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World Journal of Emergency Medicine logoLink to World Journal of Emergency Medicine
. 2024;15(2):111–120. doi: 10.5847/wjem.j.1920-8642.2024.030

Protective mechanism of quercetin in alleviating sepsis-related acute respiratory distress syndrome based on network pharmacology and in vitro experiments

Weichao Ding 1,2,3, Wei Zhang 1, Juan Chen 1,2,4, Mengmeng Wang 1, Yi Ren 1, Jing Feng 1, Xiaoqin Han 1, Xiaohang Ji 1, Shinan Nie 1,2,✉, Zhaorui Sun 1,2,✉
PMCID: PMC10925531  PMID: 38476533

Abstract

BACKGROUND:

Sepsis-related acute respiratory distress syndrome (ARDS) has a high mortality rate, and no effective treatment is available currently. Quercetin is a natural plant product with many pharmacological activities, such as antioxidative, anti-apoptotic, and anti-inflammatory effects. This study aimed to elucidate the protective mechanism of quercetin against sepsis-related ARDS.

METHODS:

In this study, network pharmacology and in vitro experiments were used to investigate the underlying mechanisms of quercetin against sepsis-related ARDS. Core targets and signaling pathways of quercetin against sepsis-related ARDS were screened and were verified by in vitro experiments.

RESULTS:

A total of 4,230 targets of quercetin, 360 disease targets of sepsis-related ARDS, and 211 intersection targets were obtained via database screening. Among the 211 intersection targets, interleukin-6 (IL-6), tumor necrosis factor (TNF), albumin (ALB), AKT serine/threonine kinase 1 (AKT1), and interleukin-1β (IL-1β) were identified as the core targets. A Gene Ontology (GO) enrichment analysis revealed 894 genes involved in the inflammatory response, apoptosis regulation, and response to hypoxia. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified 106 pathways. After eliminating and generalizing, the hypoxia-inducible factor-1 (HIF-1), TNF, nuclear factor-κB (NF-κB), and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways were identified. Molecular docking revealed that quercetin had good binding activity with the core targets. Moreover, quercetin blocked the HIF-1, TNF, NF-κB, and NOD-like receptor signaling pathways in lipopolysaccharide (LPS)-induced murine alveolar macrophage (MH-S) cells. It also suppressed the inflammatory response, oxidative reactions, and cell apoptosis.

CONCLUSION:

Quercetin ameliorates sepsis-related ARDS by binding to its core targets and blocking the HIF-1, TNF, NF-κB, and NOD-like receptor signaling pathways to reduce inflammation, cell apoptosis, and oxidative stress.

Keywords: Quercetin, Sepsis-related acute respiratory distress syndrome, Network pharmacology

INTRODUCTION

Sepsis is a fatal acute organ dysfunction secondary to an infection.[1] In sepsis-induced multiorgan injury, lung is the first and most vulnerable organ, causing acute respiratory distress syndrome (ARDS). Clinical data have revealed that sepsis-related ARDS occurs less frequently than sepsis or ARDS alone. However, individuals affected by sepsis-related ARDS tend to have poorer outcomes than those without sepsis-associated ARDS.[2] Moreover, because its high mortality rate and lacking of effective treatments, new therapeutic strategies are urgently needed for this life-threatening condition.

Quercetin is a polyphenolic flavonoid with many pharmacological activities, such as antioxidative, anti-apoptotic, and anti-inflammatory effects. Sang et al[3] reported that quercetin attenuated sepsis-related ARDS by suppressing oxidative stress-mediated endoplasmic reticulum stress through the activation of the sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK) pathway. Wang et al[4] revealed that quercetin protected against lipopolysaccharide (LPS)-induced ARDS in mice through the cyclic adenosine monophosphate (cAMP)-exchange protein activated by cAMP (Epac) pathway. Deng et al[5] showed that quercetin alleviated LPS-induced ARDS by inhibiting ferroptosis. Sul et al[6] demonstrated that quercetin prevented LPS-induced inflammation and oxidative stress by regulating nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)/reactive oxygen species (ROS)/nuclear factor kappa B (NF-κB) pathway in lung epithelial cells. Although several studies have reported that quercetin reduces sepsis-related ARDS, the detailed protective mechanism has not been fully elucidated.

Since quercetin has multiple pharmacological effects and targets, we sought to identify a method that could systematically elucidate the protective effects of quercetin against sepsis-related ARDS. Network pharmacology screening and construction of multilevel networks, the use of various database platforms, and data visualization allow the study of traditional Chinese medicinal compounds[7] and direct explanations of the correlations between compounds and diseases.[8] Thus, in this study, network pharmacology and in vitro experiments were used to investigate the mechanism of quercetin in sepsis-related ARDS.

METHODS

Target screening and network construction

Potential quercetin targets were screened using PharmMapper[9] (http://www.lilab-ecust.cn/pharmmapper/), Swiss Target Prediction[10] (http://www.swisstargetprediction.ch/), and the Comparative Toxicogenomics Database (CTD)[11] (http://ctdbase.org/). The names of corresponding target gene were subsequently obtained from the UniProt database[12] (https://www.uniprot.org). The keywords “sepsis” and “ARDS” were used for searching in the GeneCards[13] (https://www.genecards.org/), DisGeNet (https://www.disgenet.org/), Online Mendelian Inheritance in Man (OMIM)[14] (https://www.omim.org/), and Therapeutic Targets Database (TTD)[15] (http://db.idrblab.net/ttd/) databases. The intersection targets of quercetin and sepsis-related ARDS were obtained by generating a Venn diagram of quercetin and sepsis-related ARDS targets using the Weishengxin platform. The network relationships between components, diseases, and intersection targets were extracted, and a “component-intersection targets-disease” network diagram was constructed using Cytoscape 3.9.1 software (https://cytoscape.org/).

Construction of the protein-protein interaction (PPI) network

The intersection targets were imported into the STRING database[16] (https://string-db.org) for analysis, and the species “Homo sapiens” with a confidence score ≥0.4 was selected to construct the PPI network. The PPI network data were imported into Cytoscape 3.9.1 software for visualization.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses

The intersection targets were analyzed with GO and KEGG enrichment analyses using the DAVID[17] 6.8 (https://david-d.ncifcrf.gov/) database. The enrichment analysis results were visualized with the Weishengxin platform.

Construction of the KEGG relationship network

The top 20 KEGG pathways were selected to construct a KEGG relationship network. The interactions between pathways and target genes were established, followed by determination of the interactions between target genes and node attributes. A KEGG network map of quercetin protecting from sepsis-related ARDS was constructed by importing the data into Cytoscape 3.9.1 software.

Visualization of signaling pathways

The intersection targets were converted into gene IDs with the UniProt database and KEGG website (https://www.kegg.jp/). The transformed gene IDs were input into the KEGG mapper to match the signaling pathways and map them to the corresponding signaling pathways. Finally, the signaling pathways associated with the genes identified via KEGG enrichment analysis were selected, and signaling pathway diagrams of the related pathways were drawn.

Molecular docking

The 3D structure of quercetin in structure-data file (SDF) was downloaded from the PubChem database,[18] and the crystal structures of the core targets in “PDB” were downloaded from the Protein Data Bank (PDB) database. In addition, molecular docking of quercetin to core targets was performed using the CB-Dock website.[19] A binding energy ≤ -5.0 kcal/mol indicated good binding affinity between the active ingredient and the target.[20]

Cell culture and treatment

Murine alveolar macrophage (MH-S) cells (ATCC; USA) were maintained at 5% CO2 and 37 °C. The cells were cultured in RPMI-1640 culture medium (C11875500BT; Gibco, USA) supplemented with 10% fetal bovine serum (F2442; Sigma, USA) and a dual antibiotic mixture consisting of 100 U/mL penicillin and 100 μg/mL streptomycin (C0222; Beyotime, China). The cells were allowed to reach 70% to 80% confluence before exposure to LPS (L2630; Sigma, USA). Subsequently, they were classified into four groups: control (no LPS treatment), LPS (5 μg/mL LPS), LPS+L-Que (5 μg/mL LPS and 5 μmol/L quercetin), and LPS+H-Que (5 μg/mL LPS and 10 μmol/L quercetin). Finally, the cells from all groups were incubated for 24 h, after which the cells and supernatants were collected for subsequent experiments.

Cell viability assay

Cell viability was assessed using a cell counting kit-8 (CCK-8) (C0039, Beyotime, China). The cells were seeded at 2×104 cells/well in a 96-well plate, cultured for 24 h, and exposed to varying concentrations of LPS or quercetin. After 24 h, 100 μL of 10% CCK-8 reagent was added to each well, which was incubated in the dark for 1–2 h. The absorbance at 450 nm was determined with a microplate reader (Kaiao Technology Development Co. Ltd., China).

Reverse transcription-quantitative polymerase chain reaction (RT‒qPCR)

Total RNA was extracted from the cells treated for 24 h using an RNeasy Mini Kit (74104; Qiagen GmbH, Germany) according to the manufacturer’s protocol. Subsequently, 1 μg of isolated RNA was reverse transcribed into cDNA using HiScript II QRT SuperMix (KCD-M1003, Croda Co. Ltd., China). Next, cDNA was analyzed via qPCR using SYBR qPCR Master Mix (KCD-M1004; Croda Co. Ltd., China) in a LightCycler 96 RT-qPCR system (Roche Group, Switzerland). The relative mRNA expression levels were quantified using the 2-ΔΔCt method, with GAPDH serving as the reference gene. The primers used were synthesized by Jiangsu Saisofei Biotech Co., Ltd. (Wuxi, China). The primer sequences are shown in supplementary Table 1.

Western blotting

The cells were lysed in radioimmune precipitation assay (RIPA) buffer, after which the protein concentration was subsequently evaluated via a Thermo Fisher BCA protein assay kit (23227; Waltham, USA). Next, the lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation. The isolated proteins were transferred onto polyvinylidene fluoride membranes, which were subsequently blocked in 5% skim milk. Subsequently, the immunoblots were probed overnight at 4 °C with specific primary antibodies: anti-hypoxia-inducible factor-1 (HIF-1) α (48085; CST, USA), anti-TNFR1 (WL01414; Wanleibio Co. Ltd., China), anti-NF-κB p65 (phospho T254) (ab131100; Abcam, USA), anti-NF-κB p65 (8242;CST, USA), anti-NLRP3 (WL02635; Wanleibio Co. Ltd., China), anti-Bax (14796; CST, USA), anti-Bcl-2 (ab196495; Abcam, USA), anti-cleaved caspase-3 (9661; CST, USA), anti-caspase-3 (9662; CST, USA), and anti-β-actin (AB0035; Abways Technology, China; internal control). After three washes in Tris buffered saline with Tween 20 (TBST), the immunocomplexes were incubated for 2 h at ambient temperature with an anti-rabbit IgG (H+L) secondary antibody (111-035-003; Jackson ImmunoResearch Laboratories, Inc., USA). Finally, the blots were visualized using a BeyoECL Moon Kit (P0018FM; Beyotime, China), photographed utilizing a Tanon 5200 luminescent imaging workstation (Tanon Science & Technology Co. Ltd., China), and quantified with ImageJ software (version 1.8.0, NIH, USA).

Immunofluorescence staining

The cell samples were immobilized in 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 3% bovine serum albumin for approximately 1 h. The samples were incubated overnight at 4 °C with the following primary antibodies: anti-HIF-1α (48085) and anti-cleaved caspase-3 (9661). The cells were rinsed and incubated for 1 h at 37 °C in the dark with an Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) secondary antibody (A21206; Thermo Fisher Scientific, USA). The nuclei were stained with DAPI for 10 min, after which an anti-fluorescence quencher was added to the samples to decrease the fluorescence quenching speed. Images were recorded with an inverted fluorescence microscope (Leica, Germany), and the fluorescence intensity was estimated using ImageJ software.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of tumor necrosis factor (TNF)-α (1217202), interleukin (IL)-1β (1210122), and IL-6 (1210602) in the cell culture medium supernatants of MH-S cells were determined using mouse ELISA kits (Dakewe Bioengineering Co. Ltd., China). The absorbance at 450 nm was measured by a microplate reader (Kaiao Technology Development Co. Ltd., China) according to the manufacturer’s protocols. Standard curves were generated to quantify the concentrations of inflammatory factors.

Cell apoptosis assay

Cellular apoptosis was investigated using an Annexin V-FITC/PI apoptosis kit (E-CK-A211; Elabscience Biotechnology Co. Ltd., China) following the manufacturer’s protocol. The phosphate-buffered saline-rinsed cells were resuspended in 500 μL of binding buffer. A solution containing 5 μL of Annexin V-FITC and 5 μL of PI was prepared for incubating the cell suspension mentioned above. The mixture was incubated for 15 min at ambient temperature. Apoptotic cells were detected with a flow cytometer (Beckman Coulter, USA), and data analysis was performed using FlowJo 10.8.1 (FlowJo LLC, USA).

Caspase-3 activity assay

After incubating the cells, caspase-3 activity was quantified with a caspase-3 activity assay kit (C1116; Beyotime, China) based on the manufacturer’s protocols. Thereafter, the sample absorbance at 405 nm was measured using a microplate reader (Kaiao Technology Development Co. Ltd., China).

Oxidative stress analysis

ROS accumulation was assessed with a ROS assay kit (S0033M, Beyotime, China). The MH-S cells were incubated for 20 min with a 2’,7’-dichlorofluorescin diacetate (DCFH-DA) probe at 37 °C, after which ROS generation was assessed. Images were acquired with an inverted fluorescence microscope (Leica, Germany), and the fluorescence intensity was quantified using ImageJ (NIH, USA). The malondialdehyde (MDA) concentration and total superoxide dismutase (SOD) activity in MH-S cells were examined using two commercially available kits (Beyotime, China): a lipid peroxidation MDA assay kit (S0131M) and a total SOD assay kit with WST-8 (S0101M). Using a microplate reader (Kaiao Technology Development Co. Ltd., China), the absorbance of MDA was recorded at 532 nm, and that of SOD was recorded at 450 nm.

Statistical analysis

All the results were analyzed using GraphPad Prism 8.0.2 (GraphPad Software, Inc., USA). A P-value <0.05 indicated statistical significance. Normally distributed data are presented as the mean±standard error of the mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons. Moreover, all cell line experiments were performed in triplicate to ensure the accuracy and reproducibility of the results.

RESULTS

Screening targets and network construction

The structure of quercetin is shown in Supplementary Figure 1A. The screening for quercetin targets was performed on June 1, 2022, and the initial search revealed 93 targets in the PharMapper database, 100 in the Swiss Target Prediction database, and 4,148 in the CTD database. After the screening results were combined and duplicate targets were removed from the databases, 4,230 quercetin targets were predicted. In addition, 360 disease targets of sepsis-related ARDS were obtained from the GeneCards, DisGeNet, OMIM, and TTD databases on May 7, 2022. The intersection of quercetin and sepsis-related ARDS targets was mapped to construct a Venn diagram (Supplementary Figure 1B). Subsequently, 211 intersection targets of quercetin and sepsis-related ARDS were obtained to construct a “component-intersection targets-disease” network diagram (Supplementary Figure 1C).

PPI network analysis of intersection targets

A PPI network was constructed based on the degree of enrichment of 211 intersection targets (Supplementary Figure 2). Five of these targets were shown to act most closely with other targets and play crucial roles in the entire network: IL-6, TNF, ALB, AKT1 and IL-1β. Therefore, these targets were recognized as the core targets of quercetin against sepsis-related ARDS.

GO and KEGG enrichment analysis

GO enrichment analysis revealed 894 items closely related to quercetin-conferred protection against sepsis-related ARDS: 710 biological processes, 66 cellular components, and 118 molecular function items. The top 10 GO enrichment analysis terms were selected; they were related to biological processes, including the inflammatory response, negative regulation of apoptotic processes, and response to hypoxia (Supplementary Figure 3A). These data suggest that quercetin protects against sepsis-related ARDS by regulating these biological processes. Subsequent KEGG enrichment analysis revealed 106 significant pathways that constituted the potential mechanism of quercetin against sepsis-related ARDS. The results of the enrichment analysis of the top 20 KEGG pathways are listed in supplementary Figure 3B.

Construction of the KEGG relationship network

The “targets-pathways” relationship network was visualized, and the KEGG relationship network map was constructed (Supplementary Figure 4). The map confirmed that quercetin protected against sepsis-related ARDS through multiple targets and pathways.

Depiction of the signaling pathway diagram

Disease-related and generalized pathways were eliminated from the KEGG enrichment analysis, revealing 4 notable pathways strongly correlated with quercetin protection against sepsis-related ARDS: HIF-1, TNF, NF-κB, and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways. The intersection targets were mapped to the 4 signaling pathways using a KEGG mapper, and signaling pathway diagrams were drawn. These diagrams allowed us to explore the relationships between intersection targets and signaling pathways associated with quercetin protection against sepsis-related ARDS (Supplementary Figure 5).

Molecular docking

According to the degree values, the top 5 core targets, IL-6, TNF, ALB, AKT1, and IL-1β, were selected for molecular docking with quercetin using the CB-Dock website (Supplementary Figure 6). A lower binding energy suggested more stable binding. Molecular docking revealed that the binding energies between quercetin and the five core targets were ≤-5.0 kcal/mol, indicating good binding affinity.

Effect of quercetin on signaling pathways in MH-S cells

MH-S cells were incubated with varying concentrations of LPS (0.1, 0.5, 1, 2, 5, 10, 20, or 50 μg/mL) or quercetin (1, 2, 5, 10, 20, 50, 100, or 200 μmol/L) for 24 h, after which their viability was assessed using the CCK-8 assay. While LPS ≤2 μg/mL and quercetin ≤10 μmol/L had no significant effect on cell viability, while cell viability was significantly decreased by 5 μg/mL LPS and 20 μmol/L quercetin (Figure 1 A and B). Thus, 10 μmol/L quercetin (maximum treatment concentration) and 5 μg/mL LPS were applied in subsequent experiments.

Figure 1.

Figure 1

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Effect of quercetin on signaling pathways in MH-S cells. A, B: viability of MH-S cells exposed to varying concentrations of LPS or quercetin for 24 h, as detected using a CCK-8 assay. C: the relative expression of HIF-1α mRNA was quantified by RT-qPCR. D‒H: Western blotting showing HIF-1α, TNFR1, p-NF-κB p65, NF-κB p65, NLRP3, and β-actin (control) expression in MH-S cells. I‒J: immunofluorescence was used to detect HIF-1α protein expression; scale bar=50 μm, and the expression level was quantified using ImageJ software. The data are expressed as the mean±SEM (n=3). Compared with the control group, *P<0.05, **P<0.01; compared with the LPS group, #P<0.05, ##P<0.01. MH-S: murine alveolar macrophage; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; CCK-8: cell counting kit-8; SEM: standard error of the mean.

The signaling pathways was investigated in LPS-induced MH-S cells to explore the mechanism underlying the protective effect of quercetin against sepsis-related ARDS. An RT-qPCR assay was performed on total RNA isolated from MH-S cells, which revealed that the HIF-1α mRNA level was significantly higher in the LPS group than in the control group. Conversely, HIF-1α mRNA levels were reduced following quercetin treatment (Figure 1 C). Western blotting revealed that LPS stimulation increased the protein expression levels of HIF-1α, TNFR1, p-NF-κB p65, and NLRP3. In contrast, the impact significantly decreased after LPS-induced MH-S cells were stimulated with quercetin (Figure 1 D–H). Moreover, a 10 μmol/L dose was more effective than a 5 μmol/L dose of quercetin. Furthermore, semiquantitative immunofluorescence staining demonstrated that the protein expression level of HIF-1α was elevated in the LPS group but was decreased in the LPS+L-Que and LPS+H-Que groups (Figure 1 I–J). These results suggest that quercetin represses the HIF-1, TNF, NF-κB, and NOD-like receptor signaling pathways in LPS-induced MH-S cells.

Effects of quercetin on inflammation and oxidative stress in MH-S cells

ELISA was performed to assess whether quercetin treatment diminishes LPS-stimulated inflammatory responses in MH-S cells. Moreover, LPS stimulation significantly elevated the concentrations of TNF-α, IL-1β, and IL-6 in MH-S cells, whereas quercetin treatment decreased these concentrations in a dose-dependent manner (Figure 2 A–C). Next, ROS and MDA concentrations and total SOD activity were determined in MH-S cells to ascertain whether quercetin has a consistent suppressive effect on oxidative stress. Indeed, while LPS exposure significantly promoted ROS production, MDA formation, and SOD depletion, quercetin treatment reversed these effects in a dose-dependent fashion (Figure 2 D–G).

Figure 2.

Figure 2

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Effects of quercetin on inflammation and oxidative stress in MH-S cells. A–C: concentrations of TNF-α, IL-1β, and IL-6 measured via ELISA; D–E: ROS concentrations in MH-S cells; scale bar=100 μm, the expression levels quantified with ImageJ; F–G: MDA concentrations and total SOD activity in MH-S cells. The data are presented as the means±SEM (n=3). Compared with the control group, *P<0.05, **P<0.01; compared with the LPS group, #P<0.05, ##P<0.01. MH-S: murine alveolar macrophage; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6; ELISA: enzyme-linked immunosorbent assay; ROS: reactive oxygen species; MDA: malondialdehyde; SOD: superoxide dismutase; SEM: standard error of the mean.

Effect of quercetin on the apoptosis of MH-S cells

Since apoptosis is a crucial event in sepsis-related ARDS, cells were exposed to LPS or quercetin to examine how quercetin affects the apoptotic rate of MH-S cells. Flow cytometry revealed that LPS stimulation significantly improved the apoptosis rate of MH-S cells, while it decreased following quercetin treatment (Figure 3 A and B). Moreover, a caspase-3 activity assay validated the alterations in cell apoptosis in each treatment group (Figure 3C). Western blotting demonstrated that the protein levels of Bax and cleaved caspase-3 were significantly higher in the LPS group than in the control group. Conversely, Bcl-2 protein levels were lower in the LPS group than in the control group. Remarkably, quercetin administration reversed the LPS-induced effect on the MH-S cells (Figure 3 D–G). In addition, an RT-qPCR assay revealed that Bax mRNA levels were elevated, while Bcl-2 mRNA levels were decreased in the LPS group compared with those in the control group. This trend was reversed after stimulating the cells with quercetin (Figure 3 H–I), confirming that quercetin affected the expression of these genes at the RNA and protein levels. Semiquantitative immunofluorescence staining further revealed that quercetin attenuated the LPS-induced changes in cleaved caspase-3 expression in MH-S cells (Figure 3 J–K).

Figure 3.

Figure 3

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Effect of quercetin on the apoptosis of MH-S cells. A–B: flow cytometry-based assessment of the MH-S cell apoptosis rate; C: Caspase-3 activity in MH-S cells. D–G: Western blotting showing Bax, Bcl-2, cleaved caspase-3, caspase-3, and β-actin (internal control) protein levels in MH-S cells; H–I: RT-qPCR-quantified Bax and Bcl-2 mRNA levels; J-K: immunofluorescence results showing the expression level of cleaved caspase-3; scale bar=50 μm. The expression levels were quantified with ImageJ. The data are presented as the means±SEM (n=3). Compared with the control group, **P<0.01; compared with the LPS group, #P<0.05, ##P<0.01. MH-S: murine alveolar macrophage; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; SEM: standard error of the mean.

DISCUSSION

Sepsis-related ARDS is a complex and multifactorial disease whose severity is greater than that of nonsepsis-related ARDS, and it is more likely to be accompanied by severe hypoxemia and higher mortality. Unfortunately, there are no effective treatments available for sepsis-related ARDS, and the current treatment approaches primarily include mechanical ventilation, fluid resuscitation, and antibiotic therapies. Thus, researchers are investigating complementary and alternative treatments for sepsis and sepsis-related ARDS. In this study, the underlying mechanisms of quercetin against sepsis-related ARDS were investigated using network pharmacology and validated by in vitro experiments.

Using network pharmacology, 211 intersection targets of quercetin and sepsis-related ARDS were identified after screening for quercetin targets in public databases. Among the intersection targets, IL-6, TNF, ALB, AKT1, and IL-1β were highlighted as the core targets of the protective effect of quercetin against sepsis-related ARDS. In addition, the molecular docking results revealed that quercetin had good binding affinity with the core targets. Sepsis-related ARDS is initially caused by an imbalance of inflammation, and the inflammatory factors IL-6, IL-1β, and TNF-α play essential roles in this process.[21] The concentrations of these inflammatory factors in the supernatant of MH-S cells were detected using ELISA, which indicated that quercetin reduces the expression of these inflammatory factors in LPS-induced MH-S cells and alleviates the inflammatory response. Moreover, ALB is related to pulmonary vascular permeability,[22] and AKT1 is involved in the inflammatory response, apoptosis, and other processes in sepsis-related ARDS.[23]

GO enrichment analysis revealed 894 items closely related to the protective effects of quercetin against sepsis-related ARDS. The highly ranked biological processes included the inflammatory response, negative regulation of the apoptotic process, and response to hypoxia, suggesting that quercetin protects against sepsis-related ARDS by regulating these biological processes. Cell apoptosis contributes to the pathophysiology of sepsis. Sepsis induces the apoptosis of several types of cells, such as lung epithelial and immune cells, triggering ARDS.[24, 25] Taken together, these findings indicate that inhibiting apoptosis reduces pulmonary edema and ameliorates sepsis-related ARDS.[26] Moreover, oxidative stress is closely associated with the HIF-1α gene, a crucial hypoxia-regulating factor, overwhelmingly activating its transcription under hypoxia.[27] In addition, the inflammatory response and oxidative stress are critical contributors to sepsis-related ARDS pathogenesis. Oxidative stress amplifies pro-inflammatory gene expression, and inflammatory cells stimulate excessive ROS generation, creating a vicious cycle that triggers the onset of sepsis-related ARDS and subsequent progression.[28] Therefore, quercetin may protect against sepsis-related ARDS by interfering with multiple biological processes, such as the inflammatory response, cell apoptosis, and oxidative stress.

Considering the crucial role of cell apoptosis in sepsis-related ARDS pathogenesis, we used various methods to verify how quercetin affects apoptosis in MH-S cells under conditions that mimic sepsis. Our flow cytometry results suggested that quercetin decreases the apoptosis rate which is increased by LPS, which was validated by the observation that quercetin reduces caspase-3 activity in MH-S cells. Moreover, Western blotting demonstrated that quercetin reverses the expression patterns of the apoptotic proteins Bax, Bcl-2, and cleaved caspase-3 in LPS-stimulated MH-S cells. This effect was also confirmed on the mRNA level with RT-qPCR; quercetin down-regulated Bax mRNA expression but up-regulated Bcl-2 expression in LPS-induced MH-S cells. Finally, semiquantitative immunofluorescence staining proved that quercetin reduces the fluorescence intensity of cleaved caspase-3 in LPS-induced MH-S cells. We also performed experiments to verify the occurrence of oxidative stress, the results of which showed that quercetin diminishes the levels of ROS and MDA, elevates the activity of SOD, and alleviates oxidative stress in LPS-induced MH-S cells.

KEGG enrichment analysis revealed 4 signaling pathways strongly correlated with quercetin-mediated protection against sepsis-related ARDS: HIF-1, TNF, NF-κB, and NOD-like receptor signaling pathways. Sepsis-related ARDS can be interfered with by regulating HIF-1α,[29] but whether quercetin protects against sepsis-related ARDS through HIF-1 signaling has never been reported. Additionally, TNFR1-mediated inflammation is involved in sepsis-related ARDS,[30] but whether quercetin interferes with TNF signaling has not been determined. However, quercetin alleviates sepsis-related ARDS by inhibiting NF-κB[31] and NLRP3 activation.[32] Thus, Western blotting was used to verify the effects of quercetin on the expression of the above-mentioned signaling pathways. We revealed that quercetin diminishes HIF-1α, TNFR1, p-NF-κB p65, and NLRP3 protein expression in LPS-induced MH-S cells, confirming the KEGG analysis findings. Interestingly, regarding the HIF-1α pathway, RT-qPCR quantification revealed that quercetin reduces HIF-1α mRNA levels, and semiquantitative immunofluorescence staining demonstrated that quercetin depletes the fluorescence intensity of the HIF-1α protein in LPS-induced MH-S cells. Therefore, our data suggest that quercetin protects against sepsis-related ARDS by acting on multiple signaling pathways.

Our study has several limitations. For example, network pharmacology analysis is based on database screening, the differences between various databases could produce biased results. In view of this, following experiments are needed to verify the results.

CONCLUSION

This study investigated the protective mechanism of quercetin against sepsis-related ARDS using network pharmacology and in vitro experiments. Quercetin binds to the core targets of IL-6, TNF, ALB, AKT1, and IL-1β. Moreover, quercetin reduces inflammation, cell apoptosis, and oxidative stress by acting on the HIF-1, TNF, NF-κB, and NOD-like receptor signaling pathways, ameliorating sepsis-related ARDS. Importantly, this study revealed that quercetin protects against sepsis-related ARDS through multiple targets and multiple pathways, providing a basis and experimental support for future research.

Footnotes

Funding: This study was supported by the National Natural Science Foundation of China (82172182 and 82102311), Natural Science Foundation of Jiangsu Province (BK20211136), China Postdoctoral Science Foundation (2018M643890 and 2020M683718), Xuzhou Science and Technology Project (KC21215 and KC22136), and Development Fund Project of Affiliated Hospital of Xuzhou Medical University (XYFY202232).

Ethical approval: Not applicable. No animal or human subjects were involved.

Conflicts of interest: All authors declared no competing interests.

Author contributions: WCD, WZ, and JC contributed equally to this work. All authors reviewed and approved the final version.

All the supplementary files in this paper are available at http://wjem.com.cn.

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