Introduction

Cytokine storm (CS) is a systemic inflammatory syndrome with excessive hyperactivation of immune cells characterized by increased cytokine release, including interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and monocyte chemotactic protein 1 (MCP-1), which causes severe pathologic complications, such as sepsis, tissue damage, multiple organ failure, and ultimately, death1. CS might be stimulated by multiple factors such as pathogens, auto-inflammation, monogenic, or therapeutic intervention and the lungs are the main organ to be affected by CS2.

Macrophages play a pivotal role in infection and inflammation as the principal innate immune cells, exerting crucial regulatory functions in pathological inflammation. Within the tissue microenvironment, they exhibit polarization into either the classically activated M1 phenotype, characterized by pro-inflammatory properties, or the alternatively activated M2 phenotype, which demonstrates anti-inflammatory characteristics. Dysregulation in macrophage phenotypes can result in unchecked inflammatory responses, thereby precipitating CS and subsequent tissue damage3. Considering the pivotal role of macrophages in CS progression, modulating macrophage overactivation emerges as a promising strategy for CS intervention.

Quercetin, a flavonoid compound, possesses a spectrum of biological properties, including antioxidant, anti-inflammatory, antiviral, and neuroprotective effects4,5,6,7. Research indicated that quercetin exerted its anti-inflammatory effects by targeting Syk/Src/IRAK-1 to inhibit LPS-induced macrophage activation, while also preventing LPS-induced oxidative stress and inflammation through pathways NOX2/ROS/NF-κB8,9. However, the specific targets and signaling pathways through which quercetin regulates CS remain elusive. Therefore, we embarked on an exploration of new targets and potential mechanisms of quercetin for CS treatment, employing network pharmacology, molecular docking, and experimental validation techniques.

The Forkhead box O (FoxO) family of transcription factors assumes pivotal roles in diverse cellular processes encompassing cell growth, metabolism, survival, and inflammation10,11,12. Nonetheless, FoxO1’s nuclear export or phosphorylation culminates in its inactivation, abrogating its capacity to engage with target regulatory elements13. Notably, studies have underscored that elevated FoxO1 expression post-inflammatory injury prompts macrophages to unleash an array of inflammatory mediators, thereby exacerbating inflammatory damage14,15. In LPS-treated mice, macrophages exhibited heightened FoxO1 levels; transfection of FoxO1 into Raw264.7 cells markedly upregulated interleukin-1β (IL-1β) and concurrently downregulated interleukin-10 (IL-10) expression16. Furthermore, FoxO1 serves as a direct substrate of AKT, and its activity hinges on AKT phosphorylation. Notably, AKT inhibition in macrophages abolishes FoxO1 phosphorylation and nuclear exclusion, signifying AKT phosphorylation as a pivotal regulatory event governing FoxO1 activity17. Conversely, the Keap1-Nrf2 pathway constitutes a principal defense mechanism safeguarding cells and tissues against oxidative stress while upholding homeostasis. Kelch-like ECH-associated protein 1 (Keap1) serves as an electrophilic reagent and sensor of redox damage, whereas Nuclear factor erythroid 2-related factor (Nrf2) acts as a transcription factor modulating various cytoprotective genes. Oxidative stress prompts Keap1 modification, resulting in its inactivation and disassociation from Nrf2. Consequently, stabilized Nrf2 translocates to the nucleus, where it acts as a transcription factor, activating oxidative stress-responsive genes, thereby exerting antioxidant effects18.

In this study, we found that quercetin could effectively inhibit inflammatory responses and oxidative stress in vitro and exhibited anti-inflammatory activity in mice model. Likewise, the AKT1-FoxO1 and Keap1-Nrf2 signaling pathways may be involved in quercetin-mediated anti-inflammatory and antioxidant activities.

Materials and methods

Screening for target genes of quercetin and CS

The two-dimensional (2D) molecular structure and SMILES of quercetin were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/), the world's largest database of chemical information. To predict potential quercetin targets, 2D structures or SMILES were imported into the Swiss Target Prediction Database (http://swisstargetprediction.ch/). The target genes associated with CS were acquired from the OMIM (https://www.omim.org), GeneCards (https://www.genecards.org), and PharmGKB (https://www.pharmgkb.org) databases by using the following keywords; “cytokine storm” and “cytokine release syndrome.” Subsequently, to analyze and screen common target genes of CS and quercetin, the Venny.2.1.0 e-mapping tool (https://bioinfogp.cnb.csic.es/tools/venny/) was used, and then a Venn diagram was drawn.

To acquire a protein–protein interaction network (PPI), the overlapping genes were submitted to the STRING database (https://cn.string-db.org/); the species restriction was “Homo sapiens,” and the confidence level was > 4.0 for exploring their relationship.

The acquired data were imported to Cytoscape 3.9.1 software for visualization. The core target genes were screened via CytoNCA plug-in using the closeness centrality, betweenness centrality, degree centrality, eigenvector centrality, network centrality and local average connectivity.

GO function and KEGG pathway enrichment analyses

The CS and quercetin target genes intersection were converted to the corresponding gene IDs for gene ontology (GO) functional and Kyoto Protocol Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in DAVID database (https://david.ncifcrf.gov/). GO comprises biological processes (BP), molecular function (MF), and cellular components (CC). KEGG enrichment analysis can forecast some potential signaling pathways involved in biological processes19,20,21. Subsequently, for analyzing the GO and KEGG data, the Bioinformatics (http://www.bioinformatics.com.cn/) platform was employed.

Molecular docking

Molecular docking can predict the binding potential between drugs and targets. Quercetin and the eight core targets were subjected to molecular docking via SYBYL-X2.1.1 software. First, the crystal structures of the protease were retrieved in the RCSB Protein Data Bank (PDB, http://www.rcsb.org/) database as the receptors; then the ligand was docked with the receptor by first extraction the ligand, removing the water molecules, modifying the terminal residues, and hydrogenating to generate the active pocket. Lastly, the total score was used to record the strength of the interaction between the small molecule and the target.

Cell viability assay

Murine macrophage cell line Raw264.7 (SC-6005, ATCC) was cultured in 96-well plates (1 × 104 cells/well) in DMEM medium with 10% fetal bovine serum and 1% antibiotics (100 unit/ml penicillin and 100 μg/ml streptomycin) at 37 °C and 5% CO2 overnight. The next day, cells were treated with different concentrations (2.5, 5, 10, 20, and 40 μg/ml) of quercetin for 24 h. Subsequently, cell viability was tested by CCK-8 kit assay (MA0218, meilunbio), per the kits’ instructions.

Enzyme-linked immunosorbent assay (ELISA)

Raw264.7 cells were seeded in 48-well plates at a density of 5 × 104 cells overnight; then, in the Control and LPS groups, the media was replaced with 500 μl fresh complete medium, whereas in the drug groups, 500 μl medium containing the corresponding drug concentrations was added. The dexamethasone (Dex) group represented a positive control. Furthermore, LPS was added to each group except the Control to achieve a final concentration of 1 μg/ml. After 24 h of co-culture, the cell supernatant was collected, and several inflammatory mediators, including IL-6, TNF-α, IL-1β, and MCP-1, were measured using ELISA (Dakowei Biotechnology Ltd.), per the manufacturers’ instructions.

Flow cytometry analysis

Raw264.7 cells were propagated and treated as mentioned above, collected after 24 h, and co-stained after probing with CD11b (101207, BioLegend), CD40 (124612, BioLegend), CD80 (104733, BioLegend) antibodies for 20 min at room temperature, while blank and positive controls (single stained tubes for each antibody) were prepared. The cells were then washed with PBS, resuspended in 500 μl PBS, gated, and then analyzed using a flow cytometer.

Detection of nitric oxide (NO)

Raw264.7 was propagated overnight at the density 1 × 105 in 24-well plates before receiving the corresponding treatment based on the experimental groups. After 24 and 48 h of co-incubation with the drug and LPS, the cell supernatant was obtained, and the expression level of NO was detected by the Griess method using NO kit (S0021S, Beyotime).

Detection of intracellular reactive oxygen species (ROS)

Raw264.7 (1 × 106/well) were inoculated in 6-well plates and co-culture with LPS and drug for 24 and 48 h before collecting their supernatants. ROS kit (CA1420, Solarbio) detected intracellular ROS levels. The probe DCFH-DA was added to the cell precipitates and incubated at 37 °C for 20 min. DCFH-DA is a non-fluorescent substance, and the kit uses the principle that the probe can enter cells, where it will be subsequently hydrolyzed into DCFH by esterase, and the intracellular ROS will then oxidize non-fluorescent DCFH to produce fluorescent DCF. Flow cytometry and fluorescence microscopy measured intracellular ROS's fluorescence intensity.

Immunofluorescence assay

Raw264.7 cells were propagated and treated as mentioned above, Raw264.7 cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min, and after being closed with 5% BSA for 2 h at room temperature, the cells were incubated with anti-FoxO1 antibody (1:100) at 4 °C overnight. After incubation with Alexa Fluor 488-labeled secondary antibody (1:800 dilution,HA1121, HUABIO), the cellular localization of the cells to FoxO1 was assessed using a confocal microscope (Leica, German).

RNA extraction and quantitative Real-time PCR (qRT-PCR)

The total RNA of treated Raw264.7 cells was extracted using a total RNA extraction kit (RE-03111, FOREGENE). The cDNA was synthesized using the RT Easy™II (With gDNase) kit (RT-01032, FOREGENE), which was then amplified via a Real-Time PCR Easy™-SYBR Green kit (QP-01014, FOREGENE). The relative expression levels of mRNA were calculated by the 2−△△ct method.

Western blot

Cellular proteins were extracted using the RIPA lysis buffer (E-BC-R327, Elabscience), a protease inhibitor (GRF101, epizyme), and a phosphatase inhibitor (GRF102, epizyme). The proteins were quantified using the BCA protein assay kit (P0010, Beyotime), then mixed with sample loading buffer (P0295, Beyotime) and boiled for 10 min. Proteins were separated on 10% SDS–polyacrylamide gels (PG112, epizyme), transferred to PVDF membranes (IPVH00010, Millipore), which were then blocked with 5% skimmed milk at room temperature, and incubated overnight with primary antibodies at 4 °C. The primary antibodies were as follows: anti-TLR2 (ab209216, Abcam), anti-TLR4 (14358, CST), anti-MyD88 (4283, CST), anti-AKT1 (ET1609-51, HUABIO), anti-phospho-AKT1 (ET1607-73, HUABIO), anti-FoxO1 (ET1608-25, HUABIO), anti-Keap1 (10503-2-AP, Proteintech), anti-Nrf2 (16396-1-AP, Proteintech). Subsequently, the membranes were washed and then incubated with a secondary antibody (RS0002, Immunoway) for 1 h. The proteins were visualized by a supersensitive ECL kit (PD203, Oriscience) and a chemiluminescent imaging system.

Anti-CS in vivo

All procedures were conducted following ARRIVE guidelines. The Ethics Committee of West China Hospital has approved this study and confirmed the statement that all methods were performed in accordance with relevant guidelines and regulations. Male C57BL/6J mice (8 weeks old, 23 ± 2 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were first acclimatized with the environment for a week and then categorized into six groups: control, LPS, high quercetin dose (100 mg/kg), medium quercetin dose (50 mg/kg), low quercetin dose (25 mg/kg) and Dex (5 mg/kg). The mice were in 12 h fast condition before the experiment; then, four drug groups were treated with quercetin and Dex at corresponding concentrations by gavage and intraperitoneal injection, respectively. The Control and LPS groups received the same volume of normal saline. After 2 h, mice were anesthetized with an intraperitoneal injection of 0.3% pentobarbital (55 mg/kg), then 50 μl of 5 mg/kg LPS was intratracheally administered in each group (except the Control) to establish the CS model. 4 h after LPS treatment, the mice were killed, the skin on the front of the neck was cut open, the trachea was separated and exposed, the indentation needle was inserted into the trachea and fixed, and the irrigation solution was irrigated with normal saline three times, 0.6 ml each time, with a recovery rate of 80–90%. The BALF was collected for the detection of cytokines.

Histological analysis

To observe the lung's histopathological alterations, mice were sacrificed 24 h after LPS stimulation; lung tissues were dissected, fixed with 4% formaldehyde, embedded in paraffin, sectionalized, and finally stained with hematoxylin–eosin (HE) for visual analysis.

Statistical analysis

All the statistical measurements were performed using GraphPad Prism 9.0, and the acquired data are expressed as means ± standard deviation (SD). Differences between the two groups were assessed using One-way ANOVA analysis and were considered significant at P < 0.05.

Results

Acquisition of quercetin targets against CS

The 2D molecular structure (Fig. 1A) and SMILES [C1=CC(=C(C=C1C2=C(C(=O) C3=C(C=C(C=C3O2) O) O) O) O) O)] of quercetin were downloaded from PubChem. The PubChem CID of quercetin is 5280343, and the molecular formula and weight are C15H10O7 and 302.23. Subsequently, this structure was uploaded to the Swiss Targets Prediction Database, and 100 genes were identified as quercetin targets. Furthermore, 8390 CS target genes were obtained from three disease databases, including GeneCards, OMIM, and PharmGKB, after screening for disease and removing the duplication. Venn’s diagram (Fig. 1B) indicated the potential 90 CS target genes selected after matching drugs to target genes.

Fig. 1
figure 1

Acquisition of target genes for quercetin action on CS. (A) 2D molecule structure of quercetin. (B) Venn diagram of quercetin and CS target genes. (C) PPI network analysis in the common target of quercetin and CS. (D) Screening of quercetin and CS core genes by CytoNCA Plug-in.

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The PPI network (Fig. 1C), constructed using STRING, consisted of 90 nodes and 375 edges, with PPI enrichment p-value < 1.0e−16. The nodes represent target proteins, and the edges represent predicted and confirmed interactions between proteins. This network was visualized with Cytoscape 9.0 to identify core targets, which were then screened using the CytoNCA plug-in. Based on six parameters-betweenness centrality, closeness centrality, degree centrality, rigenvector centrality, network centrality, and local average connectivity, 8 core target genes, including AKT1, EGFR, SRC, MMP9, KDR, PIK3R1, CDK1, and MMP2 were filtered. These were significant genes associated with the quercetin mechanism, which regulates the occurrence and development of CS (Fig. 1D).

Potential mechanism and signal pathways of quercetin in regulating CS

GO and KEGG enrichment analyses were performed through the DAVID database to further explore the BP and potential mechanisms of 90 target genes involved in CS. The result of the GO enrichment analysis (Fig. 2A) displayed 262 BP, 54 CC, and 112 MF. The major BP included protein phosphorylation, negative regulation of the apoptotic process, and protein autophosphorylation. The target genes were mainly associated with the following determined CC: cytosol, plasma membrane, cytoplasm, nucleus, etc. Moreover, MF included ATP binding, protein serine/threonine/tyrosine kinase activity, protein kinase activity, protein serine/threonine kinase activity, etc.

Fig. 2
figure 2

GO and KEGG enrichment analysis. (A, B) Analysis of GO enrichment and KEGG potential signaling pathway enrichment for targets of action. (C) The PI3K-AKT signaling pathway was identified as the key KEGG pathway for quercetin action on CS. The above KEGG data were obtained from the KEGG database.

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The KEGG enrichment assay indicated the possible signaling pathways via which quercetin improves CS, revealing the therapeutic mechanisms of CS (Fig. 2B). It identified the CS-related key signal pathways involved in the PI3K-AKT, FoxO and ErbB. The specific signal pathways of PI3K-AKT are listed in Fig. 2C.

Molecular docking results

The interaction of eight core targets-AKT1, KDR, CDK1, EFGR, MMP2, MMP9, SRC, and PIK3R1 with quercetin in the generated active pocket regions was assessed. The higher the total score, the more stable the binding activity. The binding activity is extremely high when the total score is > 7. The docking results revealed that among the 8 core targets, the interaction between the quercetin and AKT1 was the strongest, with a total score of 8.35 (Fig. 3A–E).

Fig. 3
figure 3

Molecular docking of quercetin with target genes. (A–D) The molecular docking of quercetin with AK1, KDR, CDK1, EFGR, respectively. (E) The total score for molecular docking of quercetin to the four core targets showed the highest total score for binding to AKT1.

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Quercetin attenuated LPS-induced expression of proinflammatory factors in Raw264.7 cells

The effects of quercetin and drug solvent dimethyl sulphoxide (DMSO) on cell viability were assessed by CCK8 assay, which indicated that 2.5, 5, and 10 μg/ml concentrations do not affect cell survival (Fig. 4A), therefore, these concentrations were selected as low, medium and high doses for subsequent experiments. LPS is an essential component of the Gram-negative bacterial cell wall and induces inflammation, allowing its application in various in vivo and in vitro experiments22. Under inflammatory conditions, LPS exposure activates macrophages to produce diverse cytokines and also promotes oxidative stress23,24. Consequently, a CS model was established utilizing 1 μg/ml LPS(Fig. 4B). As shown in Fig. 4C, quercetin significantly inhibited LPS-induced proinflammatory cytokines (IL-6, TNF-α, IL-1β) and MCP-1 in a dose-dependent manner.

Fig. 4
figure 4

Quercetin inhibited LPS-induced inflammatory factors in vitro. (A) Survival of Raw264.7 cells after 24 h intervention with different concentrations of quercetin. (B) A scheme for quercetin intervention in LPS-induced macrophage activation. (C) Quercetin reduced the concentration of IL-6, TNF-α, IL-1β and MCP-1 released by LPS-activated macrophages in a concentration-dependent manner. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the LPS group.

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Upon exposure to LPS, activated M1 macrophages secrete a plethora of cytokines25. In addition, the surface markers CD40 and CD80, indicative of M1 macrophage activation, undergo alterations (Fig. 5A). To scrutinize these changes, we conducted flow cytometry analysis. As illustrated in Fig. 5B, C, the proportion of CD40+ CD80+ cells constituted approximately 70% in the LPS-untreated group, which exhibited a reduction following quercetin treatment. This observation finds validation in quantitative polymerase chain reaction (qPCR) results (Fig. 5D), wherein mRNA levels of CD40 and CD80 were augmented upon LPS stimulation, a response mitigated by quercetin intervention in a dose-dependent manner. These findings suggested that quercetin could hold promise in ameliorating LPS-induced macrophage polarization.

Fig. 5
figure 5

The ability of quercetin to modulate macrophage polarization in vitro. (A) Schematic representation of LPS-stimulated macrophage polarization. (B) Flow cytometry results indicated that quercetin treatment decreased the expression of CD40, CD80, surface markers of M1 phenotype macrophages. (C) Statistical analysis of flow cytometry results in (B). (D) Quercetin treatment also decreased mRNA expression of CD40 and CD80. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the LPS group.

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Quercetin inhibited LPS-induced NO production in Raw264.7 cells

Nitric oxide (NO) has been implicated in various cellular responses to external stimuli such as ischemia and LPS stimulation26. Our NO detection assays unveiled an absence of NO in the resting state at 24 and 48 h, but its significant induction following LPS exposure. Notably, quercetin exhibited a dose-dependent reduction in NO release in the cell supernatant, with the most pronounced effect observed at 10 μg/ml (Fig. 6A). Inducible nitric oxide synthase (iNOS) is exclusively present under inflammatory conditions and is responsible for sustained NO production. Therefore, we delved deeper into the expression of NOS2 mRNA (encoding iNOS protein) and iNOS protein. Both quantitative polymerase chain reaction (qPCR) and western blot analyses demonstrated that quercetin downregulated the expression of both in a dose-dependent manner compared to the LPS group at 24 and 48 h (Fig. 6B–F, Supplementary Fig. 2A, B).

Fig. 6
figure 6

Effects of quercetin on LPS-induced NOS2 mRNA, iNOS protein expression and NO release in Raw264.7 cells. (A) At both 24 and 48 h, quercetin reduced LPS-induced NO release in a concentration-dependent manner. (B) NOS2 mRNA levels also decreased with increasing drug concentrations after 24 and 48 h of quercetin treatment. (C, D) After 24 and 48 h of LPS attack, iNOS expression at the protein level was down-regulated in a concentration-dependent manner in response to quercetin intervention. (E, F) The iNOS expression levels at 24 and 48 h were normalized to GAPDH. Original blots are presented in Supplementary Fig. 2A, B. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the LPS group.

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Quercetin regulated the AKT1-FoxO1 signaling pathway

The molecular docking results revealed a high binding score of quercetin to AKT1, suggesting its potential to regulate AKT1 activity. In comparison to the LPS-treated group, quercetin administration led to an increase in AKT1 phosphorylation without altering its total protein levels (Fig. 7A, B, Supplementary Fig. 2C). Following LPS stimulation, the expression of FoxO1, a transcription factor, and its nuclear translocation are augmented. However, FoxO1 is subject to negative regulation by AKT, as AKT phosphorylation prompts its exclusion from the nucleus, thereby mitigating inflammation16,27. Immunofluorescence experiments depicted an intensified nuclear fluorescence upon LPS treatment, which significantly decreased following quercetin intervention (Fig. 7C, Supplementary Fig. 1A). Correspondingly, western blot analysis illustrated that LPS augmented total FoxO1 expression, a trend reversed by quercetin treatment (Fig. 7D, E, Supplementary Fig. 2D). Additionally, quercetin exhibited a dose-dependent downregulation of Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), and MyD88 expression following LPS stimulation (Supplementary Fig. 1B–G, Supplementary Fig. 2G–I). In summary, our findings suggested that quercetin could induce AKT1 activation, leading to subsequent FoxO1 inactivation.

Fig. 7
figure 7

Effects of quercetin on AKT1 and FoxO1 expression levels in response to LPS stimulation. (A) The level of phosphorylated AKT1 gradually up-regulated after quercetin intervention. (B) p-AKT1 levels were normalized to total AKT1 levels. (C) Immunofluorescence assessment of FoxO1 intranuclear expression levels in Raw264.7 cells after LPS and quercetin treatment. The FoxO1 was stained as green granular dots, while the nucleus was stained with blue. The highest expression of FoxO1 in the nucleus was observed in the LPS group, and the fluorescence of FoxO1 in the nucleus gradually decreased with the increase of quercetin concentration. (D) Western blotting analysis similarly confirmed that quercetin down-regulated FoxO1 expression in Raw264.7 cells. (E) FoxO1 levels were normalized to GAPDH. Original blots are presented in Supplementary Fig. 2C, D. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the LPS group.

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Quercetin activated Keap1-Nrf2 signaling pathway to mediate antioxidant response

Oxidative stress, emblematic of the imbalance between reactive oxygen species (ROS) and antioxidant defenses, can potentiate inflammatory responses and exacerbate tissue damage28. To confirm whether the quercetin-induced antioxidative effect involves ROS inhibition, intracellular ROS levels were assessed through flow cytometry analysis. Results revealed a gradual increase in cellular ROS levels over time in the untreated LPS cells compared to the control group, reaching approximately 70% and 90% at 24 and 48 h, respectively. Moreover, quercetin demonstrated an augmented ability to scavenge ROS with increasing time and drug dosage (Fig. 8A, B). Green fluorescence, indicative of intracellular ROS content, exhibited a progressive decline with escalating drug concentrations (Fig. 8C, D). Both flow cytometry and fluorescence microscopy analyses indicated that quercetin alleviated the high ROS levels induced by LPS. Subsequently, we investigated the expression of key factors in the Keap1-Nrf2 axis. Our results showed low or negligible expression of Nrf2 in the control group, with Nrf2 accumulation increasing with higher quercetin doses following LPS stimulation and quercetin intervention. Conversely, Keap1 expression exhibited an inverse trend compared to Nrf2 (Fig. 8E–G, Supplementary Fig. 2E, F). Hence, our findings suggested that quercetin could mitigate oxidative stress by activating the Keap1-Nrf2 signaling pathway.

Fig. 8
figure 8

Effects of quercetin on intracellular ROS levels under LPS induction at 24 and 48 h. (A, B) Flow cytometry results showed that quercetin enhanced the scavenging of intracellular ROS with increasing concentration and time under LPS induction. (C, D) The intracellular ROS content was observed by fluorescence microscopy, and green fluorescence represents ROS. Magnification is 20×. (E) Western blotting analysis revealed that Keap1 protein expression was down-regulated and Nrf2 protein expression was up-regulated in Raw264.7 cells treated with LPS and quercetin. (F, G) The expression levels of Keap1 and Nrf2 were normalized to their respective GAPDH at 24 and 48 h. Original blots are presented in Supplementary Fig. 2E, F. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the LPS group.

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Quercetin prevented LPS-induced CS in mice

To assess the in vivo anti-inflammatory activity of quercetin, four inflammatory factors in BALF, including IL-6, TNF-α, IL-1β, and MCP-1, were measured by ELISA, which indicated their rapid upregulation after LPS stimulation and quercetin reversed this effect in a concentration-dependent manner under preconditioning (Fig. 9A, B).

Fig. 9
figure 9

Quercetin inhibited CS in vivo studies. (A) Scheme for in vivo induction and intervention of CS. (B) Quercetin suppressed the LPS-induced elevation of IL-6, TNF-α, IL-1β and MCP-1 in BALF. (C) Histological study of the protective effect of quercetin against LPS-induced CS lung injury. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the LPS group.

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As H&E-staining indicates (Fig. 9C), the control group indicated normal lung structure and clear alveolar morphology, while LPS exposure distinctly caused lung tissue congestion, edema, and extensive inflammatory cell infiltration, destroying the lung structure and preventing normal lung function. Pretreated quercetin mice had improved histopathologic changes induced by LPS in a concentration-dependent manner. The examination of pathological changes in lung tissue and BALF inflammatory factors demonstrated that quercetin could effectively protect mice from LPS attacks.

Discussion

CS is a systemic immune overreaction. Under normal circumstances, pro-inflammatory and anti-inflammatory factors are in a state of mutual balance, which is disrupted when pathogens invade or medical intervention, resulting in the excessive emission of the cytokines and inducing CS. If not treated properly, it can lead to systemic damage, multi-organ failure, or even death29. In CS caused by immune-related pneumonitis and viral infection, activated macrophages produce excessive amounts of IL-6, TNF-α, and IL-1β accompanied by elevated chemokines. IL-6 is a crucial target for CS treatment and is a risk factor for assessing the severity of COVID-19 as it is associated with a high mortality rate30,31,32,33,34. Corticosteroids and cytokine monoclonal antibodies are essential for CS treatments. However, the optimal dose and duration of corticosteroids in immune-related pneumonia remains undetermined and could exacerbate the risk of opportunistic infections35,36,37. In COVID-19, despite the benefits of corticosteroids, there is some variation in different patients38. Furthermore, it has been studied that they are associated with high mortality, hyperglycemia, and infection39. Monoclonal cytokines antibodies, such as IL-6R monoclonal antibodies, TNF inhibitors, and IL-1 antagonists, are specific for specific cytokines, and unfortunately, CS comprises multiple cytokines. Some clinical trials have shown that monoclonal antibodies are only effective in some people40,41.

LPS used to model pneumonia is a classical approach that activates macrophages and monocytes to produce high levels of inflammatory cytokines (IL-6, TNF-α, IL-1β, etc.) while eliciting oxidative stress, with literature suggesting that it can also mimic the CS that occurs in the lungs42,43,44,45,46. Pneumonia frequently ensues as a consequence of CS and often stems from viral infections. In our investigation, we successfully established in vitro and in vivo CS models utilizing LPS, with in vivo modeling achieved through tracheal infusion of LPS. The groups treated solely with LPS exhibited elevated levels of inflammatory factors (IL-6, TNF-α, IL-1β, MCP-1) alongside inflammatory cell infiltration in lung tissue. Our findings highlighted quercetin's capacity to modulate the inflammatory response induced by LPS-activated macrophages, effectively suppressing the release of inflammatory factors and thereby exerting an anti-inflammatory effect.

The findings from network pharmacology unveiled quercetin's capacity to modulate CS primarily by targeting AKT1, EGFR, SRC, MMP9, KDR, PIK3R1, CDK1, and MMP2, with AKT1 being the most significantly regulated. Moreover, KEGG enrichment analysis indicated a potential association between quercetin's mechanism of action against CS and the PI3K-AKT signaling pathway. AKT1, an intracellular kinase, governs various biological processes such as cell growth, survival, and metabolism, serving as a pivotal signaling node in various tissues and cellular inflammatory responses47,56,49. Within macrophages, AKT1 represents the sole subtype. Macrophages lacking AKT1 exhibit heightened responsiveness to LPS and display a robust pro-inflammatory reaction, while AKT1 ablation induces the production of M1-type macrophages50. Furthermore, AKT1 phosphorylates and deactivates downstream GSK3β, thereby diminishing NF-κB activation and fostering the expression of the anti-inflammatory cytokine IL-1051. These findings suggest that AKT1 may play a pivotal anti-inflammatory role in inflammation. The PI3K-AKT signaling pathway indicates that AKT1 might mediate the inflammatory response through the downstream FoxO1 signaling pathway. Multiple studies have implicated FoxO1 in promoting inflammatory signaling16,17, 52. It has been observed that the TLR4/MyD88/MD2-NF-κB signaling pathway is markedly activated following FoxO1 overexpression, whereas silencing of FoxO1 downregulates levels of inflammatory pathway proteins15. However, AKT-mediated phosphorylation of FoxO1 leads to its nuclear exclusion and inhibition of its activity. Sun et al.53 demonstrated that Schisandrin substantially reversed the high expression of total FoxO1 protein in the nucleus and upregulated AKT phosphorylation following LPS stimulation. Consistent with these findings, our study revealed that LPS stimulation increased FoxO1 protein levels in Raw264.7 cells. Quercetin targeted AKT1 and significantly phosphorylated it, thereby inhibiting the entry of FoxO1 into the nucleus and reducing the expression of pro-inflammatory genes. This inhibitory effect of quercetin on FoxO1 was further confirmed using immunofluorescence assays.

CS can induce severe oxidative stress, leading to heightened production of ROS and subsequent damage to crucial organs. The Keap1-Nrf2 pathway serves as a primary defense mechanism within cells, safeguarding against oxidative stress and preserving homeostasis. Upon encountering ROS, Keap1 undergoes modification, dissociating from Nrf2. This allows Nrf2 to translocate to the nucleus, where it accumulates and counteracts oxidative stress, thereby protecting cells18,54. Curcumin, a bioactive compound found in turmeric, has been shown to scavenge ROS generated in macrophages, shielding them from oxidative stress by activating the Keap1-Nrf2 pathway55. Similarly, studies by Liu et al.56 demonstrated that Mollugin activated the Keap1-Nrf2 pathway, mitigating oxidative stress in Raw264.7 cells. Additionally, astaxanthin has been found to safeguard against LPS-induced cellular inflammation and acute lung injury in mice by suppressing iron-induced cell death through the Keap1-Nrf2 pathway57. In our investigation, we observed a quercetin dose-dependent decrease in Keap1 levels and an increase in Nrf2 protein levels, significantly inhibiting LPS-induced ROS production. This finding suggests a potential contribution of quercetin to antioxidant stress effects.

In summary, the present study demonstrated that quercetin could inhibit LPS-induced inflammation and alleviate cytokine storm in vitro and in vivo. Mechanistically, quercetin exerted its protective effects by regulating AKT-FoxO1 and Keap1-Nrf2 pathway.

Conclusion

This study employed network pharmacology and molecular docking technology to identify the effective target genes of quercetin against CS. It also preliminarily revealed that quercetin might act against CS through signaling pathways such as PI3K-AKT and FoxO. In addition, The in vitro and in vivo experiments confirmed that quercetin could play an anti-inflammatory role. Collectively, it could regulate AKT1-FoxO1 and Keap1-Nrf2 signaling pathways.