Extraction of Hyssopus cuspidatus volatile oil

H. cuspidatus was collected from Tacheng City, Xinjiang Uygur Autonomous Region of China and identified by Prof. Jin-Bo Fang at Huazhong University of Science and Technology, Wuhan, China. A voucher herbarium specimen has been deposited at the affiliated Pharmacognosy Laboratory (No. 20,191,001). The air-dried H. cuspidatus was crushed and soaked in distilled water overnight, followed by continuous extraction for 9 h in a volatile oil extractor. HVO was obtained with anhydrous sodium sulfate, and stored at 4 °C protected from light.

Animals

Female BALB/c mice (6 weeks, 20 ± 2 g body weight) were purchased from Beijing SiPeiFu Biotechnology Co., Beijing, China [SYXK (Jing) 2019-0010] and housed under Individual Ventilated Cages conditions. Pathogen-free male Sprague–Dawley rats (6 weeks, 200 ± 20 g body weight) and male C57BL/6 mice (10 weeks, 20 ± 2 g body weight) were purchased from Hubei Biont Biological Technology Co., Wuhan, China [SYXK (E) 2021-0027]. All animals were raised at the Experimental Animal Center of the Huazhong University of Science and Technology (HUST) and provided with standard rodent chow and water ad libitum under controlled environment (25 ± 2 °C; 60 ± 5%; 12 h light dark cycle). The experimental protocol was approved by the Animal Ethics Committee at Tongji Medical College of HUST (IACUC No. 3674).

Preparation of HVO-containing serum

After 1 week of adaptive feeding, the Sprague–Dawley rats were randomly divided into two groups (n = 4). The control group received 0.5% sodium carboxymethyl cellulose and the HVO-group received 2.4 mg/kg of HVO suspended in 0.5% sodium carboxymethyl cellulose solution (fivefold the clinically equivalent dose of HVO used in Uygur medicine). All drugs were administered via gavage for three consecutive days, twice a day. Consequently, rats were administered a daily dosage of 4.8 mg/kg of HVO (tenfold the clinical equivalent dose). One-hour after the last administration, rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium, and blood samples were collected from the abdominal aortas and centrifuged at 3000 rpm for 15 min. The supernatant was inactivated at 56 °C for 30 min filtered using a 0.22 μm membrane filter and then stored at − 80 °C until use [15].

Ingredients identification of the serum of HVO-treated rats by UPLC-QE-Orbitrap-MS

The serum (200 μL) was treated with 1200 μL acetonitrile (Sinopharm Chemical Reagent Co. China) and vortexed for 30 s. After centrifuging at 13,000 rpm (4 °C) for 15 min, 1300 μL of supernatant was dried under nitrogen. The residue was re-dissolved in 200 μL methanol (Sinopharm Chemical Reagent Co. China), vortexed for 30 s and centrifuged at 13,000 rpm (4 °C) for 15 min. The supernatant was analyzed by UPLC-QE-Orbitrap-MS using an Ultimate 3000 UPLC coupled with a high-resolution Q Exactive Orbitrap tandem mass spectrometer (Thermo Fisher Scientific Inc., Germany). Chromatographic separation was performed on a Hypersil GOLD column (100 × 2.1 mm, 3 μm) at 25 °C. The mobile phase consisted of acetonitrile (A) and 0.1% (v/v) formic acid (Sigma Aldrich) aqueous solution (B), and the gradient program was set as follows: 0–2 min, 5% A; 2–14 min, 5–95% A; 14–19 min, 95% A; 19–19.1 min, 95–5% A; and 19.1–25 min, 5% A. The flow rate was 0.25 mL/min, and the injection volume was 5 μL. The mass spectrometer was operated in both positive and negative ion modes, and the mass range was set to 100–1500 Da. The MS parameters were set as follows: ion source, electrospray ionization; spray voltage, 3200 V; capillary temperature, 300 °C; sheath gas, 40.00 Arb; aux gas, 8.00 Arb; max spray current, 100 µA; probe heater temperature, 275 °C. Data analysis was performed by the Xcalibur v4.1.50 software (Thermo Fisher Scientific Inc.), and compound identity was established by comparing the retention time, mass and fragment ions with literature values.

Pathways prediction based on network pharmacology

Targets of the 41 prototype components in HVO-containing serum were predicted using the SwisstargetPrediction database (http://swisstargetprediction.ch/) and normalized by UniProt (https://www.uniprot.org/). Targets associated with SRA were searched from Genecard (https://www.genecards.org/) and OMIM (https://omim.org/) databases, using the keyword “steroid resistant asthma”. The protein-protein interaction network was created by STRING database (https://cn.string-db.org/) and visualized using Cytoscape 3.9.1 software. The topological parameters in the network were calculated using Network Analyzer software plug-in for Cytoscape. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were used for functional annotations and performed in the DAVID database (https://david.ncifcrf.gov/).

Molecular docking

The PDB database (https://www1.rcsb.org/) was searched to obtain molecular structure files of the protein targets MPO (PDB ID: 5MFA), NE (PDB ID: 5ABW), IL-17 (PDB ID: 8USS) and IL-8 (PDB ID: 4XDX). 2D-structures of the 41 prototype compounds identified in HVO-containing serum as ligands were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and Chem3D software was applied to optimize molecular mechanics for the optimal conformations and conversion into 3D-structures. Subsequently, the Auto Dock Vina v.1.2.0 was used to calculate the affinity between protein targets and the ligands. Finally, the PyMOL software and Discovery Studio 2016 Client were used to visualize the molecular docking results.

SRA mouse model and drug treatment

BALB/c mice were randomly divided into the following groups (n = 5): Control, OVA/LPS, OVA/LPS + Dexamethasone (Dexa), OVA/LPS + Sivelestat (SIV), OVA/LPS + low dose of HVO (HVO-Low), OVA/LPS + middle dose of HVO (HVO-Mid), OVA/LPS + high dose of HVO group (HVO-High), OVA/LPS + SIV + middle dose of HVO group (HVO + SIV). Mice were sensitized by intraperitoneal injection of a mixture of OVA (Aladdin; 1.5 mg/kg) and Al(OH)3 adjuvant (Sigma Aldrich; 50 mg/kg) on days 0, 7, and 14, and stimulated intranasally with LPS (LPS from Escherichia coli 0111: B4, Sigma Aldrich; 0.5 mg/kg) on day 21. On days 22–26, the mice were challenged by inhalation of aerosolized 1% OVA for 30 min once a day. For drug treatment, dexamethasone (Sigma Aldrich; 5 mg/kg, i.p.), SIV (Yuanye, Shanghai, China; 100 mg/kg, i.p.) and HVO-Low (0.85 mg/kg, i.g.), HVO-Mid (1.71 mg/kg, i.g.), or HVO-High (3.42 mg/kg, i.g.) were administered 30 min before each OVA challenge, respectively. The HVO doses were determined based on the conversion formula between experimental animals and humans, 1.71 mg/kg of HVO being equivalent to the clinical dose in humans.

Measurement of airway hyper-responsiveness

To evaluate lung function, AHR was measured 24 h after the last challenge. The mice were anesthetized intraperitoneally with 1% pentobarbital sodium, followed by tracheotomy and intubation. Subsequently, the inserted tracheal tube was connected to the ventilator of a FlexiVent system (SCIREQ, Montreal, QC, Canada). Increasing doses of methacholine (0, 3.125, 6.25, 12.5, 25, and 50 mg/mL; Sigma Aldrich) were nebulized using an ultrasonic nebulizer connected to the FlexiVent system to induce bronchoconstriction. Individual peak response values of resistive resistance were recorded after each nebulization for lung function measurement.

Inflammatory cell count

Twenty-four hours after the last challenge, the mice were anesthetized intraperitoneally with 1% pentobarbital sodium. Peripheral blood samples were collected and treated with anticoagulants. Bronchoalveolar lavage fluid (BALF) was also collected and divided into two parts. The first was used for cytokine analysis, and the second was applied in cell counting using a hemocytometer under light microscope. Differential cell counts in the peripheral blood and BALF were determined using Wright–Giemsa staining. Relative percentages of eosinophils and neutrophils were recorded.

Enzyme-linked immunosorbent assay

Peripheral blood samples and the first BALF were centrifuged at 3500 rpm for 15 min and 2000 rpm for 10 min at 4 °C, respectively. The supernatant was stored at − 80 °C for cytokine measurement. Lungs were isolated and homogenized in phosphate-buffered saline, and the homogenate was centrifuged at 6000 × g for 10 min at 4 °C. The IL-17 (Mlbio), IL-8 (Mlbio), CXCL1 (Elabscience), and CXCL2 (Elabscience) levels in the BALF, serum, and lung tissues were determined using commercial ELISA kits according to the manufacturer’s protocol.

Lung histology

The left lung lobes of the mice were fixed in 4% paraformaldehyde, paraffin-embedded and 7-µm sections were cut. The extent of cell infiltration and mucus production in the lungs was evaluated using hematoxylin and eosin and periodic acid-Schiff staining, respectively.

Western blot

The lung tissues were lysed in ice-cold RIPA lysis buffer containing protease (Sigma Aldrich) and phosphatase inhibitors (Sigma Aldrich) at 4 °C for 30 min, followed by centrifugation at 14,000 g for 30 min to collect the protein supernatant. Protein content was determined by bicinchoninic acid assay (Biosharp). The samples were then separated on SDS-PAGE and transferred to PVDF membranes (Sigma Aldrich). After blocked with 5% skim milk for 2 h, the membranes were incubated with rabbit primary antibodies at 4 °C overnight: p-JNK (Beyotime), JNK (Beyotime), p-P38 (Absin), P38 (Beyotime), p-ERK (Cell Signaling Technology) and β-actin (Abbkine), ERK (Beyotime). The membranes were washed in TBST and incubated with anti-rabbit secondary antibodies (DaiAn) for 1 h at room temperature, and then washed and photographed using an imaging analysis system (GE Healthcare, Little Chalfont, UK). The mean grey values were analyzed and normalized by ImageJ software.

Immunohistochemistry staining

After de-paraffin, antigen retrieval, being blocked endogenous peroxidase nonspecific binding, lung tissue slides were incubated with anti-MPO (1:100; Beyotime) antibody, anti-citH3 antibody (1:200; Abcam), and anti-NE antibody (1:200; Abcam) at 4 °C overnight. After wash, the slides were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bosterbio) for 1 h at room temperature and stained with diaminobenzidine (ZSGB-BIO). Cell nuclei were counterstained with hematoxylin. Images were captured under light microscope, followed by analysis using the ImageJ software.

Isolation of mice neutrophils and differentiated HL-60 cell culture

Bone marrow cells from the femurs and tibias of the male C57BL/6 mice were flushed in sterile phosphate-buffered saline, and filtered through a 70 µm cell strainer. Neutrophils were then separated from bone marrow cells by plating 1 mL of the cell suspension onto a Percoll (Biosharp) gradient consisting of 3 mL of 78% Percoll, 3 mL of 62.5% Percoll, 2 mL of 55% Percoll to 2 mL of 55% Percoll, followed by centrifugation at 900 × g for 30 min at 10 °C. The neutrophil layer between 78% and 62.5% Percoll was collected, washed twice with phosphate-buffered saline, and resuspended in serum-free RPMI 1640 medium.

The HL-60 cell line was obtained from the Cell Bank of Wuhan University, China Center for Type Culture Collection (CCTCC; Wuhan, China). HL-60 cells were cultured in an RPMI-1640 medium containing 10% FBS, streptomycin (100 U/mL), and penicillin (100 U/mL), and maintained in an incubator in a humidified atmosphere containing 5% CO2 at 37 °C. Neutrophil-like differentiated HL-60 were induced by adding all-trans-retinoic acid (Sigma Aldrich; 0.1, 1, or 10 µM) to the culture media. Giemsa staining and trypan blue (0.08%) staining were used to detect the differentiation effects under different concentration of all-trans-retinoic acid and induction time.

Flowcytometry analyses of differentiated HL-60 cells

Single-cell suspension of differentiated HL-60 cells as described above was obtained. Cells were dyed with flow cytometric antibodies for neutrophils (human: CD11b + CD15 +). Samples were analyzed on a BD FACSVerse 3L8C cytometer (San Jose, CA, USA). Data were processed with FlowJo software V10 (BD Biosciences, San Jose, CA, USA). PE-anti-human-CD11b-antibody and FITC-anti-human-CD15 were obtained from Biolegend.

In vitro neutrophil extracellular traps formation

Primary mouse neutrophils and neutrophil-like differentiated HL-60 cells were seeded onto coverslips pretreated with 100 µg/mL poly-L-lysine at 4 °C overnight in 24-well plates (1 × 106 cells/well) and cultured in serum-free RPMI 1640 medium. Neutrophil extracellular traps were induced with PMA (100 nM, 4 h) in the presence or absence of HVO-rat serum (tenfold dilution with culture medium) or SIV (20 µM), and a negative control was set up.

Immunofluorescence staining

The culture supernatants were removed, and the cells were washed twice with phosphate-buffered saline. The cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Aldrich) for 15 min, and blocked with 5% BSA in PBS for 1 h at room temperature. Cells were incubated with anti-MPO (1:100; Beyotime) antibody, anti-histone H3 (citrulline R2 + R8 + R17) antibody (1:200; Abcam) and anti-Neutrophil Elastase antibody (1:200; Abcam) at 4 °C overnight, washed with PBS, and incubated with FITC -conjugated goat anti-rabbit IgG (1:200; Abbkine) for 1 h. The washed coverslips were stained with 4′,6-diamidino-2-phenylindole for 4 min and mounted onto slides using mounting media. Fluorescence images were captured using a confocal microscope (Nikon, NIS-Elements 5.4) at 400 × magnification. The integrated densities per image were quantified using the ImageJ software to determine the proportion of NETs formation induced by the neutrophils.

Statistical analysis

GraphPad Prism version 8.0 was used for depictive statistical analysis. Data were presented as mean ± SEM and analyzed by one-way analysis of variance, followed by Bonferroni post-test to evaluate the diversity between two groups. P < 0.05 were considered significance.