P. aeruginosa PAO1 (ATCC 15692) was stored in our laboratory. For all the experiments, PAO1 was grown in LB broth at 37°C with shaking, collected by centrifugation (4,000 rpm for 5 min), and diluted in the appropriate experimental medium.

RAW264.7 cells were obtained from the Cell Bank of the Chinese Academic of Sciences (Shanghai, China) and cultured in DMEM medium (HyClone, Beijing, China) with 10% fetal bovine serum (FBS, TransGen Biotech, Beijing, China) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) (Haimen, China). The cells incubated in a cell incubator at 37°C with humidified air and 5% CO₂.

The viability of RAW264.7 macrophage cells was determined using the CCK8 assay. Briefly, RAW264.7 (1 × 10⁵ cells/mL) were suspended in complete cell culture media, and 150 μL of the cell suspension was cultured in 96-well plates for 24 h. After treatment with paeonol, cell viability was evaluated using the Cell Counting Kit-8 (Sangon, Biotech, Shanghai, China).

The P. aeruginosa-infected macrophages model used in this study was performed as described previously (31). RAW264.7 cells were seeded into 12-well tissue culture plates at a density of 5×10⁵ cells/well and then reached 80-90% confluence. P. aeruginosa were collected and suspended to 10⁸ CFUs/mL in DMEM without antibiotics. RAW264.7 cells were incubated with P. aeruginosa at three multiplicity of infection (MOIs, 25, 50, or 100) in DMEM without antibiotics. After 2 h or 4 h of PAO1 infection, cells were washed with phosphate-buffered saline (PBS), then lysed with 0.5% Triton-X for 15 min at 37 °C. For invasion assay, extracellular bacteria were killed with 200 μg/mL gentamicin for 1 h. The attachment and invasion levels were assessed by bacterial plate count.

At 80%–90% confluence, cells were washed third with PBS, then subsequently incubated with P. aeruginosa of 100 (MOI) in antibiotic-free DMEM for 4 h. Cells were divided into four groups: the first part involved the control group (RAW264.7 cells group) and the P. aeruginosa infection group (PAO1). On this basis, the second part was set up as three parallel groups, namely PAO1 (MOI = 100:1, infection for 4 h) as cells model group, paeonol pretreatment (32 μg/mL, 64 μg/mL and 128 μg/mL, pretreatment for 3 h) + PAO1 group and paeonol (32 μg/mL, 64 μg/mL and 128 μg/mL, treatment for 4 h) + PAO1 treatment group. These concentrations were no bacteriostatic effect, and previous studies confirmed that the MIC of paeonol against P.aeruginosa was greater than 512 μg/ml.

Total RNA was reverse-transcribed to cDNA, and qPCR was carried out by the instruction of commercial kit. Table 1 shows the primers used to amplify lasI/R, rhlI/R, pqsA/R, LasA, LasB, rhlA, rhlC, phzm, phzM, phzH, phzS, and rpoD (reference gene). qPCR data were analyzed to calculate the relative differences in mRNA expression using the 2^-△△CT method.

The macrophages infected with P. aeruginosa (MOI=100:1) for 4 h, then treated with Paeonol at the concentrations of 32 μg/mL, 64 μg/mL and 128 μg/mL. The culture supernatants were harvested and centrifuged at 12,000 g for 5 min, and LDH activity was monitored by an LDH cytotoxicity assay kit (Jiancheng Biotech, Nanjing, China).

The effects of paeonol on the viability or cytotoxicity of macrophages cells were assayed using the Calcein-AM/PI Double Stain Kit (Solarbio, Beijing, China). In brief, RAW264.7 cells were exposed to P. aeruginosa in DMEM without antibiotics and treated with paeonol. The cells washed with PBS three times and stained with a 100 μL Calcein-AM/PI stain solution at 37°C for 15 min. Living cells with green cytoplasmic fluorescence and dead cells with red nuclei were immediately observed by the fluorescence microscope.

To further reveal the role of paeonol on macrophage-mediated phagocytosis, the intracellular killing of P. aeruginosa. Macrophages infected with P. aeruginosa (MOI=25:1 for 4 h) treated with paeonol (32, 64, and 128 μg/mL). Cells were washed with phosphate-buffered saline (PBS) and then lysed with 0.5% Triton-X for 15 min at 37°C. Extracellular bacteria were killed with 200 μg/mL gentamicin for 1 h to evaluate the intracellular bacteria. Attachment and invasion levels were assessed by bacterial plate count.

The macrophages infected with P. aeruginosa (MOI=25:1 for 2 h) were treated with Paeonol (128 μg/ml). The cells were washed with PBS three times, and fixed with 2.5% glutaraldehyde at 4°C overnight. Samples were collected and treated with osmium ferrocyanide for 1 h, dehydrated in graded ethanol concentrations (50%, 70%, 80%, 90%, 95%, and 100%) and 100% acetone, infiltrated, and embedded in epoxy resin. Subsequently, ultrathin sections (60-80 nm) were cut with a diamond knife on a microtome (Leica EM UC7; Leica Corporation, Germany) and were stained with saturated aqueous uranyl acetate and counterstained with 4% lead citrate, and finally observed using HT-7700 TEM (Hitachi, Tokyo, Japan).

Paeonol (32 μg/mL, 64 μg/mL and 128 μg/mL) treated macrophages (infected with P. aeruginosa (MOI=25:1) for 2 h) were washed with PBS, separated to single-cell suspension, and resuspended in staining buffer (BD Biosciences). The cells were stained with F4/80 (Elabscience, E-AB-F0995C), CD86 (Elabscience, E-AB-F0994E), and CD206 (Elabscience, E-AB-F1135D) fluorescently labeled antibody, then detected by a flow cytometer (Beckman coulter, CytoFLEX) and analyzed by the Flowjo software.

The mRNA expression levels of the TLR4/MyD88/NF-κB pathway of Paeonol treated RAW264.7 (infected with P. aeruginosa (MOI=100:1) for 4 h) cells were assayed by qPCR. The gene sequences of TLR4, MyD88, TRAM, NF-κB, IκB, IκBα, IKK-β, p65, p50, TNF-α, IL-1β, IL-6, IL-8, iNOS, COX-2, IL-2, IL-4, and IL-10 were retrieved from NCBI, and the primers of these genes ( Table 2 ) were synthesized by HuaDa Gene company (Beijing, China). GAPDH gene was selected as the reference gene. qPCR reaction was conducted on a LightCycler^®II real-time fluorescent quantitative PCR instrument (Roche, USA) using the PerfectStartTM Green qPCR SuperMix (TransGen Biotech, Beijing, China) following the standard steps. All data output from the qPCR experiments were analyzed using the 2^-ΔΔCT method.

Fifty SPF mice (4 weeks) were obtained from Sibeifu Biotechnology Co. Ltd. (Beijing, China). The animals were housed at 22-25°C on a 12 h day-night cycle, fed standard rodent chow and sterile water ad libitum for 1 week of acclimation. All protocols for animal studies were reviewed and approved by the Animal Ethical Committee of Sichuan Agricultural University (#20210021). The mice were randomly divided into six groups (n=10): the control group, PAO 1 group, PAO 1+Pae (25 mg/kg) group, PAO 1+Pae (50 mg/kg), PAO 1+Pae (100 mg/kg), and CIP (20 mg/kg) group (positive control). The mice were administered the same dose of saline or drugs by intragastric administration for three days. In mice, infection was induced using the previously described method with modifications (32). Briefly, mice were weighed, anesthetized, and then received an intratracheal instillation of PAO 1 (2.5 × 10⁸ CFU) in 20 μl phosphate-buffered saline (PBS) to model the acute infection of PAO 1. The mice were anesthetized and sacrificed after 24 h of infection, and lung tissue samples were harvested rapidly for subsequent analysis. Then the bacterial load, the expression of virulence, and inflammation cytokines were evaluated by PCR.

All values are expressed as mean ± standard deviation (SD). All statistical analyses were performed using SPSS 20.0. Differences between all groups were analyzed using the one-way ANOVA test, and the result with P < 0.05 or P < 0.01 was considered statistically significant.

The viability of RAW264.7 cells was performed in the presence of paeonol (0-512 µg/mL) using the CCK-8 assay. The viability of cells is more than 90% at concentrations of 0-128 µg/mL of paeonol, indicating that paeonol was not cytotoxic to RAW264.7 cells ( Figure 1 ).

The prolonged P. aeruginosa infection has been shown to cause a significant decrease in cell viability, arrest of cell growth, and morphology alternation, ultimately weakening the host’s innate immune system’s defense function and impaired bacterial clearance function. RAW264.7 cells were infected with P. aeruginosa at different MOI (25, 50, 100) to explore the effect of P. aeruginosa infection on bacterial adhesion and invasion. Figures 2A, B indicated that P. aeruginosa could adhere to and invade the cells. As the PAO1 MOI value and infection time increased, the number of P. aeruginosa attaching to and invading macrophages increased ( Figure 2 ).

P. aeruginosa secretes various virulence factors and forms biofilms regulated by the QS system. QS-related genes, including lasI, lasR, rhlI, rhlR, pqsA, and pqsR were analyzed following pretreatment or treatment with paeonol. The results showed that paeonol downregulated the expression of QS-related virulence genes of PAO1, and the therapeutic effect is significantly greater than the preventive effect. The inhibition rate in the presence of paeonol (128 μg/ml) was as follows: lasI 58.95%, lasR 59.20%, rhlI 40.19%, rhlR 41.20%, pqsA 39.33%, pqsR 58.26%. These results demonstrated the anti-infection activity of paeonol against P. aeruginosa-infected macrophages by interfering with QS-mediated related genes expression in a concentration-dependent manner ( Figure 3 ).

P. aeruginosa secretes a variety of virulence factors and forms biofilms, the expression of QS-regulated and QS-virulence genes was detected to assess the anti-infection activity of paeonol against P. aeruginosa-infected macrophages. QS system was analyzed in the macrophages cell infected P. aeruginosa following pretreatment and treatment at the same time with paeonol. The results showed that paeonol downregulated the expression of QS-related virulence genes in PAO1 infected RAW264.7 macrophages, and the therapeutic effect is significantly greater than the preventive effect. The inhibition rate in the presence of paeonol (128 μg/ml) was as follows: lasA 56.47%, lasB 61.81%, rhlA 47.65%, rhlC 56.74%, phzA 18.28%, phzM 7.73%, phzH 45.50% and phzS 35.47% ( Figure 4 ).

Bacterial infection can cause the destruction of the membrane structure of cells, resulting in the release of LDH in the cytoplasm into the culture medium. The detection of cytotoxicity can be achieved by detecting the activity of LDH in the culture medium. The LDH levels were detected by Fe²⁺ - xylenol orange-based kit. Compared with the control (RAW264.7), PAO1 significantly inhibited the proliferation of macrophages cells, but the release of LDH decreased in a concentration-dependent manner by paeonol treatment, and paeonol did not affect the proliferation of macrophages at the experimental concentrations ( Figure 5A ). To directly examine the cell viability or cytotoxicity, RAW264.7 cells were exposed to PAO1 treated without or with paeonol, then labeled with Calcein-AM and PI dyes and immediately observed by fluorescence microscope. Figure 6 illustrates that macrophages cells infected with PAO1 were mainly dead (red, propidium iodide-positive), but treated with paeonol groups were mainly viable cells (green, calcein-positive) and a few dead cells. The results indicated that paeonol within 128 μg/mL is not cytotoxic to RAW264.7 cells ( Figure 5B ).

RAW264.7 cells were infected with PAO1 to determine the bacterial adhesion and invasion. Compared with the PAO1 group, bacteria adhesion on macrophages significantly decreased after paeonol treatment. Further study revealed that paeonol reduced the invasion of PAO1 to RAW264.7 cells ( Figure 6 ).

TEM was performed to detect the ultrastructure of RAW264.7 macrophages infected by PAO1 and treated with paeonol. As shown in Figure 7 , there were no significant changes in the blank control group. In the PAO1 group, P. aeruginosa invaded macrophages, PAO1 was encapsulated by vesicles, part of the vesicle membrane was destroyed, mitochondria were swollen and vacuolized, escaped phagosomes into the cytoplasm, causing nuclear shrinkage, and even cell damage or lysis. In the paeonol(128 μg/mL) treated group, P. aeruginosa invasion was reduced and encapsulated by intact vesicles, the mycelium changed from rod-like to round, the fusion between PAO1 and lysosome increased, the mitochondria slightly swollen and more filamentous, the nucleus of the cell was intact, and the cell injury was reduced.

Compared with the control group, the relative mRNA expression level of IL-1β, IL-6, IL-8, TNF-α, COX2, and iNOS in the PAO1 group was upregulated, respectively. Compared with the PAO1 group, paeonol can attenuate the expression of inflammatory cytokines like IL-1β, IL-6, IL-8, TNF-α, various inflammatory mediators such as COX2, iNOS, and upregulated the relative mRNA expression level of IL-2, IL-4, IL-10. Paeonol treatment significantly prevented the inflammation changes induced by P. aeruginosa in a dose-dependent manner ( Figure 8 ).

Flow cytometry was used to detect macrophage polarization. F4/80 biomarker was used to label macrophages, and CD86/CD206 anti-body was used to check the surface biomarker of macrophages, respectively. The CD86 biomarker significantly was significantly upregulated when macrophages were infected with PAO1. Paeonol significantly decreased the polarization of macrophages by decreeing the CD86 biomarker when the treatment concentrations higher than 64 μg/mL ( Figure 9 ).

TLR4/MyD88/NF-κB pathway is the fetal inflammation pathway for macrophages responding to the bacteria infection. To investigate the effect of paeonol on the TLR4/MyD88/NF-κB signaling pathway, the mRNA expression levels of TLR4, MyD88, TRAM, NF-κB, IκB, IκBα, IKK-β, p65, and p50 were measured by qPCR. In Paeonol treated cells, the mRNA expression levels of TLR4, MyD88, TRAM, NF-κB, IκB, IκBα, IKK-β, p65, and p50 were significantly downregulated, and IκB was markedly higher in comparison with those in the PAO1 group. These results demonstrated that paeonol attenuated P. aeruginosa-induced inflammation as evidenced by decreased expressions of TLR4/MyD88/NF-κB pathway in RAW264.7 cells, data were included in Figure 10 .

Paeonol decreased the PAO1 load in the lung and inhibited the mRNA expression of inflammation cytokines, including IL-1β, IL-6, and TNF-α ( Figures 11 , 12 ). The mRNA expression of IL-4 was increased by IL-4. All three concentrations of paeonol decreased the expression of quorum sensing-related genes, rhlR, LasR, and pqsA ( Figure 13 ).