A fraction of Prunella vulgaris spike extract inhibited neutrophil elastase and protected mice against lipopolysaccharide-induced acute lung injury

Human neutrophil elastase (HNE) is an abundantly expressed neutrophil serine protease that promotes neutrophil invasion and neutrophil extracellular trap (NET) formation, thereby mediating lung tissue destruction and enhancing pulmonary inammation in acute lung injury (ALI). Chemical agents that target HNE and manipulate HNE level homeostasis are desired to prevent or treat ALI.

development of chemical agents that target HNE and manipulate HNE level homeostasis may be crucial for preventing or treating ALI.
Drug repositioning is a strategy that re-evaluates existing drugs for new indications, and it has the advantages of rapid incubation and reduced costs in drug research and development [9]. Traditional Chinese medicines (TCMs) have a long history of use, and considerable evidence supports their use for treating and preventing diseases [10][11][12]. Therefore, TCMs are excellent resources for drug repositioning research.
To identify new HNE inhibitors, 75 water extracts of TCMs were prepared in the present study, and their inhibitory effects on HNE activity were evaluated. Of these, the water extract of P. vulgaris spikes (PVW) exhibited inhibitory effects against HNE activity. PVW was subsequently fractionated to isolate the bioactive fractions and/or components that inhibit HNE activity. The effects of the active components of P. vulgaris on lipopolysaccharide (LPS)-induced ALI in mice were also evaluated.
2.2 Genomic identi cation of the spikes of P. vulgaris To con rm 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 identi ed 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).

Physicochemical analysis of PVAP
The carbohydrate, uronic acid, lignin, and protein contents of PVAP were modi ed and determined as described previously [25,27]. Brie y, 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. Brie y, 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.

Monosaccharide composition of PVAP
PVAP (10 mg) was transferred into a sealed ampule and then hydrolyzed using 2.0 M tri uoroacetic acid (2 mL) at 100 °C for 8 h, after which the excess tri uoroacetic 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 ddH 2 O 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 KH 2 PO 4 , pH 7.0 buffer solution : acetonitrile (83:17). The ow rate was 1 mL/min, the wavelength for UV detection was 245 nm, and injection volume was 20 μL.

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.

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%.

The animal model of LPS-induced ALI.
Male mice (6-9 weeks old) were randomly divided into ve 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 sacri ced, 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 uid (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.

BALF collection and analysis
The BALF samples were collected as described previously [25,29]. Brie y, 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 magni cation using a Zeiss PrimoStar microscope (Carl Zeiss, Gottingen, Germany). The number of neutrophils was counted in ve random elds. 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 uorescence 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]. Speci cally, 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).

H&E and IHC staining
Lung tissues were xed with 10% formalin overnight and then embedded in para n 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).

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 signi cance was indicated by P < 0.05.

Isolation and identi cation of PVAP from P. vulgaris spikes
To isolate new inhibitors of HNE from TCMs, 75 water extracts of TCMs were prepared, and their inhibitory effects on HNE activity were evaluated. Among them, PVW exhibited moderate inhibitory effects against HNE activity with an IC 50 of 8.08 ± 1.28 μg/mL. Therefore, PVW was chosen as a candidate to identify new anti-HNE agents.
Using a bioactivity-guided fractionation protocol, a crude polysaccharide fraction of PVW was precipitated, and it exerted more potent inhibitory effects on HNE. This fraction was further to fractionated via molecular weight and acetic acid precipitation. The collected precipitate (PVAP) displayed the most potent inhibitory effects against HNE (Table 1).

PVAP treatment attenuates LPS-induced lung injury in mice
Because PVAP potently and speci cally inhibited HNE in vitro, PVAP was further examined to determine its effects on an LPS-induced ALI in mice. To determine changes in capillary permeability and the occurrence of lung tissue swelling, the W/D ratio of the lungs was recorded. The results indicated that the lung W/D ratio was signi cantly increased after LPS exposure, and this value was reversed by PVAP pretreatment (Fig. 1A). The morphological and histological results also con rmed that PVAP pretreatment improved several pathological features, including in ammatory cell in ltration, alveolar cell destruction, and thickening of the alveoli, induced by LPS treatment (Fig. 1B). These results emphasize the protective effects of PVAP against LPS-induced ALI.

PVAP suppresses HNE and MPO activities and inhibits neutrophil in ltration and NET formation
Neutrophil in ltration and HNE and MPO expression are associated with the progression of ALI [1,30]. To study the effects of PVAP on ALI, BALF and lung tissues of mice were collected, and the protective effects of PVAP were determined by analyzing total protein content and MPO and HNE activities. Our data indicated that MPO and HNE activities were signi cantly augmented in the lung tissue of LPS-treated mice compared with that in control mice. By contrast, pretreatment with PVAP suppressed MPO and HNE activities in a dose-dependent manner ( Fig. 2A,B). Indeed, the total protein levels and neutrophil counts in BALF and IHC staining results suggested that PVAP treatment dramatically decreased neutrophil in ltration (Fig. 2C-E).

PVAP decreases NET formation and cytokine expression in BALF
NETs comprise an extracellular meshwork of decondensed chromatin and antimicrobial proteins that were detected during the progression of ALI, and they promoted LPS-induced synthesis and the release of IL-6 and TNF-α [4,31]. Furthermore, NET formation was modulated by MPO and HNE [32,33]. NET formation and IL-6 and TNF-α levels in BALF were measured using commercial kits. The results demonstrated that NET formation and IL-6 and TNF-α expression were meaningfully increased in the BALF of LPS-treated mice compared with the ndings in control mice, whereas these ndings were ameliorated by PVAP pretreatment (Fig. 3A-C).

Discussion
Polysaccharides, which are macromolecules composed of long chains of monosaccharide units, are the key bioactive components of TCMs, and they possess immunomodulatory, antitumor, anti-in ammatory, hypoglycemic, anti-aging, and antioxidant effects [34][35][36][37]. In our previous study, the polysaccharide KSWP was isolated from Kochia scoparia fruit. KSWP exhibited selective inhibitory effects against HNE with an IC 50 of 3.74 ± 0.46 µg/mL, thereby attenuating LPS-mediated ALI [25]. The present study results suggest that PVAP is slightly more potent than KSWP, and it also appears to have stronger protective effects in vivo. Therefore, TCMs could serve as sources for isolating HNE inhibitors.
Many polysaccharides or polysaccharide-containing fractions have been isolated from P. vulgaris. Among them, three water-soluble polysaccharides (PV-P1-P3) and a polysaccharide (P1) that enhanced NO, TNF-α, and IL-6 production in RAW264.7 cells were isolated by Li and co-workers [38,39]. Subsequently, a zinc-conjugating derivative of P1 (P1-Zn) was synthesized. P1-Zn signi cantly inhibited the proliferation of a human hepatocellular carcinoma cell line (HepG2) by inducing apoptosis [40]. Two polysaccharides, namely PW-S1 and PW-S2, inhibited anti-complement activity through suppressing the classical and alternative pathways [23]. Another polysaccharide (P32) exhibited anti-lung adenocarcinoma activity via immunostimulatory effects [24]. Additionally, polysaccharides or polysaccharide-containing fractions of P. vulgaris (PSP-2B, PPV, and PPS-2b) displayed inhibitory effects against herpes virus in vitro and in vivo [41][42][43]. In the present study, a polysaccharide-containing fraction (PVAP) was obtained using a bioactivity-guided fractionation protocol. This is the rst polysaccharide from P. vulgaris that prevented ALI through the selective inhibition of HNE. Therefore, it could represent a candidate for manipulating HNE level homeostasis and preventing ALI.

Conclusion
PVAP was obtained as a bioactive fraction from the PVW using a bioactivity-guided fractionation protocol. Our data demonstrated that PVAP could prevent LPS-induced ALI in vivo through selectively inhibiting HNE activity. Accordingly, PVAP can be a candidate for developing HNE inhibitors.

Declarations
Availability of data and materials All datasets used in this study are available through the corresponding author on reasonable application.
Ethics approval and consent to participate 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).

Consent for publication
Not applicable.

Competing interests
All authors declare that they have no competing interests.

Supplementary Files
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