cytoscape.org/). The top 100 genes were identified as core targets using the Cytohubba (https://apps.cytoscape.org/apps/cytohubba) (30), a plugin in Cytoscape software.

The overlapping targets were analyzed for GO term and KEGG pathway enrichment using the DAVID database (https://david.ncifcrf.gov). GO terms and KEGG pathways with P<0.05 were considered significantly enriched. Notably, no specific gene count thresholds were applied in the analysis. Component and target molecular docking were performed using AutoDock Tools 1.5.6 software (The Scripps Research Institute, La Jolla, CA, USA).

The data from cell experiments are from three independent experiments consisting of three replicates per experiment. All experimental data were analyzed using SPSS 23.0 software (IBM Corp.). Data are presented as the mean ± SEM. Non-parametric data were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison test. The differences among multiple groups were analyzed by one-way ANOVA, followed by Dunnett's T3 for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the therapeutic effects of baicalein (Fig. 1A) on ALI, lung tissue pathology, W/D weight ratio, the number of cells and the total protein concentration in the BALF of mice were assessed. As a well-accepted drug in treating lung injury with strong clinical evidence, DEX was used as a positive control. H&E staining showed that baicalein could markedly inhibit LPS-induced inflammatory cell infiltration, alveolar wall edema and thickening (Fig. 1B and C). Furthermore, baicalein reduced the lung W/D weight ratio (Fig. 1D), the total number of cells and the total protein concentration in the BALF of mice (Fig. 1E) compared with those in the model group. These results suggested that baicalein could improve LPS-induced lung injury, alveolar-capillary barrier dysfunction and pulmonary edema.

Bioinformatics analysis using the SwissTargetPrediction and GeneCards databases predicted 293 targets of baicalein and 8,701 ALI-related genes, respectively. The intersection of baicalein-related targets and ALI-related genes revealed a total of 248 overlapping targets (Fig. 2A). The PPI of the top 100 overlapping targets was analyzed using PPI network analysis (Fig. 2B). KEGG enrichment analysis with P<0.05 showed that the aforementioned overlapping targets were enriched in ‘MAPK signaling pathway’, ‘HIF-1 signaling pathway’ and ‘Glycolysis/Gluconeogenesis’ (Fig. 2C). The GO functional enrichment analysis results with P<0.05 indicated that the overlapping targets were associated with ‘cellular response to lipopolysaccharide’ and ‘response to lipopolysaccharide’ (Fig. 2D).

The inflammatory response is a key pathological process in ALI; therefore, the level of inflammation in mice with ALI was first evaluated. The results showed that baicalein significantly reduced the levels of IL-6 and TNF-α in the serum and BALF of mice compared with those in the model group mice (Fig. 3A and B). Additionally, cell viability was measured to assess the cytotoxicity of baicalein on MH-S cells. The result showed that the cell viability was not significantly affected by baicalein treatment (Fig. 3C). Furthermore, baicalein significantly inhibited the levels of IL-6 and TNF-α in LPS-induced MH-S cells (Fig. 3D and E).

Glycolysis serves a significant role in the inflammatory response; therefore, the present study aimed to explore whether baicalein could attenuate the inflammatory response via inhibiting glycolysis. The results showed that baicalein could inhibit the expression of key glycolysis-related enzymes (HK2, PFK1 and PKM2) in the lungs of mice with LPS-induced ALI and in LPS-induced macrophages (Fig. 4A-D and F). In addition, baicalein reduced lactate content in the lung tissues of LPS-induced ALI mice and in LPS-induced macrophages (Fig. 4E and G).

HIF-1α is considered a significant activator of glycolysis and inflammatory responses. In the present study, the expression levels of HIF-1α were detected in the lung tissues of mice with ALI and in LPS-induced macrophages. Notably, baicalein could significantly inhibit the levels of HIF-1α in the lungs of LPS-induced ALI mice (Fig. 5A-C) and in LPS-induced macrophages (Fig. 5D). Furthermore, molecular docking experiments predicted that baicalein could interact with HIF-1α through the ASP-222 and ALA-312 residues (Fig. 5E).