The following materials and reagents were purchased: MRSA strain ATCC43300 (General Microbiological Culture Collection Center, Beijing, China); 2,3,5-triphenyltetrazolium chloride (TTC) (Beijing Wanjing Lizhi I Biotechnology Ltd., Beijing, China); linezolid (Aladdin Industrial Corporation, Los Angeles, United States); SB225002 and 4-aminobenzoic hydrazide (ABAH) (TargetMol Chemicals Inc., Boston, United States); APC anti-mouse CD45 antibody (Clone: 30-F11), PE anti-mouse/human CD11b antibody (Clone: M1/70), PerCP/Cyanine5.5 anti-mouse Ly-6G antibody (Clone: 1A8), FITC anti-mouse CXCR2 antibody (Clone: SA044G4), and RBC lysis buffer (BioLegend Inc., San Diego, United States); Alexa Fluor 488 anti-mouse PSGL-1 antibody (Clone: 4RA10) (Thermo Fisher Scientific, Inc., New York, United States); rabbit monoclonal MPO antibody (Abcam Ltd., Cambridge, United Kingdom); rabbit neutrophil elastase antibody (Novus Biologicals Inc., Littleton, United States); Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and DAPI (Wuhan Servicebio Technology Ltd., Wuhan, China); DCFH-DA (Beyotime Biotechnology Ltd., Shanghai, China); Mouse PSGL-1 and CXCR2 ELISA kits (Jingmei Biotechnology Ltd., Yancheng, China); MPO ELISA kit (Jiubang Science & Technology Ltd., Quanzhou, China); RIPA lysis buffer (Lablead Biotechnology Ltd., Beijing, China); and Mouse StarScript III RT Mix with gDNA Remover and 2 × RealStar Fast SYBR qPCR Mix (GenStar Biotech Ltd., Beijing, China).

The Lanata herb was collected from the Dalad Banner of Inner Mongolia Province, China, and identified by Professor Chun-Lin Long (College of Life and Environmental Sciences, Minzu University of China). The herb (10 kg) was reflux-extracted with 80 L 75% ethanol (1:8, w/v) for 2 h. The extracting liquid was transferred to the concentration tank; then, the herb remnant was reflux-extracted for 1 h. The two extraction solutions were concentrated under reduced pressure, and 1.25 kg extract was obtained. The extract rate was 12.5%. The extract was stored at −20°C and dissolved in distilled water to the indicated concentrations before use.

Male ICR mice (23–25 g, 5 weeks old) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (SCXK 2019-0010) and housed in a room with stable temperature (22°C ± 2°C), humidity (50% ± 10%), and 12 h/12 h light/dark cycle. All mice had access to free water and food and underwent one-week accommodation before experiments. All procedures were approved by the Biological and Medical Ethics Committee, Minzu University of China (approval no. ECMU2023001AO).

After the mice were anesthetized with 1% pentobarbital sodium (50 mg/kg), hair was removed from a 3 cm × 3 cm area on the back of anesthetized mice and a skin area of 5-mm-diameter area was cut. Then, 5 μL of the 2 × 10⁸ CFU/mL MRSA bacterial solution (0.9% saline) was added to the wound. Mice in the sham group were subjected to the same surgical procedures without MRSA.

The skin wound MRSA infection mice were divided into the following five groups: MRSA (model, vehicle), MRSA + Lanata-L (Lanata low-dose, 2 g/kg herb), MRSA + Lanata-M (Lanata medium-dose, 4 g/kg herb), MRSA + Lanata-H (Lanata high-dose, 8 g/kg herb), and MRSA + Linezolid (linezolid, 0.12 g/kg). All drugs with distilled water as the vehicle were administered by oral gavage immediately after infecting with MRSA. Mice in the sham and the control groups were given an equal volume of vehicle. Mice in all groups were administered five times (0 day, 1 day, 2 days, 3 days, and 4 days).

After the mice were anesthetized, 3 mL air sterilized through a 0.22-μM filter was injected subcutaneously on the dorsal region of anesthetized mice to generate an air pouch, which was then injected with 1 mL 1 × 10⁶ CFU/mL MRSA bacterial solution (0.9% saline) or 1 mL of 0.9% saline (control group).

The air pouch MRSA infection mice were divided into two groups: MRSA (model, vehicle) and MRSA + Lanata (Lanata, 4 g/kg herb). Lanata and distilled water as the vehicle were administered by oral gavage immediately after infecting with MRSA. Mice in the control group were given an equal volume of vehicle. Mice in all groups were administered once.

A metal foil with an inner diameter of 9 mm was placed over the wound as a reference for area measurement, and ImageJ software was used to calculate the area of photographed wounds. The healing rate of infected wounds was calculated according to the following formula: healing rate of infected wound = (wound area 0 day post-operation − wound area n days post-operation)/wound area 0 day post-operation × 100%.

The zone of growth inhibition test was performed according to previous reports (Moussa et al., 2013). The concentrated extract of Lanata was dissolved in the tryptic soy broth (TSB) liquid medium to a concentration of 200 mg/mL, and serial dilutions of Lanata (100, 50, 25, 12.5, and 6.25 mg/mL) were prepared in a 96-well plate in a maximum volume of 100 μL of TSB. The 100 μL 1 × 10⁶ CFU/mL MRSA bacterial solution was added to each well. Here, 200 μL TSB was used as negative control, while 100 μL of the TSB +100 μL 1 × 10⁶ CFU/mL MRSA bacterial solution was used as positive control. After incubation at 37°C for 24 h, 20 μL of the 0.5% TTC solution was added to each well, followed by incubation at 37°C for 24 h and OD measurement at 485 nm.

Blood was collected from the orbital sinus of anesthetized mice at 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h (skin wound) or at 4 h, 6 h, 12 h, and 24 h (air pouch) post-MRSA infection. A volume of 20 μL blood was added to 1 mL of 1 × red blood cell (RBC) lysis buffer and incubated at 37°C protected from light for 5 min, followed by centrifugation at 350 g/min for 5 min. The supernatant was discarded, and the cell pellet was washed with PBS and incubated with the APC anti-mouse CD45 antibody, PE anti-mouse CD11b antibody, and PerCP/Cyanine5.5 anti-mouse Ly-6G antibody. After washing with PBS, the cells were analyzed on a FACSCelesta (BD, America), and data were analyzed with FlowJo v10.

Mice were sacrificed following blood collection, and hind limbs were separated. The bone marrow cavity was rinsed with 1 mL PBS for three times to obtain the bone marrow cells, which were analyzed by flow cytometry, as described above.

At 4 h, 6 h, 12 h, and 24 h post-infection, 1 mL solution (PBS containing 5.4 mM EDTA-Na₂) was injected into the air pouch for lavage, and the lavage fluid was collected and centrifuged at 500 g/min for 10 min. The supernatant was discarded, and the cells in the air pouch were collected and analyzed by flow cytometry, as described above.

At 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h post-infection, the mice were sacrificed and a 2-cm × 2-cm wound skin was obtained to perform H&E staining and immunohistochemistry, according to previous reports (Yuan et al., 2014).

At 6 h, 12 h, 24 h, and 48 h post-MRSA infection, skin samples of 2 cm × 2 cm around the wound were obtained and added to 1 mL of 0.9% saline and immediately homogenized. At 4 h, 6 h, 12 h, and 24 h post-infection, the lavage fluid in the air pouch was collected and serially diluted in saline.

Serially diluted solutions of skin homogenates and air pouch lavage fluids were spread on a TSA plate and incubated at 37°C for 24 h to conduct colony count.

At 6 h post-infection, skin samples of 2 cm × 2 cm around the wound were obtained, and RIPA lysis buffer was added and homogenized immediately. Lysates were centrifuged at 12,000 g/min for 10 min and sent to RayBiotech Guangzhou Co., Ltd. to detect the concentration of 200 proteins. A p-value < 0.05 and fold change >2 or <0.5 were set as thresholds for significantly differentially expressed proteins (DEPs). Enrichment analysis of DEPs was conducted using the GO (http://www.geneontology.org/) and KEGG (http://www.kegg.jp/) databases.

At 4 h, 6 h, 12 h, and 24 h post-MRSA infection, cells in the air pouch were washed with PBS and incubated with the APC anti-mouse CD45 antibody, PE anti-mouse CD11b antibody, PerCP/Cyanine5.5 anti-mouse Ly-6G antibody, and DCFH-DA. After washing with PBS, the cells were analyzed by flow cytometry, as described above.

At 4 h, 6 h, 12 h, and 24 h post-infection, 5 μL cell suspension was evenly distributed on a slide in an area of 1 cm², blocked with 5% BSA overnight at 4°C and incubated with the rabbit anti-neutrophil elastase (NE) antibody for 4 h at room temperature, washed with PBS, and incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody for 1 h and DAPI for 15 min at room temperature. Images were captured with the FV3000 confocal system (Olympus, Japan).

At 6 h post-infection, neutrophils in the air pouch were collected in both the MRSA and MRSA + Lanata groups. Cells were washed with PBS, and total RNA was extracted and quantified. The sequencing library was sequenced on a NovaSeq 6000 platform by Beijing Qinglian Biotech Co., Ltd. Differential expression analysis was performed using the Love et al. (2024) R package. A p-value < 0.05 and fold change >2 or <0.5 were set as thresholds for significantly differentially expressed genes (DEGs). Enrichment analysis of DEGs was conducted using GO and KEGG databases.

Neutrophils in the air pouch and blood were collected at 4 h, 6 h, 12 h, and 24 h post-infection. According to the RNA extraction method, total mRNA was extracted with TRIzol, chloroform, isopropanol, ethanol, and other reagents. The total RNA concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific, United States), and reverse transcription was performed with StarScript III RT Mix with gDNA Remover. Results were analyzed using 2 × RealStar Fast SYBR qPCR Mix on LightCycler^® 96. Primers used are presented in Supplementary Table S1.

Skin samples of 2 cm × 2 cm around the wound were obtained at 6 h post-infection. At 4 h, 6 h, 12 h, and 24 h post-infection, neutrophils in the air pouch and blood were collected. P-selectin glycoprotein ligand-1 (PSGL-1) and CXC chemokine receptor 2 (CXCR2) in the skin and myeloperoxidase (MPO) in neutrophils were determined, following the manufacturer’s instructions.

At 4 h, 6 h, 12 h, and 24 h post-infection, cells in peripheral blood and air pouch were washed with PBS and incubated with the APC anti-mouse CD45 antibody, PE anti-mouse CD11b antibody, PerCP/Cyanine5.5 anti-mouse Ly-6G antibody, and FITC anti-mouse CXCR2 antibody or Alexa Flour 488 (AF488) anti-mouse PSGL-1 antibody. After washing with PBS, the cells were analyzed on a FACSCelesta instrument, and data were analyzed with FlowJo v10.

At 0.5 h before MRSA was injected into the air pouch, the CXCR2 inhibitor SB225002 (10 mg/kg) or MPO inhibitor ABAH (40 mg/kg) was intraperitoneally injected into mice. The air pouch MRSA infection mice were divided into the following six groups: the model group (MRSA, vehicle), MRSA + Lanata group (MRSA + Lanata, 4 g/kg herb), MRSA + SB225002 group (MRSA + SB225002, vehicle), MRSA + ABAH group (MRSA + ABAH, vehicle), MRSA + Lanata + SB225002 group (MRSA + Lanata + SB225002, 4 g/kg herb), and MRSA + Lanata + ABAH group (MRSA + Lanata + ABAH, 4 g/kg herb). At 4 h post-infection, immunofluorescence of NETs in the air pouch was performed. At 6 h post-infection, the number of neutrophils and ROS levels in neutrophils from the air pouch were determined.

Statistical analysis was conducted with SPSS 26.0. Comparisons between two groups were assessed using Student’s t-test. Comparisons for three or more groups were analyzed using one-way analysis of variance (ANOVA), followed by a least significant difference (LSD) post hoc test. All data were expressed as mean ± standard deviation (SD). p < 0.05 was considered to be statistically significant.

Considering the therapeutic effect of Lanata on abscess infection, we established a skin wound MRSA infection mouse model to investigate the healing effect of Lanata on infected wounds (Figure 1A). Wound healing of mice in the Sham group was the fastest, being almost completely healed at 72 h post-infection (Figures 1B, C), while healing in the MRSA group was significantly slower due to bacterial infection. Compared with the MRSA group, the healing rate in MRSA + Linezolid and MRSA + Lanata groups increased in different degrees. The healing effect in the MRSA + Lanata-M and MRSA + Lanata-H groups was most prominent, with the largest difference observed at 24 h. However, Lanata had no direct antibacterial activity against MRSA in vitro (Figures 1D, E).

We observed the spatiotemporal dynamics of neutrophil migration during MRSA infection to investigate whether mice treated with Lanata could resist MRSA infection by regulating neutrophils (Figure 2A). At 2 h post-infection, there was no neutrophil infiltration in the infected wound of mice in the MRSA and MRSA + Lanata groups (Figure 3A), and the proportion and number of neutrophils in the bone marrow and the peripheral blood remained homeostatic (Figures 2B–F).

At 6 h, a considerable number of neutrophils began to migrate into the infected wound in the MRSA group (Figures 3A–C) and the proportion of neutrophils in the bone marrow significantly decreased, while the number of neutrophils in the peripheral blood significantly increased (Figures 2B–F), indicating that neutrophils in the bone marrow had been rapidly mobilized into the peripheral blood. Compared with the MRSA group, the number of neutrophils in the infected wound in the MRSA + Lanata group significantly increased, while simultaneously, MRSA and the number of neutrophils in the peripheral blood significantly decreased (Figures 2F, 3A–D).

At 12 h, the number of neutrophils in the infected wound in the MRSA group significantly increased compared with 6 h (Figure 3A), while the proportion and number of neutrophils in the bone marrow and peripheral blood were significantly reduced (Figures 2B–F). Interestingly, the peak in the number of neutrophils in the infected wound in the MRSA + Lanata group was observed in the acute stage of infection, and MRSA and the number of neutrophils in the peripheral blood were significantly lower than those in the MRSA group (Figures 2F, 3A–D).

At 24 h, the number of neutrophils in the infected wound in the MRSA group reached the peak (Figure 3A), while the number of neutrophils in the bone marrow and peripheral blood further decreased (Figures 2B–F). Meanwhile, the number of neutrophils in the infected wound in the MRSA + Lanata group was significantly reduced compared with 12 h and that in the MRSA group, and MRSA was almost completely eradicated, and the number of neutrophils in the bone marrow and peripheral blood began to return to a steady state.

At 48 h, MRSA was almost completely eradicated in the MRSA group, the number of neutrophils was significantly reduced compared with 24 h (Figure 3A), and the number of neutrophils in the peripheral blood and the bone marrow began to return to the steady state (Figures 2B–F). In the MRSA + Lanata group, there was no obvious neutrophil infiltration in the infected wound, and the number of neutrophils in the bone marrow and peripheral blood had returned to a homeostatic state.

Together, the above results suggest that Lanata promoted the rapid migration of neutrophils from the peripheral blood to the infected wound to eradicate MRSA quickly in the acute stage of infection, thus promoting healing of the infected wound.

To explore the mechanism by which Lanata resists MRSA infection, protein expression in the infected wound skin at 6 h post-infection was analyzed by protein chip technology (Figure 4A). Compared with the Sham group, 145 and 55 proteins were upregulated and downregulated in the MRSA group, respectively (Figure 4B). Compared with the MRSA group, 133 and 67 proteins were upregulated and downregulated in the MRSA + Lanata group, respectively (Figure 4C). As shown in Figure 4D, 17 DEPs were identified to be upregulated in the MRSA group compared with the Sham group, and GO enrichment analysis indicated that these proteins were mainly involved in neutrophil migration and leukocyte adhesion to vascular endothelial cells (Figure 4E). Meanwhile, 10 DEPs were identified to be upregulated in the MRSA + Lanata group compared with the MRSA group (Figure 4F), which were mainly involved in leukocyte and neutrophil migration (Figure 4G).

Some DEPs identified in the comparison MRSA vs. Sham overlapped with those identified in the MRSA vs. MRSA + Lanata group, with four main upregulated DEPs (PSGL-1, CXCR2, CXCL2, and CXCL1) identified as the action target of Lanata (Figure 4H). Upregulation of PSGL-1 and CXCR2 in the infected skin was confirmed by ELISA (Figures 4I, J).

In order to obtain the accurate number of neutrophils in the MRSA-infected site and directly study neutrophils, we established an air pouch MRSA infection mouse model. At 4 h, 6 h, 12 h, and 24 h post-infection (Figure 5A), there were almost no neutrophils migrating into the air pouch of mice in the control group (Supplementary Table S2). Compared with the MRSA group, Lanata promoted the migration of neutrophils from the peripheral blood to the air pouch (Figures 5B–G) to quickly eradicate MRSA (Figure 5H).

Next, we measured the ROS levels in neutrophils and NET formation in the air pouch at 4 h, 6 h, 12 h, and 24 h post-infection to explore the effect of Lanata on activation of neutrophils (Figure 6A). As shown in Figures 6B, C, ROS levels in neutrophils in both MRSA + Lanata and MRSA groups showed the same trend of an initial increase, followed by a decrease (of note, levels in the MRSA + Lanata group were higher at 6 h and lower at 12 h post-infection than those in the MRSA group), which was related to the continuous elimination of MRSA. As shown in Figures 6D–F, Lanata promoted NET formation in the air pouch at 4 h and 6 h post-infection. At 24 h, there were almost no NETs left, which was related to the eradication of MRSA (Figure 5H).

We conducted RNA-seq analysis on neutrophils in the air pouch at 6 h post-infection to understand the mechanism by which Lanata regulates neutrophils (Figure 7A). Compared with the MRSA group, 501 and 97 genes were significantly upregulated and downregulated in the MRSA + Lanata group, respectively (Figure 7B). The top 10 upregulated genes with Lanata included CXCR2, PSGL-1, MPO, CXCL2, CXCL1, CXCL5, CXCL3, LFA-1, PAD4, and IL-17A (Figure 7C). GO and pathway enrichment analysis indicated that the DEGs were mainly involved in neutrophil chemotaxis and migration (Figures 7D, E).

To validate the RNA-seq findings, we measured the mRNA and protein expressions of CXCR2, PSGL-1, and MPO in neutrophils regulated by Lanata (Figure 8A). As shown in Figures 8B–H, mRNA and protein expression of CXCR2, PSGL-1, and MPO in neutrophils in the peripheral blood in both MRSA + Lanata and MRSA groups first increased post-infection, followed by a decrease. Of note, mRNA and protein expression of the three genes increased significantly at 6 h post-infection and were lower at 12 h in the MRSA + Lanata group compared with the MRSA group.

Similarly, the expression of CXCR2, PSGL-1, and MPO in neutrophils in the air pouch in the MRSA + Lanata group increased significantly at 6 h post-infection and were lower than those in the MRSA group at 12 h (Figures 9A–G).

To further investigate the role of CXCR2 and MPO in Lanata-promoted migration and activation of neutrophils, we used ABAH and SB225002 to inhibit MPO and CXCR2, respectively (Figure 10A). As shown in Figure 10B, in the absence of treatment, there was no NET formation in the air pouch in the MRSA group at 4 h post-infection, while NETs had already begun to be released in the MRSA + Lanata group. However, after administration of ABAH, there was almost no NET formation in the MRSA + Lanata group. Similarly, in the absence of treatment, ROS levels in neutrophils in the air pouch in the MRSA + Lanata group were significantly higher than that in the MRSA group at 6 h post-infection (Figures 10C, D). However, after ABAH treatment, ROS levels in neutrophils in the two groups were significantly reduced without significant differences between groups.

As shown in Figures 10E, F, the number of neutrophils in the air pouch in the MRSA + Lanata group was significantly higher than that in the MRSA group at 6 h post-infection. However, after administration of SB225002, the number of neutrophils in the two groups was significantly reduced, and no significant differences were observed between groups.