Network pharmacology and experimental verification of the mechanism of licochalcone A against Staphylococcus aureus pneumonia

Staphylococcus aureus strains cause the majority of pneumonia cases and are resistant to various antibiotics. Given this background, it is very important to discover novel host-targeted therapies. Licochalcone A (LAA), a natural plant product, has various biological activities, but its primary targets in S. aureus pneumonia remain unclear. Therefore, the purpose of this study was to identify its molecular target against S. aureus pneumonia. Network pharmacology analysis, histological assessment, enzyme-linked immunosorbent assays, and Western blotting were used to confirm the pharmacological effects. Network pharmacology revealed 33 potential targets of LAA and S. aureus pneumonia. Enrichment analysis revealed that these potential genes were enriched in the Toll-like receptor and NOD-like receptor signaling pathways. The results were further verified by experiments in which LAA alleviated histopathological changes, inflammatory infiltrating cells and inflammatory cytokines (TNF, IL-6, and IL-1β) in the serum and bronchoalveolar lavage fluid in vivo. Moreover, LAA treatment effectively reduced the expression levels of NF-κB, p-JNK, p-p38, NLRP3, ASC, caspase 1, IL-1β, and IL-18 in lung tissue. The in vitro experimental results were consistent with the in vivo results. Thus, our findings demonstrated that LAA exerts anti-infective effects on S. aureus-induced lung injury via suppression of the Toll-like receptor and NOD-like receptor signaling pathways, which provides a theoretical basis for understanding the function of LAA against S. aureus pneumonia and implies its potential clinical application.


Introduction
Staphylococcus aureus (S.aureus) is a momentous Gram-positive human opportunistic pathogen.Research has shown that it is a serious etiology of community-acquired pneumonia in the last 20 years, with severe community-acquired pneumonia caused by methicillin-resistant S. aureus (MRSA) (Self et al., 2016;Ensinck et al., 2021;Sakamoto et al., 2021).MRSA pneumonia patients had greater in-hospital mortality, longer hospital

Identification of S. aureus pneumonia-related targets of LAA
PharmMapper is an open-source web server that identifies potential drug targets via reversed pharmacophore matching of the query compound against an in-house pharmacophore model database (Wang et al., 2017).The Comparative Toxicogenomics Database (CTD) harmonizes cross-species heterogeneous data for chemical exposures and their biological repercussions by manually curating and interrelating chemical, gene, phenotype, anatomy, disease, taxa, and exposure content from the published literature (Davis et al., 2023).The PharmMapper server 1 and CTD 2 were used to collect the LAA-related targets.The S. aureus pneumoniarelated targets were collected from GeneCards 3 (Relevance score ≥5) and CTD (see text footnote 2) (Inference score ≥5) with "Staphylococcus aureus pneumonia" as the search term.The interactions between the potential targets of LAA and S. aureus pneumonia were gathered using the STRING database 4 with a combined score ≥0.4.The compound-target network was created based on the protein-protein interaction (PPI) network and displayed using Cytoscape-v3.7.2 software.The network characteristics were calculated by the plug-in Network Analyzer of Cytoscape-v3.7.2 software.The degree of freedom was applied as a topological index to indicate the importance of the network node.The larger the value in the network is, the more vital the node.Shen et al. 10.3389/fmicb.2024.1369662Enrichment analysis of LAA and S. aureus pneumonia-related target pathways Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were executed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) program.5 Specifically, we identified 33 potential targets, selected official_gene_symbol and Homo sapiens, and then performed GO analysis and KEGG pathway enrichment analysis.The GO items associated with biological process (BP), cellular component (CC), molecular function (MF), and KEGG signaling pathways were analyzed, and the mapped senior bubbles of the top 15 BP, CC, MF terms and the top 41 KEGG signaling pathways were visualized by Weishengxin6 according to the p values.

Molecular docking verification
The chemical structure of LAA was obtained from the Zinc database7 and saved in its mol2 format.The three-dimensional structures of intersecting targets were obtained from the RCSB PDB database8 in PDB format.The corresponding protein molecular docking was executed using AutoDock Tools 1.5.7.The twodimensional plan of the docking results was visualized by Discovery Studio 2016 Client.

Bacterial strains
The standard S. aureus strain ATCC 29213 was obtained from the China Medical Culture Collection Center (Beijing, China).S. aureus Newman was provided courtesy of Prof. Timothy J. Foster.Five MRSA strains (MRSA 3183, MRSA 2961, MRSA 2047, MRSA 41122, andMRSA 3335) were provided courtesy of Prof. Guo Na of Jilin University, Changchun, China.

Planktonic antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of LAA and seven antibiotic drugs against the seven S. aureus strains were measured by microdilution in cation-adjusted CA-MHB using the Clinical and Laboratory Standards Institute protocol (Clinical and Laboratory Standards Institute [CLSI], 2012a).Oxacillin (2% NaCl) was added to the wells.The MIC was defined as the lowest concentration at which no visible bacterial growth was observed.On the basis of CLSI standards and guidelines, MIC breakpoints were used to define resistant (R), intermediate (I), and susceptible (S) strains (Clinical and Laboratory Standards Institute [CLSI], 2012b).The minimum bactericidal concentrations (MBCs) was defined as the lowest concentration which no microbial growth was detected on agar plates after 24 h at 37 • C.

S. aureus challenge
THP-1 cells were differentiated to the macrophage (M ) phenotype at 160 ng/mL PMA and then challenged with S. aureus (1 × 10 7 CFU/mL, 2 mL/well) in the absence or presence of LAA for 6 h.The control group did not receive S. aureus.

Mouse model of S. aureus pneumonia
Six-to eight week old male BALB/c mice were obtained from Skbex Biotechnology (Henan, China), housed on a 12/12 h light/dark cycle and allowed to eat freely.The S. aureus pneumonia model was established by intranasal administration of 1 × 10 9 CFU of S. aureus 29213 in 30 µl of sterile isotonic saline, as previously described with modifications (Liu et al., 2017).The mice were randomly divided into the following five groups: control group, S. aureus group, and S. aureus + LAA (10, 20, and 30 mg/kg) group.The animals were treated intraperitoneally with DMSO or LAA (10, 20, or 30 mg/kg) two times a day.The first LAA injection was 1 h before S. aureus infection.Serum, BALF, and lung tissues were obtained 72 h after S. aureus infection.The BALF was obtained using a 1 mL syringe with cold PBS.The BALF was loaded onto a slide and stained with Giemsa stain.

Hematoxylin and eosin staining
Lung, heart, liver, spleen, and kidney samples were obtained and fixed in 4% (w/v) formalin, embedded in paraffin, sliced into 5-mm sections, and stained with hematoxylin and eosin kits according to the manufacturer's instructions.The specimens were observed and collected with Motic DSAssistant Lite 1.0 software or fluorescence microscopy (Nikon, Japan).

Lactate dehydrogenase release and cytokine measurements
The serum and BALF from S. aureus-induced pneumonia and culture supernatant from THP-1-derived M cells were collected.A lactate dehydrogenase (LDH) cytotoxicity assay kit was used to measure LDH release, and the optical density of the samples was measured with a microplate reader set at 450 nm.The inflammatory cytokines IL-1β, IL-6, and TNF-α were examined using ELISA kits according to the manufacturer's instructions.Then, the absorbance of each sample was analyzed at 450 nm with a microplate reader (Thermo Scientific, China).

Western blotting
Total protein from the THP-1 cells and lung tissues was extracted on ice for 10 min with RIPA buffer and then centrifuged at 4 • C for 10 min.The protein concentration was calculated using a BCA protein quantification kit.The samples were separated by 8%-12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (300 mA, 90 min).Then, the membranes were incubated in 5% skim milk for 2 h at Frontiers in Microbiology 03 frontiersin.orgroom temperature.The membranes were washed with TBST.Subsequently, the membranes were incubated with primary antibodies (1:500) overnight at 4 • C. The membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature.The target protein was visualized using ECL Plus detection reagents.

Statistical analysis
All the statistical analyses in this study were analyzed using SPSS 21 and GraphPad Prism 8 software.The data are presented as the mean ± standard deviation (SD).One-way analysis of variance followed by Dunnett's post-hoc test was used with p < 0.05 considered to indicate statistical significance.

Activity of LAA against S. aureus
The MICs, MBCs, and MIC breakpoints are displayed in Table 1 according to Clinical and Laboratory Standards Institute [CLSI] (2012a,b).S. aureus 29213 was sensitive to seven common antibiotics.Five clinical strains of S. aureus were sensitive to vancomycin and resistant to penicillin G, oxacillin, ciprofloxacin, levofloxacin, tetracyclines, and gentamicin except for MRSA 41122 (Shen et al., 2020).S. aureus Newman strains were sensitive to four common antibiotics (vancomycin, oxacillin, levofloxacin, and gentamicin) and resistant to three common antibiotics (penicillin G, ciprofloxacin, and tetracyclines).
The MICs and MBCs of LAA for the target strains were 2-4 and 8 µg/ml, respectively.This suggests that LAA has antimicrobial activity against MRSA indicating that it is a very competitive anti-S.aureus drug candidate.

LAA alleviated pulmonary morphological damage in S. aureus pneumonia mice
To prove the effect of LAA on S. aureus pneumonia, we constructed a mouse model of S. aureus pneumonia.Histopathological examination showed that the S. aureus-infected lungs were slightly or weakly red after LAA treatment.H&E staining of the sliced lung tissues was then performed.The lung tissues of S. aureus-infected mice showed thickening of alveolar walls, accumulation of inflammatory cells, edema within alveolar spaces and no intact alveolar structure in some areas.Notably, LAA protected lung tissue with reduced infiltration of inflammatory cells, alveolar wall thickening and edema after infection (Figure 1A).Compared with that in untreated mice, the abundance of S. aureus bacteria in the lungs of mice was significantly reduced by LAA (p < 0.05; Figure 1B).Moreover, the number of total cells and neutrophils in the BAL fluid increased during S. aureus infection.LAA substantially reduced the number of total cells and neutrophils, except at 10 mg/kg (Figures 1C, D).This suggests that LAA alleviated pulmonary morphological damage in S. aureus pneumonia mice.LAA reduced the expression of IL-6, TNF-α, and IL-1β To investigate the anti-inflammatory effects of LAA, we detected the levels of IL-6, TNF-α, and IL-1β in culture supernatants from macrophages, serum and BALF by ELISA.As shown in Figures 2A-C, LAA treatment suppressed the S. aureus-triggered expression of IL-6, TNF-α, and IL-1β, but not that of IL-6, in THP-1-derived M s treated with 1 µg/mL LAA.Similarly, the production of IL-6, TNF-α, and IL-1β was significantly reduced by various concentrations of LAA in the serum and BALF of the mice (p < 0.05 and p < 0.01; Figures 2D-I).The results suggested that LAA reduced the expression of inflammatory mediators in S. aureus-induced macrophages and pneumonia mice.

Network pharmacology
To determine the mechanism underlying the anti-S.aureus pneumonia effects of LAA, we used CTD, PharmMapper and GeneCards to identify the S. aureus pneumonia-related targets  of LAA via text mining.The targets of LAA were identified by PharmMapper and CTD.After deleting duplicates, we ultimately obtained 203 targets (Supplementary Table 1).The S. aureus pneumonia-related genes were identified from the GeneCards database and CTD, and 354 S. aureus pneumonia-related genes were identified (Supplementary Table 2).The common targets of LAA and S. aureus pneumonia-related genes were analyzed using the online software Venny 2.1.0,and Venn diagrams were generated.In this study, 33 genes overlapped as targets of both LAA and S. aureus pneumonia (Figure 3B and Supplementary Table 3).

PPI network analysis
Based on the potential pharmacodynamics of LAA against S. aureus pneumonia, the interacting proteins were screened using the STRING online tool.We found 33 nodes with 233 PPI relationships in the network (Figure 3C).The PPI network was established using Cytoscape 3.7.2software for further visualization and analysis (Figure 3D).A bar chart of the top 16 intersecting targets (including IL1B, AKT1, TNF, EGFR, MAPK3, SRC, IL2, IFNG, PPARG, MMP2, MAPK14, CASP8, MAPK8, CASP1, HMOX1, and SYK) was exhibited according to the degree value, which was greater than or equal to the average score of 13.5 (Figure 3E).All of these results implied that LAA might play an effective role against S. aureus pneumonia through these targets.

GO and KEGG analysis
Gene Ontology enrichment analysis was performed with the common potential target genes of LAA and S. aureus pneumonia.The top 15 significantly enriched terms in BP, CC, and MF categories are shown in Figure 4A.BP analysis indicated that these targets were interconnected with biological processes, including positive regulation of MAP kinase activity, positive regulation of interleukin-6 production, and positive regulation of the inflammatory response.CC analysis showed that markedly enriched terms were concentrated in the extracellular region, cytosol, and cytoplasm.MF analysis showed that the enriched functional terms included enzyme binding, MAP kinase activity, protein serine/threonine kinase activity, and protease binding.
Furthermore, the 33 targets were enriched in 151 pathways according to KEGG analysis.We observed that these targets participated in pathways including the C-type lectin receptor signaling pathway, the relaxin signaling pathway, the Toll-like receptor signaling pathway, the T cell receptor signaling pathway, Frontiers in Microbiology 06 frontiersin.orgthe Fc epsilon RI signaling pathway, the TNF signaling pathway, neutrophil extracellular trap formation, and the NOD-like receptor signaling pathway (Figure 4B).In particular, two major pathways, the Toll-like receptor signaling pathway and the NOD-like receptor signaling pathway, are the known therapeutic pathways of S. aureus infection.Therefore, the Toll-like receptor and NODlike receptor signaling pathway-related targets were selected as candidate targets of LAA against S. aureus pneumonia for further experimental validation.

Molecular docking display
Based on these results, the five targets were selected for molecular docking and visualization by AutoDock Vina and Discovery Studio 2016.The details of the docking results are shown in Figure 5 and Table 2.
The binding affinities (kcal/mol) of the 5 targets were all < −6 kcal/mol, indicating that LAA had good binding affinity with these receptor proteins.According to the twodimensional diagram, LAA bound to 3ELJ (MAPK8) through conventional hydrogen bonds with GLU-109 and ASN156.Other forces, including unfavorable donor-donor, pi-sigma, alkyl, and pialkyl bonds, were also found.LAA formed 1 conventional hydrogen bond with ASP-168 of 7BDO (MAPK14).In addition, pi-sigma and pi-alkyl bonds also existed.LAA was attracted to 2AZ5 (TNF) by 2 conventional hydrogen bonds with AEU-120 and SER-60, as well as pi-pi stacked and pi-alkyl bonds.When encountering 6PZP (CASP1), LAA formed 1 hydrogen bond with ILE-155, and pi-pi stacked, pi-pi T-shaped, alkyl, and pi-alkyl bonds were also found.When the target was 6Y8I (IL1B), LAA could form unfavorable acceptor-acceptor, pi-donor, pi-sigma, and pi-alkyl hydrogen bonds at the corresponding positions.Combining the results of the free binding energy score and chemical bond distribution showed that LAA might be an inhibitor of these five targets; however, this still needs to be experimentally verified.

LAA reduced NLRP3 inflammasome activation
To survey the inhibitory effect of LAA on activated M s, THP-1-derived M s were used for in vitro experiments.In vitro, we investigated the activation of the NLRP3 inflammasome (NLRP3, ASC, caspase-1, IL-1β, and IL-18) in M s after treatment with S. aureus and LAA.As shown in Figure 6A, the levels of NLRP3, ASC, pro-caspase-1, caspase-1 p20, pro-IL-1β, mature-IL-1β, and IL-18 were decreased by LAA in S. aureus-treated THP-1-derived M compared to S. aureus alone.The concentrations of LDH released from THP-1-derived M s were determined using an LDH assay kit to evaluate cell integrity.As shown in Figure 6B, S. aureus increased LDH release compared to control group (p < 0.05), and 2 µg/mL LAA co-treatment decreased LDH release in S. aureustreated THP-1-derived M s (p < 0.05); however, the effect of 1 µg/mL LAA was not obvious.These findings suggested that LAA reduced S. aureus-induced NLRP3 inflammasome activation in vitro.Moreover, LAA treatment reduced ASC, pro-caspase-1, caspase-1 p20, pro-IL-1β, mature-IL-1β, and IL-18 activation in the lungs of pneumonia mice in comparison with S. aureus-treated mice (Figure 6C).These results suggest that LAA reduced the activation of the NLRP3 inflammasome induced by S. aureus in macrophages and in mice with pneumonia.
To demonstrate that the anti-inflammatory effect of LAA is not solely dependent on its antibacterial effect, we further examined the effect of LAA on ATP and nigericin-induced NLRP3 inflammasome in vitro.As shown in Figure 6D, compared with those in the control group, the levels of NLRP3, pro-caspase-1, caspase-1 p20, pro-IL-1β, and mature-IL-1β in the THP-1-derived M were increased by ATP or nigericin, and the levels of these proteins were decreased with LAA treatment compared to ATP or nigericin in THP-1-derived M .Meanwhile, Figure 6E indicated that LAA treatment suppressed ATP-or nigericin-induced expression of IL-1β in the culture supernatant of THP-1-derived M s (p < 0.01).LAA treatment alone was not significant for these indicators (Figures 6D, E).This finding suggested that LAA can inhibit NLRP3 inflammasome activation independently of antibacterial activity.

LAA inhibited TLR signaling activation
Staphylococcus aureus can activate the TLR2 signaling pathway through NF-κB and MAPK to upregulate inflammatory gene expression.Next, we assessed the in vitro anti-inflammatory activities of LAA in THP-1-derived M s that were activated by S. aureus.As shown in Figure 7A, the p-JNK and p-p38 levels, Frontiers in Microbiology 08 frontiersin.orgbut not the p-ERK level, in THP-1-derived macrophages were increased by S. aureus, whereas LAA inhibited the S. aureusinduced phosphorylation of JNK and p38 MAPK in THP-1-derived M (Figure 7A).As shown in Figure 7B, S. aureus treatment increased p-JNK and p-p38 levels but not ERK levels in the lungs of the mice.In contrast, LAA treatment markedly reduced JNK and p38 phosphorylation, but not ERK phosphorylation (Figure 7B).These results showed that LAA reduced JNK and p38MAPK signaling activation in macrophages and in S. aureus-induced pneumonia mice.We found that the phosphorylation of NF-κB was induced by S. aureus, suggesting that the NF-κB signaling axis was activated (Figure 7C).Interestingly, LAA inhibited the S. aureus-induced phosphorylation of NF-κB in S. aureus-induced THP-1-derived M s (Figure 7C).Moreover, LAA treatment inhibited the phosphorylation of NF-κB in the lungs of pneumonia mice compared with that in the lungs of S. aureus-treated mice  (Figure 7D).These results showed that LAA had an antiinflammatory effect by reducing NF-κB in S. aureus-induced macrophages and in mice with pneumonia.
To further prove the inhibitory effect of LAA on the TLR signaling pathway, the TLR2 activator Pam3CSK4 was used for in vitro experiments.As shown in Figures 7E-G, the levels of IL-1β, TNF-α, and IL-6 in the culture supernatant of THP-1-derived M s were greater in the Pam3CSK4 group than in the control group, and the levels of these proinflammatory cytokines were lower in the LAA group than in the Pam3CSK4 group (p < 0.01).Moreover, as shown in Figure 7H, LAA treatment suppressed the Pam3CSK4induced increase in p-p65 and TNF-α levels in the THP-1-derived M s.LAA treatment alone was not significant for these indicators (Figures 7E-H).This finding suggested that LAA has an inhibitory effect on the S. aureus-induced TLR signaling pathway in vitro.

Effects of LAA on organ damage
To investigate the organ damage of the LAA during the time of the subject, the histology of vital organs (heart, liver, spleen, lung, and kidney) was examined by H&E.No obvious signs of damage, such as inflammation, necrosis, pyknosis, polymorphonuclear infiltration, or interstitial hemorrhage, were observed in any of the examined organs, including the heart, liver, spleen, lung, and kidney, after LAA treatment (Figure 8).Our Frontiers in Microbiology 10 frontiersin.orgresults suggest that LAA has no significant organ damage to mice within 3 days.

Discussion
Natural compounds have the advantages of biological activity and diversity and have been historically recognized as major sources of traditional medicines and new drug discovery (Boufridi and Quinn, 2018;Ekiert and Szopa, 2020).Natural products have been used since antiquity as traditional medicines and in the modern era.Increasing evidence indicates that natural products play a crucial role in the treatment of S. aureus pneumonia (Shaukat et al., 2019;Yao and Sun, 2019;Wu Y. X. et al., 2021).Notably, these drugs displayed multitargeting properties and lower systemic toxicity.Hematoxylin and eosin staining images of organs including the heart, liver, spleen, lung, and kidney (×20).The mice were intraperitoneally injected with either 0.9% saline or LAA (at dosages of 10, 20, or 30 mg/kg) twice daily, consistent with the drug administration protocol used in the infection model.Seventy-two hours after administration, the heart, liver, spleen, lung, and kidney tissues of the mice were collected, fixed, sliced, and stained.
Licochalcone A is isolated from Glycyrrhiza inflata, which is widely used clinically in traditional Chinese medicine.A previous study demonstrated that 20-80 mg/kg LAA had obvious antiinflammatory effects on LPS-induced acute lung injury (Chu et al., 2013).Our previous studies and other previous studies have found that LAA inhibits S. aureus activity and the secretion of enterotoxins A and B by S. aureus (Qiu et al., 2010;Shen et al., 2015).However, it is unclear whether LAA plays a vital role in the treatment of S. aureus infection, especially S. aureus pneumonia; this leads to the following question, 10.3389/fmicb.2024.1369662

Conclusion
In summary, network pharmacology and experimental results showed that LAA protected against S. aureus pneumonia in mice by inhibiting NF-κB, JNK, p38MAPK, and NLRP3-mediated inflammation.The results showed that LAA is a potential agent for the treatment of S. aureus pneumonia.

FIGURE 1
FIGURE 1 Effects of LAA on S. aureus-induced mouse pneumonia.(A) The effects of LAA on the pathology and histopathology of mice with S. aureus pneumonia (stained with HE, ×10 or ×40).(B) CFU counts obtained from lung tissue homogenates.(C,D) Inflammatory cellular responses in the lungs of S. aureus-infected mice.BALB/c mouse lungs were lavaged.Total inflammatory cells (C) and the number of neutrophils (D) were enumerated in the BALF.*p < 0.05 and **p < 0.01 between the indicated groups.

FIGURE 2
FIGURE 2 Effects of LAA on proinflammatory cytokines in THP-1 cells, blood and BALF after S. aureus infection.THP-1 cells were treated with S. aureus in the absence or presence of LAA (1 or 2 µg/mL) for 6 h.The levels of TNF-α (A), IL-6 (B), and IL-1β (C) in the culture supernatant of THP-1 cells were tested by ELISA.The levels of TNF-α (D,G), IL-6 (E,H), and IL-1β (F,I) in the BALF and serum were detected by ELISA at 72 h after LAA treatment.n = 4 in each group.*p < 0.05 and **p < 0.01 between the indicated groups.

FIGURE 3
FIGURE 3The targets of LAA were collected to explore drug-disease relationships.(A) Two-dimensional structure of the LAA structure diagram.(B) Venn diagram of LAA and S. aureus pneumonia.The blue part represents LAA, and the yellow part represents S. aureus pneumonia.(C) Diseasedrug-active ingredient-key target network.(D) PPI network of potential targets of LAA for S. aureus pneumonia treatment.(E) Bar chart of 16 core genes.

FIGURE 4 Gene
FIGURE 4 Gene Ontology and KEGG pathway enrichment of candidate targets for LAA in S. aureus pneumonia.(A) Bubble graph of the GO functional annotation of potential targets of LAA.The top 15 biological process BP, CC, and MF categories were ranked on the basis of -logP values.(B) Bubble graph of the top 41 pathways based on KEGG enrichment analysis for potential targets of LAA.

FIGURE 6
FIGURE 6 Licochalcone A reduced S. aureus induced activation of the NLRP3 inflammasome in THP-1 cells and mouse lung tissue.(A) NLRP3, ASC, caspase-1, IL-18, and IL-1β protein expression levels were evaluated by Western blotting in THP-1 cells.(B) The concentrations of LDH released by THP-1-derived M s were determined using an LDH assay kit to evaluate cell integrity.n = 3 in each group.*p < 0.05 between the indicated groups.(C) Mice were treated with LAA for 1 h before S. aureus infection.The protein expression of ASC, caspase-1, IL-18, and IL-1β in the lung tissue was measured by Western blotting.THP-1-derived M s were primed with LPS and then stimulated with LAA for 6 h with or without the NLRP3 inflammasome activator nigericin or ATP for 0.5 h before the end of the experiment.(D) NLRP3, caspase-1 and IL-1β protein expression levels were evaluated by Western blotting.(E) The levels of IL-1β were tested by ELISA.n = 4 in each group.**p < 0.01 between the indicated groups.

FIGURE 7
FIGURE 7 The effect of LAA on MAPK and NF-κB pathway activation.(A) The levels of p-JNK, p-p38, and p-ERK in THP-1 cells were evaluated by Western blotting.(B) The levels of p-JNK, p-p38, and p-ERK in the lung tissue.(C) p-p65 protein expression in THP-1 cells.(D) p-p65 protein expression in lung tissues.(E-G)THP-1-derived M s were treated with the TLR2 activator Pam3CSK4 in the absence or presence of LAA for 6 h.The levels of IL-1β (E), TNF-α (F), and IL-6 (G) were tested by ELISA.n = 4 in each group.**p < 0.01 between the indicated groups.(H)The expression levels of p-p65 and TNF-α were evaluated by Western blotting.

TABLE 1
Minimum inhibitory concentrations and minimum bactericidal concentrations of LAA and seven common antibiotics for seven strains of S. aureus.

TABLE 2
Different binding energies of LAA with five selected targets.