The RAW264.7 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. DMEM and FBS were from Gibco (Thermo Fisher Scientific, Inc.). Lipopolysaccharide (LPS; cat. no. L2630) was obtained from Sigma-Aldrich (Merck-KGaA). The Cell Counting Kit (CCK)-8 assay kit (cat. no. C0037) was purchased from Beyotime Institute of Biotechnology. The RNA Purification kit (cat. no. B0004DP), RNA reverse transcription (RT) kit (cat. no. EZB-RT2GQ) and the fluorescence quantitative PCR (qPCR) kit (cat. no. A0001-R2) were from EZBioscience. The TNF-α (cat. no. EM3311S), IL-6 (cat. no. EM3201S) and IL-1β (cat. no. EM3184S) ELISA duo set kits were purchased from Biotechwell. Antibodies against phosphorylated (p-)Akt (cat. no. T55561) and Akt (cat. no. T40067) were obtained from Abmart. Antibodies against TNF-α (cat. no. 11948), IL6 (cat. no. 12912), β-tubulin (cat. no. 2148), GAPDH (cat. no. 5174) and rabbit horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074) were purchased from Cell Signaling Technology, Inc. The antibody against IL-1β (cat. no. 26048-1-AP) was obtained from ProteinTech Group, Inc. The Akt inhibitor (cat. no. HY-108232) was purchased from MedChemExpress.

The RAW264.7 cell line was maintained in DMEM containing 10% FBS and 100 U/ml streptomycin/penicillin at 37˚C in a humidified incubator with 5% CO₂. Cells in the logarithmic phase were used for the experiments.

Cell viability was analyzed using the CCK-8 assay according to the manufacturer's protocol. In brief, the cells were seeded into 96-well plates (1x10⁴ cells/ml) for 12 h and subsequently, quercetin was added to the cells at various concentrations (0, 5, 10, 20, 40 and 80 µM), followed by incubation at 37˚C for 24 h. Subsequently, 10 µl CCK-8 solution was added into each well of the plate and cells were incubated for 3 h at 37˚C. The optical density was measured at 450 nm using a microplate reader (BioTek Instruments, Inc.).

The RAW264.7 cell line was treated with various concentrations of quercetin (5, 10 and 20 µM) at 37˚C for 1 h in 6-well microplates and stimulated with LPS (1 µg/ml) at 37˚C for 24 h. The total RNA was extracted according to the manufacturer's specification (EZBioscience). The concentration of RNA was evaluated using spectrophotometric analysis. Total RNA (1 µg) was reverse transcribed into cDNA using 4X EZscript Reverse Transcription Mix Ⅱ. RT was performed at 42˚C for 15 min. The fluorescence quantitative PCR was performed using a 2X SYBR-Green qPCR Master Mix (ROX2 plus). Relative gene expression levels were obtained following normalization to β-actin. The following thermocycling conditions were used: Initial denaturation at 95˚C for 5 min; followed by 40 cycles of 95˚C for 10 sec and 60˚C for 30 sec and the reaction was performed in a 7500 Real Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2^-ΔΔCq method was used to quantify the results (11). The primer sequences used for qPCR were purchased from Sangon Biotech Co., Ltd. and the sequences of the primers are presented in Table I.

Raw264.7 cells (3x10⁵ cells/well) were cultured in 6-well microplates at 37˚C for 12 h. Thereafter, the RAW264.7 cell line was treated with various concentrations of quercetin (5, 10 and 20 µM) at 37˚C for 1 h and stimulated with LPS (1 µg/ml) at 37˚C for 24 h. The cell culture supernatant was then obtained to measure the levels of TNF-α, IL-6 and IL-1β using the ELISA kits according to the manufacturer's protocol.

Raw264.7 cells (3x10⁵ cells/well) were cultured in 6-well microplates at 37˚C for 12 h. Thereafter, the RAW264.7 cell line was treated with various concentrations of quercetin (5, 10 and 20 µM) at 37˚C for 1 h and stimulated with LPS (1 µg/ml) at 37˚C for 6 h. Ice-cold PBS was used to wash the cells three times and lysis solution (cat. no. P0013B; Beyotime Institute of Biotechnology) was used to dissociate the cells on ice for 30 min. The protein concentration was determined using the bicinchoninic acid method. Total protein (20 µg) was separated using 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (cat. no. IPVH00010 PORE; Merck Millipore) for 1 h at 350 mA. After blocking with 5% bovine serum albumin (BSA; cat. no. 4240GR100; BioFroxx) for 1 h, the protein was incubated with the primary antibodies [p-Akt (1:1,000 dilution), Akt (1:1,000 dilution), TNF-α (1:500), IL6 (1:500), IL-1β (1:500), β-tubulin (1:1,000 dilution) or GAPDH (1:1,000 dilution)] overnight at 4˚C. The membranes were washed four times with Tris-buffered saline-Tween-20 (TBST) solution for 10 min and then incubated with rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution) in the presence of BSA for 1 h at room temperature. Subsequently, the membranes were washed four times with TBST for 10 min. ImageJ software 1.46 (National Institutes of Health) was used for grey value analysis.

The targets of quercetin were obtained from the Traditional Chinese Medicine database system pharmacology (TCMSP) and analysis platform (http://tcmspw.com/tcmsp.php) using the key word ‘quercetin’. The ‘Canonical SMILES’ of quercetin was searched using the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) with the key word of ‘quercetin’ and then imported into Swiss Target Prediction (http://www.swisstargetprediction.ch/index.php) to obtain the targets of quercetin. The targets screened from the two databases were combined and the overlapped targets were removed. Subsequently, the targets associated with inflammation were obtained from the GeneCards database (https://www.genecards.org/), with ‘inflammation’ as the key word. The targets with a relevance score greater than the median digit were screened.

The targets of quercetin were mapped to those of inflammation. The overlapped targets were imported into the Search Tool for the Retrieval of Interacting Genes/proteins database (https://string-db.org/) to construct the PPI network. The host species was limited to ‘Homo sapiens’ and the confidence score for the correlation degree was set to ≥0.4. Subsequently, Cytoscape v3.7.2 was used for topology analysis.

Gene set enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID; https://david.ncifcrf.gov/). Gene Ontology (GO) and Kyoto Encyclopedia Genes and Genomes enrichment (KEGG) analyses were performed using the DAVID v6.7 database. The species was set as ‘Homo sapiens’ and the significance threshold was set as P<0.05.

The molecular docking analysis was performed using the TCM Network Pharmacology Analysis System (TCMNPAS; no. 2019SR1127090; http://54.223.75.62:3838/npa4/). The PSOVina algorithm was used for molecular docking. First, the ‘Canonical SMILES’ of quercetin was searched using the PubChem database and the ‘protein database (PDB) ID’ of Akt (3O96) was obtained from the PDB database (https://www.rcsb.org/). Subsequently, the ‘Canonical SMILES’ of quercetin and ‘PDB ID’ of Akt were added to the molecular docking module of TCMNPAS. ‘From ligand’ is an online method to obtain protein docking pocket parameters based on the ligand position. The ‘From ligand’ button was then selected. Finally, the conformation and docking results of the compounds were downloaded and imported into Pymol3.8 (https://pymol.org) to visualize their three-dimensional structure.

The RAW264.7 cell line was treated with various concentrations of quercetin (5, 10 or 20 µM) at 37˚C for 1 h in 6-well microplates and stimulated with LPS (1 µg/ml) at 37˚C for 6 h. Other experimental steps were mentioned previously in the western blot analysis subsection.

The RAW264.7 cell line was preincubated with quercetin (20 µM) and the AKT inhibitor (MK-2206; 5 µM) at 37˚C for 1 h in 6-well microplates and stimulated with LPS (1 µg/ml) at 37˚C for 6 or 24 h. Other experimental steps were mentioned previously in the RT-qPCR and western blot analysis subsections.

All the experiments were performed at least 3 times. Data in this study were statistically processed by SPSS Statistics 22.0 (IBM Corporation). Values are expressed as the mean ± standard deviation. Significant differences were analyzed using one-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The results of the CCK-8 assay indicated that treatment with quercetin (5-20 µM) for 24 h exerted no notable effects on cell viability (Fig. 1). Therefore, 5-20 µM quercetin was used for the subsequent experiments.

As presented in Fig. 2A, after treatment with LPS, the mRNA expression levels of TNF-α, IL-6 and IL-1β were significantly increased compared with those in the blank group. In comparison, pre-treatment with quercetin decreased the mRNA expression levels of TNF-α, IL-6 and IL-1β in the RAW264.7 cell line in a dose-dependent manner.

The results of the ELISA suggested that after stimulation with LPS, the concentrations of TNF-α, IL-6 and IL-1β were significantly increased and this increase was attenuated in the groups pre-treated with quercetin (Fig. 2B).

Western blot analysis also indicated that after stimulation with LPS, the protein expression levels of TNF-α, IL-6 and IL-1β were significantly increased. However, in comparison, pre-treatment with quercetin decreased the protein expression levels of TNF-α, IL-6 and IL-1β in the RAW264.7 cell line (Fig. 2C and D).

A total of 226 potential targets of quercetin were obtained using the TCMSP and Swiss Target Prediction databases. A total of 10,454 differentially expressed genes were obtained from the GeneCards database and 5,210 targets with a Relevance Score >0.947288 were screened. Finally, 188 targets were selected for enrichment analyses from an overlap in the Venn diagram (Fig. 3).

Fig. 4A presents the PPI network of the potential targets of quercetin treatment to counteract inflammation. The PPI network was constructed and visualized using Cystoscape. The nodes and edges are presented in Fig. 4B. All the Akt targets are displayed in the figure. The average node degree value in the PPI network was 34.556. The top 10 nodes with the largest degree of connectivity values were AKT1, TP53, IL6, VEGFA, IL1B, CASP3, JUN, MYC, EGFR and PTGS2.

A total of 188 selected key targets were submitted for GO and KEGG enrichment analyses and screened with a cutoff of P<0.05. A total of 651 targets were identified in the category biological process (BP), 65 in cellular component (CC) and 144 in the category molecular function (MF), while there were 122 enriched KEGG pathways. The top 10 enriched GO and KEGG pathway terms are presented in Fig. 5. The BP targets included positive regulation of transcription from RNA polymerase II promoter, signal transduction and negative regulation of apoptotic process. The CC targets included nucleus, cytoplasm and cytosol. The MF targets included protein binding, ATP blinding and identical protein binding. The KEGG pathways included cancer, PI3K/AKT signaling pathway and the Hepatitis B signaling pathway.

Molecular docking analysis was performed to estimate the binding between quercetin and Akt1. The results indicated that the binding force of quercetin with Akt1 was-8.9 kJ/mol, indicating that they had a good affinity. The three-dimensional structure of the molecular docking diagram is provided in Fig. 6.

As presented in Fig. 7A and B, quercetin dose-dependently inhibited the phosphorylation of Akt, suggesting that quercetin counteracted inflammation by repressing Akt phosphorylation.

As indicated in Fig. 7C-E, MK-2206 and quercetin decreased the expression levels of TNF-α, IL-6 and IL-1β, and quercetin had a stronger efficacy than MK-2206.

As presented in Fig. 7F and G, MK-2206 and quercetin inhibited the phosphorylation of Akt and quercetin had a stronger efficacy than MK-2206.