All animal experiments were performed according to the “Guide for the Care and Use of Laboratory Animals” and were approved by the Institutional Animal Care and Use Committee of Xinxiang Medical University.

Licochalcone A (>98%) was purchased from Must Bio-Technology Co., Ltd. (Chengdu, China). Seven antibiotic drugs (vancomycin, ciprofloxacin, penicillin G, oxacillin, tetracyclines, levofloxacin, and gentamicin) and hematoxylin and eosin (H&E) kits were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). A BCA protein quantitation kit, eECL Western blot kit, RIPA lysis buffer, and LPS were obtained from Beyotime Biotechnology (Haimen, China). The NLRP3 activator ATP (HY-B2176) and TLR2 activator Pam3CSK4 (HY-P1180A) were purchased from MedChemExpress (Shanghai, China). Lactate dehydrogenase assay kit (A020-2-2) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The NLRP3 activator nigericin (Abs819747) was purchased from Absin (Shanghai, China). Phorbol myristate acetate (PMA) was purchased from Sigma-Aldrich. Antibodies against NLRP3 (No. DF7438), IL-1β (No. AF5103), and IL-18 (No. DF6252) were obtained from Affbiotech; antibodies against caspase-1 (14F468) (No. sc-56036) and ASC (F-9) (No. sc-271054) were obtained from Santa Cruz Biotechnology, Inc. (USA), and antibodies against phospho-NF-κB p65 (Ser536, No. AN371), TNF-α (No. AF8208), p-Erk1/2 (No. AF1891), p-JNK (No. AJ516), and p-p38 (No. AM063) were obtained from Beyotime Biotechnology (Haimen, China). Antibodies against GAPDH (No. bs-10900R) were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Mueller-Hinton broth (CA-MHB) was obtained from Hope Bio-Technology Co., Ltd. (Qingdao, China). ELISA kits for the inflammatory cytokines TNF-α (REF 88-7324-22), IL-1β (REF 88-7013-22) and IL-6 (REF 88-7064-22) were purchased from Thermo Fisher Scientific.

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 server¹ and CTD² were used to collect the LAA-related targets. The S. aureus pneumonia-related targets were collected from GeneCards³ (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 database⁴ 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.

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.

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 two-dimensional plan of the docking results was visualized by Discovery Studio 2016 Client.

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, and MRSA 3335) were provided courtesy of Prof. Guo Na of Jilin University, Changchun, China.

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.

THP-1 cells were differentiated to the macrophage (MΦ) phenotype at 160 ng/mL PMA and then challenged with S. aureus (1 × 10⁷ CFU/mL, 2 mL/well) in the absence or presence of LAA for 6 h. The control group did not receive S. aureus.

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 × 10⁹CFU 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.

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).

The serum and BALF from S. aureus-induced pneumonia and culture supernatant from THP-1-derived MΦ 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).

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 room 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.

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.

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.

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.

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 MΦs 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.

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).

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 3.7.2 software 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.

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, the 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 NOD-like receptor signaling pathway-related targets were selected as candidate targets of LAA against S. aureus pneumonia for further experimental validation.

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 two-dimensional 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 pi-alkyl 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.

To survey the inhibitory effect of LAA on activated MΦs, THP-1-derived MΦs 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 MΦs 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 MΦ compared to S. aureus alone. The concentrations of LDH released from THP-1-derived MΦs 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. aureus-treated THP-1-derived MΦs (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 MΦ 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 MΦ. Meanwhile, Figure 6E indicated that LAA treatment suppressed ATP- or nigericin-induced expression of IL-1β in the culture supernatant of THP-1-derived MΦs (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.

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 MΦs that were activated by S. aureus. As shown in Figure 7A, the p-JNK and p-p38 levels, but not the p-ERK level, in THP-1-derived macrophages were increased by S. aureus, whereas LAA inhibited the S. aureus-induced phosphorylation of JNK and p38 MAPK in THP-1-derived MΦ (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 MΦs (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 anti-inflammatory 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 MΦs 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 Pam3CSK4-induced increase in p-p65 and TNF-α levels in the THP-1-derived MΦs. 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.

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 results suggest that LAA has no significant organ damage to mice within 3 days.