2.1 Materials and reagents
Glucose, mannose, galacturonic acid, rhamnose, galactose, xylose, sodium hydroxide, acetic acid, myeloperoxidase (MPO), LPS (L2280, Escherichia coli 055:B5), dexamethasone (DEX), heparin, hydrogen peroxide, bovine serum albumin, o-dianisidine dihydrochloride, and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl), sodium citrate dihydrate, and HEPES sodium salt were purchased from J.T Baker (PA, USA). Fucose was procured from Acros Organics (NJ, USA). Arabinose was purchased from Tokyo Chemical Industry (Tokyo, Japan). 1-Phenyl-3-methyl-5-pyrazolone (PMP, A11161) was obtained from Alfa Aesar (MA, USA). HNE, an HNE substrate (methoxysuccinyl-Ala-Ala-Pro-Val-pNA), human CG, a human CG substrate (suc-Ala-Ala-Pro-Phe-pNA), and a human PR3 substrate (Boc-Ala-Ala-Vna-Sbzl) were acquired from Enzo Life Sciences (NY, USA). PR3 (539483) was procured from Merck (NY, USA). Zoletil® 50 was purchased from Virbac (Carros, France). Xylazine was obtained from Bayer (Seoul, Korea). Isoflurane was acquired from AbbVie (IL, USA). A protein assay kit (#500-0006) was procured from Bio-Rad (CA, USA). ELISA kits for inflammatory cytokines were purchased from eBioscience (CA, USA). Quant-iT PicoGreen was obtained from Invitrogen (MA, USA). Dextran T standards were acquired from Pharmacosmos (Holbaek, Denmark). Anti-MPO antibody (ab9535) was procured from Abcam (Cambridge, UK).
2.2 Genomic identification of the spikes of P. vulgaris
To confirm the origin of a TCM that was purchased from a Chinese medicine store (Huang-De-An, New Taipei city, Taiwan), the dried spikes of P. vulgaris were identified via ITS sequence and psbA-trnH sequence analyses (see supplementary material). Then, the sequence similarity (99%) was determined and compared with that in the NCBI genome database (accession # JQ669130 and KX347037).
2.3 Preparation of PVAP
Initially, the bioactive crude polysaccharide fraction (10.5 g) of the dried spikes of P. vulgaris (400 g) was prepared as descried previously [25, 26]. Specifically, the crude polysaccharide was dissolved in ddH2O (1:20, w/v) and sequentially filtrated using Vivaspin 20 (MWCO 100 and 300 kDa, GE Healthcare, Little Chalfont, UK), and then three sub-fractions (<100 kDa, 5.2 g; 100–300 kDa, 3.0 g; and >300 kDa, 2.3 g) were obtained. The middle fraction (2.8 g) was re-dissolved in ddH2O (50 mg/mL) and centrifuged at 8000 × g for 10 min, and then the supernatant was adjusted to pH 3.0 using acetic acid (99.8%). The subsequent precipitate (220 mg, PVAP) was obtained via centrifugation at 8000 × g for 10 min. HNE activity assays were then used to monitor the effect of each fraction.
2.4 Physicochemical analysis of PVAP
The carbohydrate, uronic acid, lignin, and protein contents of PVAP were modified and determined as described previously [25, 27]. Briefly, a mixture of 5% (w/v) phenol aqueous solution (30 μL) and concentrated sulfuric acid (150 μL) was added to PVAP or standard (glucose) solution (0.5 mg/mL, 50 μL) and then incubated at 90 °C for 20 min. The products (200 μL) were transferred to 96-well microplates, and their absorbance at 492 nm was monitored. To determine the uronic acid content of PVAP, a sodium tetraborate solution (12.5 mM, 600 μL) was mixed with the PVAP solution (0.5 mg/mL, 100 μL) or with standard (galacturonic acid) solutions (7.81–125 μg/mL, 100 μL) and then incubated at 90 °C for 5 min. Each sample was added into a solution of 0.15% m-hydroxydiphenyl and 0.5% sodium hydroxide aqueous solution (10 μL). The mixture was reacted for 5 min, and its absorbance at 520 nm was measured. The amounts of protein in PVAP were determined following the protocol of the Bio-Rad assay kit, and bovine serum albumin (BSA) was used as standard. Briefly, the PVAP solution (0.5 mg/mL, 10 μL) was mixed with Coomassie brilliant blue G-250 (200 μL) and incubated for 5 min at room temperature. The mixture was analyzed at 595 nm using the ELISA reader. To determine its lignin content, PVAP (6 mg) was dissolved in 30% acetyl bromide solution (in glacial acetic acid, 1.5 mL) and incubated at 70 °C for 1 h. The sample was mixed with 2 M sodium hydroxide (2.7 mL), 0.5 M hydroxylamine HCl (0.3 mL), and glacial acetic acid (10.5 mL), and then the UV absorbance of the mixture was recorded at 280 nm. Alkali lignin (Sigma-Aldrich) was used the standard.
2.5 Monosaccharide composition of PVAP
PVAP (10 mg) was transferred into a sealed ampule and then hydrolyzed using 2.0 M trifluoroacetic acid (2 mL) at 100 °C for 8 h, after which the excess trifluoroacetic acid was removed by vacuum. The product and monosaccharide standards (galactose, glucose, galacturonic acid, arabinose, mannose, rhamnose, xylose, fucose, and glucosamine hydrochloride) were dissolved in ddH2O and conjugated to PMP [26]. The PMP-labeled monosaccharides were analyzed using a Thermo SN4000 HPLC system (MA, USA) equipped with a C18 column (Hypersil™ BDS 5 μm, 4.6 mm × 250 mm, Thermo Fisher) and eluted with a mobile phase of 0.1 M KH2PO4, pH 7.0 buffer solution : acetonitrile (83:17). The flow rate was 1 mL/min, the wavelength for UV detection was 245 nm, and injection volume was 20 μL.
2.6 Amino acid analysis
To determine the amino acid residues to which the carbohydrate was linked, PVAP was subjected to reductive alkaline degradation followed by amino acid analysis [28]. Initially, PVAP (20 mg) was cleaved using an alkaline solution (0.25 M sodium hydroxide containing 0.5 M sodium borohydride, 4 mL) at 45 °C for 6 h. The residue (21 mg) was hydrolyzed using 4 N sulfonic acid at 115 °C for 24 h. After cooling, the mixture was neutralized with pyridine, and its pH was adjusted to 6.80. Then, 4 μM dithiothreitol solution (2 mL) was added to the solution, which was incubated at 37 °C for 1 h, and then sodium tetrathionate (120 mg) was added followed by incubation at 25 °C. After 5 h, the mixture was dried by vacuum, and 0.02 N HCl buffer solution (pH 2.2) was added, after which the amino acid residues of protein were established using a Hitachi L-8900 high-speed amino acid analyzer.
2.7 In vitro NSPs activities assay
The HNE enzyme and selectivity assays (CG and PR3) were conducted using 96-well plates as described previously [25, 29]. Briefly, sample or vehicle solution (50 μL) was mixed with 25 μL of enzyme solution (HNE, 5 μM; CG, 10 μM; and PR3, 30 μM), after which 25 μL substrate solution (HNE, 100 μM; PR3, 50 mM; and CG, 10 mM) was added, and the mixture was incubated for up to 30 min (for NE and PR3) or 60 min (for CG). Chromogenic absorbance at 405 nm was monitored used a Thermo Labsystems Multiskan Ascent reader (MA, USA).
2.8 Animal
Male ICR mice (30–35 g, 5–9 weeks old) were purchased from BioLASCO (Ilan, Taiwan). All animal research procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University (Taoyuan, Taiwan, IDs CGU105-019 and CGU16-079). All mice were acclimated for at least 1 week. Animals were granted ad libitum access to a commercial rodent diet and drinking water in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility of Chang Gung University. Mice were housed under constant light conditions (12-h/12-h light/dark). The room temperature was kept at approximately 25 °C, and the relative humidity was maintained at approximately 60%.
2.9 The animal model of LPS-induced ALI.
Male mice (6–9 weeks old) were randomly divided into five groups (n ≥ 6 in each group) as follows: vehicle, LPS (5 mg/kg), PVAP (125 mg/kg) + LPS, PVAP (250 mg/kg) + LPS, and DEX (10 mg/kg) + LPS. The DEX and LPS solutions were prepared with 10% Tween 80 in sterile PBS. PVAP was dissolved in PBS (100 μL) at a dose equivalent to 125 or 250 mg/kg. Initially, mice were administered PBS, PVAP, or DEX through gavage. After 30 min, mice were anesthetized using a mixture solution of Zoletil® 100 and xylazine (Bayer, Germany) through intraperitoneal injection, and then 50 μL of PBS or LPS (E. coli O55:B5, 5 mg/kg) was administrated via intratracheal instillation. After 6 h, mice were sacrificed, and the left lobes of lung tissues were obtained for MPO and HNE activities assays, as well as immunohistochemical (IHC) and hematoxylin and eosin (H&E) staining. The right lobes were harvested for bronchoalveolar lavage fluid (BALF) collection and wet/dry (W/D) ratio analysis.
2.10 Measurement of lung wet to dry (W/D) weight ratios:
The right lobes of lung tissues were harvested and weighted immediately (wet weight). Tissues were transferred to the oven and dried at 80 °C for 72 h, and the dried tissue weight was recorded.
2.11 BALF collection and analysis
The BALF samples were collected as described previously [25, 29]. Briefly, the right lung tissues were lavaged with PBS (1.5 mL) and then centrifuged at 1000 rpm for 15 min at 4 °C, and the supernatant was collected and stored at −80 °C. The total protein concentration of BALF was determined using Bradford protein assay dye (Bio-Rad, 500-0006) with BSA as the standard. Cytokines (IL-6 and TNF-α) levels in BALF were measured using ELISA kits following the manufacturer's instructions. Cell pellets from BALF were re-suspended in PBS (100 μL) and used to prepare cytospin slides. The neutrophil pictures and counts were recorded at ×100 magnification using a Zeiss PrimoStar microscope (Carl Zeiss, Gottingen, Germany). The number of neutrophils was counted in five random fields. To quantify NET DNA, the amount of dsDNA in BALF was measured using a Quant-iT PicoGreen assay kit per the manufacturer's protocols. In brief, BALF supernatant (100 μL) and standard (lambda DNA) were transferred to a 96-well plates, and then dsDNA staining reagent (100 μL) was added. The mixtures were incubated in the dark for 5 min. The fluorescence absorbance of the mixture was recorded at excitation and emission wavelengths of 485 and 535 nm, respectively.
2.12 Measurement of HNE and MPO activities in the lungs
HNE and MPO activities in lung homogenates were determined as previously described [25, 29]. Lung tissues were homogenized, and the supernatant was collected via centrifugation at 12,000 rpm for 10 min at 4 °C. The supernatant was mixed with substrate solution (0.0005% hydrogen peroxide and 0.167 mg/mL o-dianisidine dihydrochloride in pH 6.0 phosphate buffer) and incubated for 15 min. The absorbance of the mixtures at 460 nm was recorded. The protein concentration of the supernatant was determined using a Bio-Rad assay kit. The protein concentration and MPO activity of samples were calculated using a standard curve derived from commercial BSA and MPO, respectively. The results are presented as MPO units/sample protein concentration. Neutrophil elastase activity was determined as described previously [25]. Specifically, supernatants (20 μL) were transferred to 96- well plates, and substrate solution (500 μM, 80 μL) was added immediately. Following incubation at 37 °C for 6 h, the absorbance at 405 nm was recorded. The amount of HNE in tissues was calculated using the calibration curve prepared with HNE and presented as neutrophil elastase (μg)/sample protein concentration (mg).
2.13 H&E and IHC staining
Lung tissues were fixed with 10% formalin overnight and then embedded in paraffin wax. The lung microsections (six microns) were then processed via H&E staining. IHC staining was conducted by an automatic IHC staining device (Vision BioSystems, Australia) using anti-MPO antibody (1:200, dilution) following the manufacturer's protocols. Images were obtained using an Olympus IX81 microscope (Tokyo, Japan).
2.14 Statistical analysis
All results are presented as the mean ± SEM. Differences between control and treatment groups were evaluated using Student's t-test or one-way ANOVA as appropriate with GraphPad Prism 5 (San Diego, CA, USA). Statistical significance was indicated by P < 0.05.