- Email: [email protected]
Inhibitory effects of collismycin C and pyrisulfoxin A on particulate matter-induced pulmonary injury
Phytomedicine 62 (2019) 152939
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Original Article
Inhibitory effects of collismycin C and pyrisulfoxin A on particulate matterinduced pulmonary injury Hyukjae Choia,1, Wonhwa Leeb,1, Eonmi Kima, Sae-Kwang Kuc, , Jong-Sup Baed, ⁎⁎
T
⁎
a
College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea c Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany University, Gyeongsan-si 38610, Republic of Korea d College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics based Creative Drug Research Team, Kyungpook National University, Daegu 41566, Republic of Korea b
ARTICLE INFO
ABSTRACT
Keywords: Collismycin C Pyrisulfoxin A Particulate matter Vascular permeability Akt
Background: Inhalation of fine particulate matter (PM2.5) is associated with elevated pulmonary injury caused by the loss of vascular barrier integrity. Marine microbial natural products isolated from microbial culture broths were screened for pulmonary protective effects against PM2.5. Two 2,2′-bipyridine compounds isolated from a red alga-associated Streptomyces sp. MC025—collismycin C (2) and pyrisulfoxin A (5)—were found to inhibit PM2.5-mediated vascular barrier disruption. Purpose: To confirm the inhibitory effects of collismycin C and pyrisulfoxin A on PM2.5-induced pulmonary injury Study design: In this study, we investigated the beneficial effects of collismycin C and pyrisulfoxin A on PMinduced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Methods: Permeability, leukocyte migration, proinflammatory protein activation, reactive oxygen species (ROS) generation, and histology were evaluated in PM2.5-treated ECs and mice. Results: Collismycin C and pyrisulfoxin A significantly scavenged PM2.5-induced ROS and inhibited the ROSinduced activation of p38 mitogen-activated protein kinase as well as activated Akt, which helped in maintaining endothelial integrity, in purified pulmonary endothelial cells. Furthermore, collismycin C and pyrisulfoxin A reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the bronchoalveolar lavage fluid of PM-treated mice. Conclusion: These data suggested that collismycin C and pyrisulfoxin A might exert protective effects on PMinduced inflammatory lung injury and vascular hyperpermeability.
Introduction Particulate matter (PM) has become the focus of public health research; epidemiological studies have revealed the negative biological effects of PM on several major organs, including the lungs, and an inverse correlation between PM inhalation and average life span (Liu et al., 2017; Xing et al., 2016). Fine particulate matter (PM2.5; aerodynamic diameter, <2.5 μm) is a well-known air pollutant that threatens public health. Exposure to high concentrations of airborne PM2.5
has been associated with a risk of arteriosclerosis, respiratory diseases, and lung cancer (de Kok et al., 2005; Sun et al., 2008; Wu et al., 2014). The toxicity of PM2.5 is mainly attributed to its small size that allows it to bypass the human innate defense mechanisms and penetrate deeper into the bronchi, reaching the alveoli; in addition, the adsorbed toxic substances, including endotoxins, polycyclic aromatic hydrocarbons, sulfate, and heavy metals, contribute to the toxic effects (FalconRodriguez et al., 2016). Since environmental problems cannot be immediately addressed, identifying novel preventive and therapeutic
Abbreviations: BAL, bronchoalveolar lavage fluid; IL, interleukin; MAPK, mitogen-activated protein kinase; MLMVEC, mouse lung microvascular endothelial cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PM, particulate matter; ROS, reactive oxygen species; TNF, tumor necrosis factor ⁎ Corresponding author at: College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea. ⁎⁎ Corresponding author at: Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany University, 1 Haanydaero, Gyeongsan-si 38610, Republic of Korea. E-mail addresses: [email protected] (S.-K. Ku), [email protected] (J.-S. Bae). 1 These authors contributed equally. https://doi.org/10.1016/j.phymed.2019.152939 Received 23 January 2019; Received in revised form 3 April 2019; Accepted 22 April 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Collismycin B (3): white powder; 1H NMR (CDCl3, 250 MHz) and C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S6 and S7); LR-ESIMS m/z 276.2 [M + H]+ (calcd. for C13H14N3O2S, 276.1); purity, 100.00%. SF2738 F (4): white powder; 1H NMR (CDCl3, 250 MHz) and 13C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S8 and S9); LR-ESIMS m/z 244.1 [M + H]+ (calcd. for C12H10N3OS, 244.1); purity, 100.00%. Pyrisulfoxin A (5): white powder; 1H NMR (CDCl3, 250 MHz) and 13 C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S10 and S11); LR-ESIMS m/z 292.1 [M + H]+ (calcd. for C13H14N3O3S, 292.1); purity, 100.00%. SF2738 D (6): white powder; 1H NMR (CDCl3, 250 MHz) and 13C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figures S12 and S13); LR-ESIMS m/z 257.1 [M + H]+ (calcd. for C13H12N3OS, 258.1); purity, 100.00%.
strategies to protect the human respiratory system against PM-mediated pulmonary injury is necessary. Collismycin C and pyrisulfoxin A are 2,2′-bipyridine-class natural products isolated from Streptomyces sp. SF2738 and Streptomyces californicus (Gomi et al., 1994; Tsuge et al., 1999). Although collismycin C has poor antimicrobial activity and cytotoxicity compared to collismycin B, it was recently shown to be a potent inhibitor of biofilm formation of methicillin-resistant Staphylococcus aureus (Lee et al., 2017). Collismycin B is an inhibitor of dexamethasone−glucocorticoid receptor binding, which is responsible for the anti-inflammatory activity (Shindo et al., 1994). Pyrisulfoxins A and B were shown to induce cytotoxicity against P388 murine leukemia (Tsuge et al., 1999). Caerulomycin A, a representative bipyridine compound, is a known antibiotic (Funk and Divekar, 1959), anti-asthmatic (Kujur et al., 2015), and immunosuppressive agent (Kujur et al., 2017); hence, we hypothesized that 2,2′-bipyridines, including collismycin C, might show beneficial effects against PM-induced pulmonary injury. In this study, we used a mouse model to evaluate the effects of collismycin C and pyrisulfoxin A on pulmonary histology, lung inflammation, and oxidative stress after PM2.5 exposure and determined whether they could repress disruptive responses mediated by PM2.5-induced vascular barrier in an in vitro model.
13
Animals and husbandry Male Balb/c mice (7 weeks old, approximately 27 g) were purchased from Orient Bio Co. (Sungnam, Republic of Korea) and used after 12 days of acclimatization. The mice were housed (5 per polycarbonate cage) under controlled temperature (20–25 °C) and humidity (40–45%) conditions and a 12:12 h light:dark cycle. All mice were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Kyungpook National University (IRB No., KNU 2017101). After the peroral administration of the tested compounds (0.2–10 mg/kg) or hyaluronan (0.1%) for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline), as previously described (Wang et al., 2017a). Mice were killed after 10 days of intratracheal instillation of PM2.5, and bronchoalveolar lavage fluid (BAL) and lung tissue were harvested for further analysis.
Methods Reagents Diesel PM NIST 1650b (PM2.5) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO), prepared in saline, and sonicated for 30 min to avoid agglomeration of the suspended PM2.5 particles. High-molecularweight hyaluronan (Sigma-Aldrich, Inc.) was used as a positive control (Xu et al., 2018). All other chemicals and reagents were obtained from Sigma-Aldrich, unless otherwise stated.
Primary culture of mouse lung microvascular endothelial cells
Fermentation of microbial strains and isolation of 1–6
Mouse lung microvascular endothelial cells (MLMVECs) were harvested, as previously described (Kovacs-Kasa et al., 2017). Briefly, lung tissues were minced and digested with collagenase A (1 mg/ml) for 45–60 min at 37 °C. The ECs were purified using an anti-PECAM-1 monoclonal antibody magnetic bead (BD Pharmingen, San Diego, CA) separation technique and allowed to grow for 2 days in a growth medium. For monolayer cultures, the cells were plated on fibronectincoated dishes in EC basal medium supplemented with EGM-2 MV Bulletkit™ (Lonza, MD) and incubated at 37 °C under a humidified atmosphere of 5% CO2 and 95% air.
A red alga-associated Streptomyces sp. MC025 was cultured in 35 l of SYP-SW liquid medium (soluble starch, 10 g; yeast extract, 4 g; peptone, 2 g; filtered seawater, 1 l) for 7 days at 25 °C with shaking at 150 rpm. The culture broth was extracted twice with the same volume of EtOAc, and the combined extract was evaporated under reduced pressure to yield crude material (2.3 g). The extract was fractionated into six fractions (A-F) by using normal-phase vacuum liquid chromatography (silica gel) and step-gradient elution with CH2Cl2 and MeOH. Fractions D and E were subjected to reversed-phase high-performance liquid chromatography (HPLC; Hector C18; 250 × 21.2 mm; 6 ml/min) with an acetonitrile-H2O gradient from 48:52 to 58:42 (v/v), and five 2,2′bipyridine compounds—pyrisulfoxin B (1, 47 min, 6.2 mg), collismycin C (2, 30 min, 18.8 mg), collismycin B (3, 17 min, 8.0 mg), SF2738 F (4, 50 min, 6.6 mg), and SF2738 D (6, 35 min, 4.3 mg)—were obtained. Fraction F was subjected to reversed-phase HPLC (Hector C18; 250 × 21.2 mm, 25% acetonitrile in H2O; 6 ml/min) to yield pure pyrisulfoxin A (5, 25 min, 40.7 mg). The chemical structures of the isolated compounds were confirmed by comparing the mass spectrometry and nuclear magnetic resonance spectroscopic data with the reported values (Gomi et al., 1994; Tsuge et al., 1999). The purity of the isolated compounds was calculated based on HPLC-ELSD chromatograms (Fig. S1). Pyrisulfoxin B (1): white powder; 1H NMR (CDCl3, 250 MHz) and 13 C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S2 and S3); LR-ESIMS m/z 274.1 [M + H]+ (calcd. for C13H12N3O2S, 274.1); purity, 95.33%. Collismycin C (2): white powder; 1H NMR (CDCl3, 250 MHz) and 13 C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figures S4 and S5); LR-ESIMS m/z 263.1 [M + H]+ (calcd. for C13H15N2O2S, 263.1); purity, 100.00%.
Permeability assay For spectrophotometric quantification of EC permeability in response to increasing concentrations of the tested compounds in vitro, the flux of Evans blue-bound albumin across functional cell monolayers was measured using a modified 2-compartment chamber model, as previously described (Kim et al., 2019; Lee and Bae, 2019). MLMVECs were plated (5 × 104 cells/well) in transwells (pore size, 3 μm; diameter, 12 mm) for 3 days. Confluent monolayers of MLMVECs were first treated with the tested compounds for 6 h, followed by subsequent treatment with PM2.5 (1 mg/ml) for 6 h. Permeability-related data were recorded using an ELISA plate reader, as previously described (Kim et al., 2019; Lee and Bae, 2019). For the in vivo permeability assay, mice were allowed to breathe spontaneously during the procedure. Mice were treated with PM2.5 and each compound for 10 days and then anesthetized with 2% isoflurane (Forane; JW Pharmaceutical, South Korea) in oxygen delivered first via a small rodent gas anesthesia machine (RC2; Vetequip, Pleasanton, CA) in a breathing chamber and then via a facemask, following which 1% Evans blue dye solution in normal saline was injected intravenously 2
Phytomedicine 62 (2019) 152939
H. Choi, et al.
into each mouse. After 6 h, mice were euthanized by cervical dislocation, and peritoneal exudates were collected for recording permeabilityrelated data, as previously described (Kim et al., 2019; Lee and Bae, 2019; Lee et al., 2019).
Western blot analysis For western blot analysis, the cells were first rinsed with ice-cold phosphate-buffered saline (PBS) and treated with lysis buffer containing 0.5% sodium dodecyl sulfate, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors. Regular western blot analysis was used to detect phospho- and total-Akt MAPK by using the corresponding antibodies (Cell Signaling, Danvers, MA).
Leukocyte migration assay Leukocyte migration was assessed by euthanizing mice after 6 h and washing BAL with 5 ml of normal saline. BAL (20 μl) was mixed with 0.38 ml of Turk's solution (0.01% crystal violet in 3% acetic acid), and the number of leukocytes was counted under a light microscope.
Hematoxylin and eosin staining The phenotypic changes in the lungs were analyzed by removing these organs and washing them three times with PBS (pH 7.4) to remove the remaining blood; the organs were then fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS for 20 h at 4 °C. After fixation, the samples were dehydrated using an ethanol series, embedded in paraffin, sectioned into 4 μm slices, and placed on a slide. The slides were deparaffinized in a 60 °C oven, rehydrated, and stained with hematoxylin (Sigma). Excess stain was removed by rapidly dipping the slides three times in 0.3% acid alcohol and counterstaining with eosin (Sigma). Excess stain was then removed by washing in an ethanol series and xylene, and the samples were placed under a coverslip. A blinded observer performed light microscopic analysis of the lung specimens and evaluated the pulmonary architecture, tissue edema, and inflammatory cell infiltration by using a previously defined method (Ozdulger et al., 2003).
ELISA for the assay of phosphorylated p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-6 The expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) was quantified using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA), according to manufacturer's instructions. The concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in BAL were determined using ELISA kits (R&D Systems, Minneapolis, MN). For all assays, the values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria). Cell viability assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was used as an indicator of cell viability (Jang et al., 2018; Zhang and Wang, 2018). Purified MLMVECs isolated from collismycin C-treated mice were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 h, the cells were washed, and 100 μl of MTT (1 mg/ml) was added to each well and incubated for 4 h. Finally, dimethyl sulfoxide (150 μl) was added to solubilize the formed formazan salt. The amount of formazan salt was determined by measuring the optical density at 540 nm by using a microplate reader (Tecan Austria GmbH, Austria).
Statistical analysis All experiments were independently performed at least three times, and data are expressed as the means ± standard deviation. Student's ttest was used to detect significant differences. p-values < 0.05 were considered statistically significant. SPSS for Windows, version 16.0 (SPSS, Chicago, IL) was used to perform all statistical analyses. Results
Detection of intracellular ROS
Effects of collismycin C on PM2.5-mediated vascular barrier disruption
The ROS in MLMVECs were quantified using fluorescence microscopy, as previously described (Piao et al., 2018). Isolated primary MLMVECs grown on a 4-well glass chamber slide (>90% confluent) were loaded with 2′,7′-dichlorofluorescein diacetate (DCFDA, 10 μM; Molecular Probes, Eugene, OR, USA) for 30 min. The medium containing DCFDA was aspirated, and the cells were rinsed. Next, the stained cells were imaged using fluorescence microscopy.
Since PM2.5 is known to disrupt vascular barrier integrity (Wang et al., 2010, 2017b), a vascular permeability assay was used to evaluate the effects of the six 2,2′-bipyridine compounds (1–6) on barrier integrity in MLMVECs (Fig. 1). Purified MLMVECs were treated with each bipyridine compound (1–6) for 6 h following the administration of PM2.5 (1 mg/ml, 6 h). Compounds 2–5, in particular, collismycin C (2)
Fig. 1. Structures of compounds 1−6. Pyrisulfoxin B (1), collismycin C (2), collismycin B (3), SF2738 F (4), pyrisulfoxin A (5), SF2738 D (6). 3
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 2. Effects of the tested compounds on particulate matter (PM)-induced endothelial cell (EC) barrier disruptive responses and p38 MAPK activation. (A, B) Effects of various concentrations of the tested compounds (1−6) or hyaluronan (HA; 0.1%) on PM2.5 (1 mg/ml, 6 h)-induced barrier disruption were monitored by the flux of Evans blue-bound albumin across mouse lung microvascular endothelial cells (MLMVECs). (C, D) After the peroral administration of the tested compounds (1−6) or HA (0.1%) for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline, 10 days). Effects of the tested compounds (1−6) on PM2.5induced vascular permeability were assessed based on the flux of Evans blue in mice (expressed as μg/mouse, n = 5). (E, F) The same as (A, B) except that the effects of various concentrations of tested compounds (1−6) or HA (0.1%) on PM2.5 (1 mg/ml, 6 h)-induced phospho-p38 expression was monitored using ELISA. (G, H) The same as (C, D) except that phospho-p38 expression in purified MLMVECs isolated from each mouse was measured using ELISA. (I) Effects of PM (1 mg/ml) with or without SB203580 (p38 MAPK inhibitor, 10 μM) on the permeability of MLMVECs. (J) Effect of the tested compounds (1−6) on cellular viability was measured using the MTT assay. Results are expressed as the means ± SD of three independent experiments. CNTL, control. *p < 0.05 vs. PM2.5 challenge.
and pyrisulfoxin A (5), dose-dependently inhibited PM2.5-mediated hyperpermeability (Fig. 2A and B). These results were verified by assessing the in vivo effects of 2 and 5 on vascular permeability. Compounds 2 and 5 also induced marked inhibition of peritoneal dye leakage (Fig. 2C and D). Since the vascular disruptive responses caused by inflammatory proteins are mediated by the p38 MAPK signaling pathways (Qin et al., 2009; Sun et al., 2009), next, we determined
whether the activation of p38 MAPK was affected by PM2.5 and treatment with 2,2′-bipyridine compounds (1–6). MLMVECs were treated with 2,2′-bipyridines after activation with PM2.5. PM2.5 upregulated the expression of phosphorylated p38, which was clearly reduced by collismycin C or pyrisulfoxin A treatment in purified MLMVECs (Fig. 2E and F) and mice (Fig. 2G and H). Moreover, PM2.5-activated p38 MAPK mediated PM2.5-induced endothelial cell hyperpermeability in 4
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 3. Effects of collismycin C and pyrisulfoxins A on PM-induced generation of reactive oxygen species (ROS). After the peroral administration of the tested compounds (1−6) or hyaluronan (HA; 0.1%) for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline, 10 days). (A) Purified mouse lung microvascular endothelial cells (MLMVECs) isolated from treated mice (>90% confluent on 35 mm dishes) were pretreated with 10 μM 2′,7′–dichlorofluorescein diacetate (DCFDA) for 30 min. (B, C) ROS generation was quantified using immunofluorescence microscopy. Representative images from each group are shown (n = 5). Values represent the means ± SD of three independent experiments. *p < 0.05 vs. PM2.5 challenge. Scale bar, 100 μm.
MLMVECs (Fig. 2I). Cell viability assays were performed to probe the toxicity of collismycin C in purified MLMVECs. At the tested concentrations (up to 1000 μM), collismycin C did not affect cell viability (Fig. 2J).
and Poulogiannis, 2018; Manna and Jain, 2015; Schade et al., 2006; Singleton et al., 2009). Therefore, we determined whether 2 and 5 could activate Akt, and whether collismycin C- or pyrisulfoxin A-induced Akt activation was maintained even under PM challenge. Both 2 and 5 significantly induced Akt phosphorylation, which was attenuated by the PI3 kinase inhibitor, LY294002 in MLMVECs (Fig. 4A). Further, Akt phosphorylation persisted despite PM treatment (1 mg/ml, 6 h). The addition of LY294002 to MLMVECs significantly abolished the protective ability of 2 and 5 against PM2.5-induced endothelial cell barrier disruption (Fig. 4C). To define the specific role of collismycin Cand pyrisulfoxin A-induced Akt activation, we knocked down Akt1, the major Akt isotype in the endothelium (Shiojima and Walsh, 2002). Akt1 small interfering RNA reduced Akt protein expression (Fig. 4B) and significantly prevented 2- or 5-mediated protective effects against PM challenge (Fig. 4C). Taken together, these results indicated that collismycin C and pyrisulfoxin A induced Akt phosphorylation, which contributed to the attenuation of PM-induced MLMVEC barrier disruption.
Effects of collismycin C on PM2.5-stimulated ROS generation in MLMVECs Previous studies have shown that intracellular oxidative stress increases remarkably owing to mitochondrial dysregulation after PM exposure (Gualtieri et al., 2012; Wang et al., 2010; Xu et al., 2018; Zhao et al., 2009). In this study, compounds 2 and 5 abolished PM2.5-induced substantial ROS production, as evidenced by the results of DCFDA oxidation assay (Fig. 3). Thus, 2 and 5 acted as ROS scavengers in the endothelial cells, preventing PM-induced ROS accumulation and significantly inhibiting PM-mediated endothelial cell barrier disruption. Effects of collismycin C and pyrisulfoxin A on Akt phosphorylation and endothelial cell barrier function Phosphatidylinositol 3-phosphate (PI3K)/Akt signaling is one of the most critical pathways in the regulation of cellular survival, proliferation, and metabolism in mammals (Bellacosa et al., 1998; Koundouros 5
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 4. Effects of collismycin C (2) and pyrisulfoxin A (5) on Akt activation. (A) Mouse lung microvascular endothelial cells (MLMVECs) were pretreated with 2 or 5 (500 μM, 6 h) and challenged with PM2.5 (1 mg/ml) for 6 h. Phospho-Akt MAPK and total Akt MAPK expression were determined in cell lysates by using western blot analysis. Representative blots are shown. Values represent the means ± SD of three independent experiments. (B) Akt MAPK and β-actin expression was measured using western blot analysis in control siRNA (siCNTL, 100 μM)- and Akt1 siRNA (siAkt1, 100 μM)-treated MLMVECs. (C) MLMVECs were treated with LY294002 (LY, 10 μM, 1 h), Akt1 siRNA, or control siRNA (100 μM, 3 h). A permeability assay was performed after treatment with 2 or 5 (500 μM, 6 h) and PM2.5 (1 mg/ml). Values represent the mean ± SD of three independent experiments. *p< 0.05. NS, not significant.
Effects of collismycin C and pyrisulfoxin A on PM2.5-induced pulmonary inflammation
several vascular disorders (de Kok et al., 2005; Sun et al., 2008; Wu et al., 2014). The vascular endothelium acts as a dynamic barrier that selectively restricts the passage of plasma and cells from the blood into the adjacent tissues. Reversible alterations in barrier function typically occur during inflammatory responses, during which inflammatory mediators cause a transient increase in vascular permeability (Komarova et al., 2007; Mehta et al., 2014). Further, inflammatory mediators irreversibly disrupt vascular integrity and cause excessive loss of fluids from the circulation in inflammatory diseases (Komarova et al., 2007; Mehta et al., 2014). Therefore, the prevention of vascular disruptive responses could result in improved survival of patients with inflammatory diseases. The present study showed that collismycin C and pyrisulfoxin A inhibited PM2.5-induced endothelial barrier disruption and attenuated PM2.5-induced lung vascular leakage via the p38 MAPK pathway, indicating their potential barrier protective functions in guarding endothelial integrity against PM challenge. Growing epidemiological evidence supports the deleterious cardiopulmonary health effects, mortality, and morbidity associated with the exposure to ambient pollutant particles (Dominici et al., 2006; Peng et al., 2008). Various mechanisms have been proposed to explain the cardiopulmonary health effects of PM, including pulmonary and systemic oxidative stress and inflammation (Schicker et al., 2009), coagulation pathway activation (Mutlu et al., 2007), and cardiac autonomic function alteration (Baccarelli et al., 2008). Recent in vivo and in vitro studies revealed that PM triggered significant lung inflammation and vascular hyperpermeability responses, including endothelial barrier disruption, vascular protein leakage, neutrophil infiltration, and proinflammatory cytokine release (Wang et al., 2010, 2008; Zhao et al., 2009). These pathophysiological alterations in pulmonary cells and tissues lead to systemic inflammation, which is one of the primary
Since collismycin C and pyrisulfoxin A suppressed PM2.5-induced vascular barrier disruptive responses in vivo (Fig. 2), we determined their effects on PM-induced pulmonary inflammatory responses and injury in vivo. PM2.5 caused an increase in inflammatory leukocyte infiltration and leukocyte count in BAL, which was significantly prevented by 2 and 5 (Fig. 5A). Furthermore, 2 and 5 attenuated PM2.5stimulated release of inflammatory cytokines, IL-6 (Fig. 5B) and TNF-α (Fig. 5C), into the BAL fluid. Histological studies confirmed 2- and 5mediated reduction of PM2.5-induced remarkable increase in leukocyte infiltration in murine lung tissue (Fig. 5D). These data indicated that collismycin C and pyrisulfoxin A attenuated PM-induced pulmonary vascular hyperpermeability, inflammatory leukocyte infiltration, and proinflammatory cytokine release, thereby protecting against PMmediated pulmonary inflammatory injury. Discussion Endothelial integrity disruption and vascular hyperpermeability are prominent features of vascular inflammatory diseases, including severe inflammatory response syndrome, sepsis, pulmonary diseases, and atherosclerosis (Curry and Adamson, 2013; Komarova et al., 2007). Therefore, the prevention of endothelial barrier disruption has obvious therapeutic applications. Airborne particulates containing high concentrations of transition metals and polycyclic aromatic hydrocarbons stimulate the excessive generation of ROS in lung tissues, particularly in the vascular endothelium, which is readily susceptible to PM-induced oxidative stress and plays a critical role in the pathophysiology of 6
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 5. Effects of collismycin C (2) and pyrisulfoxin A (5) on particulate matter-induced pulmonary inflammation and injury. After the peroral administration of 2 or 5 for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline, 10 days). (A−C) Total leukocyte counts (A) as well as IL-6 (B) and TNF-α (C) concentrations in bronchoalveolar lavage (BAL) fluids were measured. (D) Lung histology was examined using hematoxylin and eosin staining. Representative images from each group are shown (n = 5). Scale bar, 200 μm. Arrows indicate leukocyte infiltration. Values represent the mean ± SD of three independent experiments. *p < 0.05 vs. PM2.5 challenge.
aggravators of existing cardiopulmonary conditions such as asthma, chronic obstructive pulmonary disease, cardiac arrhythmias, and congestive heart failure. In this study, collismycin C and pyrisulfoxin A scavenged PM2.5-induced ROS; inhibited oxidative stress-induced p38 MAPK and Akt activation; and reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the BAL fluid of PM-treated mice. Therefore, 2 and 5 might attenuate ROSmediated pulmonary inflammation caused by PM air pollution. Among the six tested 2,2′-bipyridines tested, compounds 2–5 showed does-dependent inhibition of PM2.5-mediated vascular barrier disruption and PM2.5-stimulated ROS generation, whereas pyrisulfoxin B (1) and SF2738D (6), which possess 6-nitrile groups, did not. Previously, the aldoxime moiety of caerulomycins was speculated to be the pharmacophore (Fu et al., 2011), and the bioactive 2,2′-bipyridines, including pyrisulfoxins A and B as well as collismycins A and B, have the 6-aldoxime group (Gomi et al., 1994; Tsuge et al., 1999). However, collismycin C (2) possesses 6-hydroxymethyl rather than 6-aldoxime. Therefore, the 6-aldoxime unit does not seem to be critical for the attenuation of airborne PM-induced ROS-dependent pulmonary inflammation by the 2,2′-bipyridines. The influences of other components such as 4-O-methyl, 5-S-methyl, and 5-methyl sulfoxide on the activities of 2,2′-bipyridines are not yet clear. The hydroxyl radical is a harmful byproduct of oxidative metabolism and can cause molecular damage in living systems; it plays a critical role in initiating and catalyzing various radical reactions (Sun et al., 2008). ROS formation exceeds the detoxification capacity of the antioxidant defense system, thereby producing a change in the cell redox status and causing a cascade of events that is closely involved in the inflammatory responses (Garcon et al., 2006; Pozzi et al., 2003). In addition, ROS are thought to play an active role in inducing airway inflammation and hyper-responsiveness (Hulsmann et al., 1994). Our findings suggest that collismycin C might exhibit beneficial effects in the respiratory system against PM-induced ROS formation. In addition, inhaled PM, particularly fine and ultrafine particles with diameters less than 2.5 mm, was shown to move from the alveolar space into the pulmonary circulation and to trigger amplified endothelial dysfunction (Karoly et al., 2007; Kreyling et al., 2002). Moreover, PM challenge results in the migration of activated neutrophils from the blood into the lung interstitium, which can lead to the production of significant amounts of ROS, thereby exacerbating inflammation (Walters et al., 2001). In this study, we showed that PM induced significant ROS
production in the pulmonary endothelial cells, thereby activating p38 MAPK. Activated p38 MAPK is involved in actin filament reorganization in response to oxidative stress, to facilitate stress actin fiber synthesis and paracellular gap formation, which are essential markers of endothelial cell integrity disruption (Wang et al., 2010), as was evident by the disruption of vascular barrier integrity in this study (Fig. 2). Collismycin C inhibited PM2.5-induced endothelial barrier disruption and attenuated PM2.5-induced lung vascular leakage via the p38 MAPK pathway; therefore, it could act as an endogenous ROS scavenger and maintain endothelial integrity during oxidative stress. Our findings showed that collismycin C and pyrisulfoxin A prevented PM-induced endothelial integrity disruption and vascular hyperpermeability via the dual actions of ROS scavenging and Akt pathway activation. Thus, they might attenuate PM air pollution-induced ROS-mediated pulmonary inflammation. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [Grant nos. 2018R1A5A2025272 and 2017R1A2B4006110]. Conflict of interest The authors declare no conflicts of interest. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2019.152939. References Baccarelli, A., Cassano, P.A., Litonjua, A., Park, S.K., Suh, H., Sparrow, D., Vokonas, P., Schwartz, J., 2008. Cardiac autonomic dysfunction: effects from particulate air pollution and protection by dietary methyl nutrients and metabolic polymorphisms. Circulation 117, 1802–1809. Bellacosa, A., Chan, T.O., Ahmed, N.N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J.N., Tsichlis, P., 1998. Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17, 313–325. Curry, F.R., Adamson, R.H., 2013. Tonic regulation of vascular permeability. Acta Physiol. (Oxf) 207, 628–649. de Kok, T.M., Engels, L.G., Moonen, E.J., Kleinjans, J.C., 2005. Inflammatory bowel disease stimulates formation of carcinogenic N-nitroso compounds. Gut 54, 731.
7
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Peng, R.D., Chang, H.H., Bell, M.L., McDermott, A., Zeger, S.L., Samet, J.M., Dominici, F., 2008. Coarse particulate matter air pollution and hospital admissions for cardiovascular and respiratory diseases among Medicare patients. JAMA 299, 2172–2179. Piao, M.J., Ahn, M.J., Kang, K.A., Ryu, Y.S., Hyun, Y.J., Shilnikova, K., Zhen, A.X., Jeong, J.W., Choi, Y.H., Kang, H.K., Koh, Y.S., Hyun, J.W., 2018. Particulate matter 2.5 damages skin cells by inducing oxidative stress, subcellular organelle dysfunction, and apoptosis. Arch. Toxicol. 92, 2077–2091. Pozzi, R., De Berardis, B., Paoletti, L., Guastadisegni, C., 2003. Inflammatory mediators induced by coarse (PM2.5-10) and fine (PM2.5) urban air particles in RAW 264.7 cells. Toxicology 183, 243–254. Qin, Y.H., Dai, S.M., Tang, G.S., Zhang, J., Ren, D., Wang, Z.W., Shen, Q., 2009. HMGB1 enhances the proinflammatory activity of lipopolysaccharide by promoting the phosphorylation of MAPK p38 through receptor for advanced glycation end products. J. Immunol. 183, 6244–6250. Schade, A.E., Powers, J.J., Wlodarski, M.W., Maciejewski, J.P., 2006. Phosphatidylinositol-3-phosphate kinase pathway activation protects leukemic large granular lymphocytes from undergoing homeostatic apoptosis. Blood 107, 4834–4840. Schicker, B., Kuhn, M., Fehr, R., Asmis, L.M., Karagiannidis, C., Reinhart, W.H., 2009. Particulate matter inhalation during hay storing activity induces systemic inflammation and platelet aggregation. Eur. J. Appl. Physiol. 105, 771–778. Shindo, K., Yamagishi, Y., Okada, Y., Kawai, H., 1994. Collismycins A and B, novel nonsteroidal inhibitors of dexamethasone-glucocorticoid receptor binding. J. Antibiot. (Tokyo). 47, 1072–1074. Shiojima, I., Walsh, K., 2002. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ. Res. 90, 1243–1250. Singleton, P.A., Chatchavalvanich, S., Fu, P., Xing, J., Birukova, A.A., Fortune, J.A., Klibanov, A.M., Garcia, J.G., Birukov, K.G., 2009. Akt-mediated transactivation of the S1P1 receptor in caveolin-enriched microdomains regulates endothelial barrier enhancement by oxidized phospholipids. Circ. Res. 104, 978–986. Sun, C., Liang, C., Ren, Y., Zhen, Y., He, Z., Wang, H., Tan, H., Pan, X., Wu, Z., 2009. Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways. Basic Res. Cardiol. 104, 42–49. Sun, Y., Yin, Y., Zhang, J., Yu, H., Wang, X., Wu, J., Xue, Y., 2008. Hydroxyl radical generation and oxidative stress in Carassius auratus liver, exposed to pyrene. Ecotoxicol. Environ. Saf. 71, 446–453. Tsuge, N., Furihata, K., Shin-Ya, K., Hayakawa, Y., Seto, H., 1999. Novel antibiotics pyrisulfoxin A and B produced by Streptomyces californicus. J. Antibiot. (Tokyo). 52, 505–507. Walters, D.M., Breysse, P.N., Wills-Karp, M., 2001. Ambient urban Baltimore particulateinduced airway hyperresponsiveness and inflammation in mice. Am. J. Respir. Crit. Care Med. 164, 1438–1443. Wang, H., Song, L., Ju, W., Wang, X., Dong, L., Zhang, Y., Ya, P., Yang, C., Li, F., 2017a. The acute airway inflammation induced by PM2.5 exposure and the treatment of essential oils in Balb/c mice. Sci. Rep. 7, 44256. Wang, T., Chiang, E.T., Moreno-Vinasco, L., Lang, G.D., Pendyala, S., Samet, J.M., Geyh, A.S., Breysse, P.N., Chillrud, S.N., Natarajan, V., Garcia, J.G., 2010. Particulate matter disrupts human lung endothelial barrier integrity via ROS- and p38 MAPKdependent pathways. Am. J. Respir. Cell Mol. Biol. 42, 442–449. Wang, T., Moreno-Vinasco, L., Huang, Y., Lang, G.D., Linares, J.D., Goonewardena, S.N., Grabavoy, A., Samet, J.M., Geyh, A.S., Breysse, P.N., Lussier, Y.A., Natarajan, V., Garcia, J.G., 2008. Murine lung responses to ambient particulate matter: genomic analysis and influence on airway hyperresponsiveness. Environ. Health Perspect. 116, 1500–1508. Wang, T., Shimizu, Y., Wu, X., Kelly, G.T., Xu, X., Wang, L., Qian, Z., Chen, Y., Garcia, J.G.N., 2017b. Particulate matter disrupts human lung endothelial cell barrier integrity via Rho-dependent pathways. Pulm. Circ. 7, 617–623. Wu, S., Deng, F., Hao, Y., Wang, X., Zheng, C., Lv, H., Lu, X., Wei, H., Huang, J., Qin, Y., Shima, M., Guo, X., 2014. Fine particulate matter, temperature, and lung function in healthy adults: findings from the HVNR study. Chemosphere 108, 168–174. Xing, Y.F., Xu, Y.H., Shi, M.H., Lian, Y.X., 2016. The impact of PM2.5 on the human respiratory system. J. Thorac. Dis. 8, E69–E74. Xu, C., Shi, Q., Zhang, L., Zhao, H., 2018. High molecular weight hyaluronan attenuates fine particulate matter-induced acute lung injury through inhibition of ROS-ASK1p38/JNK-mediated epithelial apoptosis. Environ. Toxicol. Pharmacol. 59, 190–198. Zhang, L., Wang, M.C., 2018. Growth inhibitory effect of mangiferin on thyroid cancer cell line TPC1. Biotechnol. Bioprocess Eng. 23, 649–654. Zhao, Y., Usatyuk, P.V., Gorshkova, I.A., He, D., Wang, T., Moreno-Vinasco, L., Geyh, A.S., Breysse, P.N., Samet, J.M., Spannhake, E.W., Garcia, J.G., Natarajan, V., 2009. Regulation of COX-2 expression and IL-6 release by particulate matter in airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 40, 19–30.
Dominici, F., Peng, R.D., Bell, M.L., Pham, L., McDermott, A., Zeger, S.L., Samet, J.M., 2006. Fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases. JAMA 295, 1127–1134. Falcon-Rodriguez, C.I., Osornio-Vargas, A.R., Sada-Ovalle, I., Segura-Medina, P., 2016. Aeroparticles, composition, and lung diseases. Front. Immunol. 7, 3. Fu, P., Wang, S., Hong, K., Li, X., Liu, P., Wang, Y., Zhu, W., 2011. Cytotoxic bipyridines from the marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6. J. Nat. Prod. 74, 1751–1756. Funk, A., Divekar, P.V., 1959. Caerulomycin, a new antibiotic from Streptomyces caeruleus Baldacci. I. Production, isolation, assay, and biological properties. Can. J. Microbiol. 5, 317–321. Garcon, G., Dagher, Z., Zerimech, F., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., Shirali, P., 2006. Dunkerque city air pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells (L132) in culture. Toxicol. In Vitro 20, 519–528. Gomi, S., Amano, S., Sato, E., Miyadoh, S., Kodama, Y., 1994. Novel antibiotics SF2738A, SF2738B and SF2738C, and their analogs produced by Streptomyces sp. J. Antibiot. (Tokyo). 47, 1385–1394. Gualtieri, M., Longhin, E., Mattioli, M., Mantecca, P., Tinaglia, V., Mangano, E., Proverbio, M.C., Bestetti, G., Camatini, M., Battaglia, C., 2012. Gene expression profiling of A549 cells exposed to Milan PM2.5. Toxicol. Lett. 209, 136–145. Hulsmann, A.R., Raatgeep, H.R., den Hollander, J.C., Stijnen, T., Saxena, P.R., Kerrebijn, K.F., de Jongste, J.C., 1994. Oxidative epithelial damage produces hyperresponsiveness of human peripheral airways. Am. J. Respir. Crit. Care Med. 149, 519–525. Jang, M.H., Kang, N.H., Mukherjee, S., Yun, J.W., 2018. Theobromine, a methylxanthine in cocoa bean, stimulates thermogenesis by inducing white fat browning and activating brown adipocytes. Biotechnol. Bioprocess Eng. 23, 617–626. Karoly, E.D., Li, Z., Dailey, L.A., Hyseni, X., Huang, Y.C., 2007. Up-regulation of tissue factor in human pulmonary artery endothelial cells after ultrafine particle exposure. Environ. Health Perspect. 115, 535–540. Kim, J.E., Lee, W., Yang, S., Cho, S.H., Baek, M.C., Song, G.Y., Bae, J.S., 2019. Suppressive effects of rare ginsenosides, Rk1 and Rg5, on HMGB1-mediated septic responses. Food Chem. Toxicol. 124, 45–53. Komarova, Y.A., Mehta, D., Malik, A.B., 2007. Dual regulation of endothelial junctional permeability. Sci. STKE 2007, re8. Koundouros, N., Poulogiannis, G., 2018. Phosphoinositide 3-kinase/Akt signaling and redox metabolism in cancer. Front. Oncol. 8, 160. Kovacs-Kasa, A., Varn, M.N., Verin, A.D., Gonzales, J.N., 2017. Method for the culture of mouse pulmonary microvascular endothelial cells. Sci. Pages Pulmonol. 1, 7–18. Kreyling, W.G., Semmler, M., Erbe, F., Mayer, P., Takenaka, S., Schulz, H., Oberdorster, G., Ziesenis, A., 2002. Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low. J. Toxicol. Environ. Health A 65, 1513–1530. Kujur, W., Gurram, R.K., Haleem, N., Maurya, S.K., Agrewala, J.N., 2015. Caerulomycin A inhibits Th2 cell activity: a possible role in the management of asthma. Sci. Rep. 5, 15396. Kujur, W., Gurram, R.K., Maurya, S.K., Nadeem, S., Chodisetti, S.B., Khan, N., Agrewala, J.N., 2017. Caerulomycin A suppresses the differentiation of naive T cells and alleviates the symptoms of experimental autoimmune encephalomyelitis. Autoimmunity 50, 317–328. Lee, I.C., Bae, J.S., 2019. Pelargonidin protects against renal injury in a mouse model of sepsis. J. Med. Food 22, 57–61. Lee, J.H., Kim, E., Choi, H., Lee, J., 2017. Collismycin C from the Micronesian Marine Bacterium Streptomyces sp. MC025 Inhibits Staphylococcus aureus Biofilm Formation. Mar. Drugs 15 (12), 387. Lee, W., Cho, S.H., Kim, J.E., Lee, C., Lee, J.H., Baek, M.C., Song, G.Y., Bae, J.S., 2019. Suppressive effects of Ginsenoside Rh1 on HMGB1-mediated septic responses. Am. J. Chin. Med. 47, 119–133. Liu, Q., Xu, C., Ji, G., Liu, H., Shao, W., Zhang, C., Gu, A., Zhao, P., 2017. Effect of exposure to ambient PM2.5 pollution on the risk of respiratory tract diseases: a metaanalysis of cohort studies. J. Biomed. Res. 31, 130–142. Manna, P., Jain, S.K., 2015. Phosphatidylinositol-3,4,5-triphosphate and cellular signaling: implications for obesity and diabetes. Cell. Physiol. Biochem. 35, 1253–1275. Mehta, D., Ravindran, K., Kuebler, W.M., 2014. Novel regulators of endothelial barrier function. Am. J. Physiol. Lung Cell Mol. Physiol. 307, L924–L935. Mutlu, G.M., Green, D., Bellmeyer, A., Baker, C.M., Burgess, Z., Rajamannan, N., Christman, J.W., Foiles, N., Kamp, D.W., Ghio, A.J., Chandel, N.S., Dean, D.A., Sznajder, J.I., Budinger, G.R., 2007. Ambient particulate matter accelerates coagulation via an IL-6-dependent pathway. J. Clin. Invest. 117, 2952–2961. Ozdulger, A., Cinel, I., Koksel, O., Cinel, L., Avlan, D., Unlu, A., Okcu, H., Dikmengil, M., Oral, U., 2003. The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and puncture-induced sepsis model. Shock 19, 366–372.
8
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Original Article
Inhibitory effects of collismycin C and pyrisulfoxin A on particulate matterinduced pulmonary injury Hyukjae Choia,1, Wonhwa Leeb,1, Eonmi Kima, Sae-Kwang Kuc, , Jong-Sup Baed, ⁎⁎
T
⁎
a
College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea c Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany University, Gyeongsan-si 38610, Republic of Korea d College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics based Creative Drug Research Team, Kyungpook National University, Daegu 41566, Republic of Korea b
ARTICLE INFO
ABSTRACT
Keywords: Collismycin C Pyrisulfoxin A Particulate matter Vascular permeability Akt
Background: Inhalation of fine particulate matter (PM2.5) is associated with elevated pulmonary injury caused by the loss of vascular barrier integrity. Marine microbial natural products isolated from microbial culture broths were screened for pulmonary protective effects against PM2.5. Two 2,2′-bipyridine compounds isolated from a red alga-associated Streptomyces sp. MC025—collismycin C (2) and pyrisulfoxin A (5)—were found to inhibit PM2.5-mediated vascular barrier disruption. Purpose: To confirm the inhibitory effects of collismycin C and pyrisulfoxin A on PM2.5-induced pulmonary injury Study design: In this study, we investigated the beneficial effects of collismycin C and pyrisulfoxin A on PMinduced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Methods: Permeability, leukocyte migration, proinflammatory protein activation, reactive oxygen species (ROS) generation, and histology were evaluated in PM2.5-treated ECs and mice. Results: Collismycin C and pyrisulfoxin A significantly scavenged PM2.5-induced ROS and inhibited the ROSinduced activation of p38 mitogen-activated protein kinase as well as activated Akt, which helped in maintaining endothelial integrity, in purified pulmonary endothelial cells. Furthermore, collismycin C and pyrisulfoxin A reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the bronchoalveolar lavage fluid of PM-treated mice. Conclusion: These data suggested that collismycin C and pyrisulfoxin A might exert protective effects on PMinduced inflammatory lung injury and vascular hyperpermeability.
Introduction Particulate matter (PM) has become the focus of public health research; epidemiological studies have revealed the negative biological effects of PM on several major organs, including the lungs, and an inverse correlation between PM inhalation and average life span (Liu et al., 2017; Xing et al., 2016). Fine particulate matter (PM2.5; aerodynamic diameter, <2.5 μm) is a well-known air pollutant that threatens public health. Exposure to high concentrations of airborne PM2.5
has been associated with a risk of arteriosclerosis, respiratory diseases, and lung cancer (de Kok et al., 2005; Sun et al., 2008; Wu et al., 2014). The toxicity of PM2.5 is mainly attributed to its small size that allows it to bypass the human innate defense mechanisms and penetrate deeper into the bronchi, reaching the alveoli; in addition, the adsorbed toxic substances, including endotoxins, polycyclic aromatic hydrocarbons, sulfate, and heavy metals, contribute to the toxic effects (FalconRodriguez et al., 2016). Since environmental problems cannot be immediately addressed, identifying novel preventive and therapeutic
Abbreviations: BAL, bronchoalveolar lavage fluid; IL, interleukin; MAPK, mitogen-activated protein kinase; MLMVEC, mouse lung microvascular endothelial cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PM, particulate matter; ROS, reactive oxygen species; TNF, tumor necrosis factor ⁎ Corresponding author at: College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea. ⁎⁎ Corresponding author at: Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany University, 1 Haanydaero, Gyeongsan-si 38610, Republic of Korea. E-mail addresses: [email protected] (S.-K. Ku), [email protected] (J.-S. Bae). 1 These authors contributed equally. https://doi.org/10.1016/j.phymed.2019.152939 Received 23 January 2019; Received in revised form 3 April 2019; Accepted 22 April 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Collismycin B (3): white powder; 1H NMR (CDCl3, 250 MHz) and C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S6 and S7); LR-ESIMS m/z 276.2 [M + H]+ (calcd. for C13H14N3O2S, 276.1); purity, 100.00%. SF2738 F (4): white powder; 1H NMR (CDCl3, 250 MHz) and 13C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S8 and S9); LR-ESIMS m/z 244.1 [M + H]+ (calcd. for C12H10N3OS, 244.1); purity, 100.00%. Pyrisulfoxin A (5): white powder; 1H NMR (CDCl3, 250 MHz) and 13 C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S10 and S11); LR-ESIMS m/z 292.1 [M + H]+ (calcd. for C13H14N3O3S, 292.1); purity, 100.00%. SF2738 D (6): white powder; 1H NMR (CDCl3, 250 MHz) and 13C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figures S12 and S13); LR-ESIMS m/z 257.1 [M + H]+ (calcd. for C13H12N3OS, 258.1); purity, 100.00%.
strategies to protect the human respiratory system against PM-mediated pulmonary injury is necessary. Collismycin C and pyrisulfoxin A are 2,2′-bipyridine-class natural products isolated from Streptomyces sp. SF2738 and Streptomyces californicus (Gomi et al., 1994; Tsuge et al., 1999). Although collismycin C has poor antimicrobial activity and cytotoxicity compared to collismycin B, it was recently shown to be a potent inhibitor of biofilm formation of methicillin-resistant Staphylococcus aureus (Lee et al., 2017). Collismycin B is an inhibitor of dexamethasone−glucocorticoid receptor binding, which is responsible for the anti-inflammatory activity (Shindo et al., 1994). Pyrisulfoxins A and B were shown to induce cytotoxicity against P388 murine leukemia (Tsuge et al., 1999). Caerulomycin A, a representative bipyridine compound, is a known antibiotic (Funk and Divekar, 1959), anti-asthmatic (Kujur et al., 2015), and immunosuppressive agent (Kujur et al., 2017); hence, we hypothesized that 2,2′-bipyridines, including collismycin C, might show beneficial effects against PM-induced pulmonary injury. In this study, we used a mouse model to evaluate the effects of collismycin C and pyrisulfoxin A on pulmonary histology, lung inflammation, and oxidative stress after PM2.5 exposure and determined whether they could repress disruptive responses mediated by PM2.5-induced vascular barrier in an in vitro model.
13
Animals and husbandry Male Balb/c mice (7 weeks old, approximately 27 g) were purchased from Orient Bio Co. (Sungnam, Republic of Korea) and used after 12 days of acclimatization. The mice were housed (5 per polycarbonate cage) under controlled temperature (20–25 °C) and humidity (40–45%) conditions and a 12:12 h light:dark cycle. All mice were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Kyungpook National University (IRB No., KNU 2017101). After the peroral administration of the tested compounds (0.2–10 mg/kg) or hyaluronan (0.1%) for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline), as previously described (Wang et al., 2017a). Mice were killed after 10 days of intratracheal instillation of PM2.5, and bronchoalveolar lavage fluid (BAL) and lung tissue were harvested for further analysis.
Methods Reagents Diesel PM NIST 1650b (PM2.5) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO), prepared in saline, and sonicated for 30 min to avoid agglomeration of the suspended PM2.5 particles. High-molecularweight hyaluronan (Sigma-Aldrich, Inc.) was used as a positive control (Xu et al., 2018). All other chemicals and reagents were obtained from Sigma-Aldrich, unless otherwise stated.
Primary culture of mouse lung microvascular endothelial cells
Fermentation of microbial strains and isolation of 1–6
Mouse lung microvascular endothelial cells (MLMVECs) were harvested, as previously described (Kovacs-Kasa et al., 2017). Briefly, lung tissues were minced and digested with collagenase A (1 mg/ml) for 45–60 min at 37 °C. The ECs were purified using an anti-PECAM-1 monoclonal antibody magnetic bead (BD Pharmingen, San Diego, CA) separation technique and allowed to grow for 2 days in a growth medium. For monolayer cultures, the cells were plated on fibronectincoated dishes in EC basal medium supplemented with EGM-2 MV Bulletkit™ (Lonza, MD) and incubated at 37 °C under a humidified atmosphere of 5% CO2 and 95% air.
A red alga-associated Streptomyces sp. MC025 was cultured in 35 l of SYP-SW liquid medium (soluble starch, 10 g; yeast extract, 4 g; peptone, 2 g; filtered seawater, 1 l) for 7 days at 25 °C with shaking at 150 rpm. The culture broth was extracted twice with the same volume of EtOAc, and the combined extract was evaporated under reduced pressure to yield crude material (2.3 g). The extract was fractionated into six fractions (A-F) by using normal-phase vacuum liquid chromatography (silica gel) and step-gradient elution with CH2Cl2 and MeOH. Fractions D and E were subjected to reversed-phase high-performance liquid chromatography (HPLC; Hector C18; 250 × 21.2 mm; 6 ml/min) with an acetonitrile-H2O gradient from 48:52 to 58:42 (v/v), and five 2,2′bipyridine compounds—pyrisulfoxin B (1, 47 min, 6.2 mg), collismycin C (2, 30 min, 18.8 mg), collismycin B (3, 17 min, 8.0 mg), SF2738 F (4, 50 min, 6.6 mg), and SF2738 D (6, 35 min, 4.3 mg)—were obtained. Fraction F was subjected to reversed-phase HPLC (Hector C18; 250 × 21.2 mm, 25% acetonitrile in H2O; 6 ml/min) to yield pure pyrisulfoxin A (5, 25 min, 40.7 mg). The chemical structures of the isolated compounds were confirmed by comparing the mass spectrometry and nuclear magnetic resonance spectroscopic data with the reported values (Gomi et al., 1994; Tsuge et al., 1999). The purity of the isolated compounds was calculated based on HPLC-ELSD chromatograms (Fig. S1). Pyrisulfoxin B (1): white powder; 1H NMR (CDCl3, 250 MHz) and 13 C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figs. S2 and S3); LR-ESIMS m/z 274.1 [M + H]+ (calcd. for C13H12N3O2S, 274.1); purity, 95.33%. Collismycin C (2): white powder; 1H NMR (CDCl3, 250 MHz) and 13 C NMR (CDCl3, 63 MHz) data, see supplementary materials (Table S1, Figures S4 and S5); LR-ESIMS m/z 263.1 [M + H]+ (calcd. for C13H15N2O2S, 263.1); purity, 100.00%.
Permeability assay For spectrophotometric quantification of EC permeability in response to increasing concentrations of the tested compounds in vitro, the flux of Evans blue-bound albumin across functional cell monolayers was measured using a modified 2-compartment chamber model, as previously described (Kim et al., 2019; Lee and Bae, 2019). MLMVECs were plated (5 × 104 cells/well) in transwells (pore size, 3 μm; diameter, 12 mm) for 3 days. Confluent monolayers of MLMVECs were first treated with the tested compounds for 6 h, followed by subsequent treatment with PM2.5 (1 mg/ml) for 6 h. Permeability-related data were recorded using an ELISA plate reader, as previously described (Kim et al., 2019; Lee and Bae, 2019). For the in vivo permeability assay, mice were allowed to breathe spontaneously during the procedure. Mice were treated with PM2.5 and each compound for 10 days and then anesthetized with 2% isoflurane (Forane; JW Pharmaceutical, South Korea) in oxygen delivered first via a small rodent gas anesthesia machine (RC2; Vetequip, Pleasanton, CA) in a breathing chamber and then via a facemask, following which 1% Evans blue dye solution in normal saline was injected intravenously 2
Phytomedicine 62 (2019) 152939
H. Choi, et al.
into each mouse. After 6 h, mice were euthanized by cervical dislocation, and peritoneal exudates were collected for recording permeabilityrelated data, as previously described (Kim et al., 2019; Lee and Bae, 2019; Lee et al., 2019).
Western blot analysis For western blot analysis, the cells were first rinsed with ice-cold phosphate-buffered saline (PBS) and treated with lysis buffer containing 0.5% sodium dodecyl sulfate, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors. Regular western blot analysis was used to detect phospho- and total-Akt MAPK by using the corresponding antibodies (Cell Signaling, Danvers, MA).
Leukocyte migration assay Leukocyte migration was assessed by euthanizing mice after 6 h and washing BAL with 5 ml of normal saline. BAL (20 μl) was mixed with 0.38 ml of Turk's solution (0.01% crystal violet in 3% acetic acid), and the number of leukocytes was counted under a light microscope.
Hematoxylin and eosin staining The phenotypic changes in the lungs were analyzed by removing these organs and washing them three times with PBS (pH 7.4) to remove the remaining blood; the organs were then fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS for 20 h at 4 °C. After fixation, the samples were dehydrated using an ethanol series, embedded in paraffin, sectioned into 4 μm slices, and placed on a slide. The slides were deparaffinized in a 60 °C oven, rehydrated, and stained with hematoxylin (Sigma). Excess stain was removed by rapidly dipping the slides three times in 0.3% acid alcohol and counterstaining with eosin (Sigma). Excess stain was then removed by washing in an ethanol series and xylene, and the samples were placed under a coverslip. A blinded observer performed light microscopic analysis of the lung specimens and evaluated the pulmonary architecture, tissue edema, and inflammatory cell infiltration by using a previously defined method (Ozdulger et al., 2003).
ELISA for the assay of phosphorylated p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-6 The expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) was quantified using a commercially available ELISA kit (Cell Signaling Technology, Danvers, MA), according to manufacturer's instructions. The concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in BAL were determined using ELISA kits (R&D Systems, Minneapolis, MN). For all assays, the values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria). Cell viability assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was used as an indicator of cell viability (Jang et al., 2018; Zhang and Wang, 2018). Purified MLMVECs isolated from collismycin C-treated mice were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 h, the cells were washed, and 100 μl of MTT (1 mg/ml) was added to each well and incubated for 4 h. Finally, dimethyl sulfoxide (150 μl) was added to solubilize the formed formazan salt. The amount of formazan salt was determined by measuring the optical density at 540 nm by using a microplate reader (Tecan Austria GmbH, Austria).
Statistical analysis All experiments were independently performed at least three times, and data are expressed as the means ± standard deviation. Student's ttest was used to detect significant differences. p-values < 0.05 were considered statistically significant. SPSS for Windows, version 16.0 (SPSS, Chicago, IL) was used to perform all statistical analyses. Results
Detection of intracellular ROS
Effects of collismycin C on PM2.5-mediated vascular barrier disruption
The ROS in MLMVECs were quantified using fluorescence microscopy, as previously described (Piao et al., 2018). Isolated primary MLMVECs grown on a 4-well glass chamber slide (>90% confluent) were loaded with 2′,7′-dichlorofluorescein diacetate (DCFDA, 10 μM; Molecular Probes, Eugene, OR, USA) for 30 min. The medium containing DCFDA was aspirated, and the cells were rinsed. Next, the stained cells were imaged using fluorescence microscopy.
Since PM2.5 is known to disrupt vascular barrier integrity (Wang et al., 2010, 2017b), a vascular permeability assay was used to evaluate the effects of the six 2,2′-bipyridine compounds (1–6) on barrier integrity in MLMVECs (Fig. 1). Purified MLMVECs were treated with each bipyridine compound (1–6) for 6 h following the administration of PM2.5 (1 mg/ml, 6 h). Compounds 2–5, in particular, collismycin C (2)
Fig. 1. Structures of compounds 1−6. Pyrisulfoxin B (1), collismycin C (2), collismycin B (3), SF2738 F (4), pyrisulfoxin A (5), SF2738 D (6). 3
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 2. Effects of the tested compounds on particulate matter (PM)-induced endothelial cell (EC) barrier disruptive responses and p38 MAPK activation. (A, B) Effects of various concentrations of the tested compounds (1−6) or hyaluronan (HA; 0.1%) on PM2.5 (1 mg/ml, 6 h)-induced barrier disruption were monitored by the flux of Evans blue-bound albumin across mouse lung microvascular endothelial cells (MLMVECs). (C, D) After the peroral administration of the tested compounds (1−6) or HA (0.1%) for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline, 10 days). Effects of the tested compounds (1−6) on PM2.5induced vascular permeability were assessed based on the flux of Evans blue in mice (expressed as μg/mouse, n = 5). (E, F) The same as (A, B) except that the effects of various concentrations of tested compounds (1−6) or HA (0.1%) on PM2.5 (1 mg/ml, 6 h)-induced phospho-p38 expression was monitored using ELISA. (G, H) The same as (C, D) except that phospho-p38 expression in purified MLMVECs isolated from each mouse was measured using ELISA. (I) Effects of PM (1 mg/ml) with or without SB203580 (p38 MAPK inhibitor, 10 μM) on the permeability of MLMVECs. (J) Effect of the tested compounds (1−6) on cellular viability was measured using the MTT assay. Results are expressed as the means ± SD of three independent experiments. CNTL, control. *p < 0.05 vs. PM2.5 challenge.
and pyrisulfoxin A (5), dose-dependently inhibited PM2.5-mediated hyperpermeability (Fig. 2A and B). These results were verified by assessing the in vivo effects of 2 and 5 on vascular permeability. Compounds 2 and 5 also induced marked inhibition of peritoneal dye leakage (Fig. 2C and D). Since the vascular disruptive responses caused by inflammatory proteins are mediated by the p38 MAPK signaling pathways (Qin et al., 2009; Sun et al., 2009), next, we determined
whether the activation of p38 MAPK was affected by PM2.5 and treatment with 2,2′-bipyridine compounds (1–6). MLMVECs were treated with 2,2′-bipyridines after activation with PM2.5. PM2.5 upregulated the expression of phosphorylated p38, which was clearly reduced by collismycin C or pyrisulfoxin A treatment in purified MLMVECs (Fig. 2E and F) and mice (Fig. 2G and H). Moreover, PM2.5-activated p38 MAPK mediated PM2.5-induced endothelial cell hyperpermeability in 4
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 3. Effects of collismycin C and pyrisulfoxins A on PM-induced generation of reactive oxygen species (ROS). After the peroral administration of the tested compounds (1−6) or hyaluronan (HA; 0.1%) for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline, 10 days). (A) Purified mouse lung microvascular endothelial cells (MLMVECs) isolated from treated mice (>90% confluent on 35 mm dishes) were pretreated with 10 μM 2′,7′–dichlorofluorescein diacetate (DCFDA) for 30 min. (B, C) ROS generation was quantified using immunofluorescence microscopy. Representative images from each group are shown (n = 5). Values represent the means ± SD of three independent experiments. *p < 0.05 vs. PM2.5 challenge. Scale bar, 100 μm.
MLMVECs (Fig. 2I). Cell viability assays were performed to probe the toxicity of collismycin C in purified MLMVECs. At the tested concentrations (up to 1000 μM), collismycin C did not affect cell viability (Fig. 2J).
and Poulogiannis, 2018; Manna and Jain, 2015; Schade et al., 2006; Singleton et al., 2009). Therefore, we determined whether 2 and 5 could activate Akt, and whether collismycin C- or pyrisulfoxin A-induced Akt activation was maintained even under PM challenge. Both 2 and 5 significantly induced Akt phosphorylation, which was attenuated by the PI3 kinase inhibitor, LY294002 in MLMVECs (Fig. 4A). Further, Akt phosphorylation persisted despite PM treatment (1 mg/ml, 6 h). The addition of LY294002 to MLMVECs significantly abolished the protective ability of 2 and 5 against PM2.5-induced endothelial cell barrier disruption (Fig. 4C). To define the specific role of collismycin Cand pyrisulfoxin A-induced Akt activation, we knocked down Akt1, the major Akt isotype in the endothelium (Shiojima and Walsh, 2002). Akt1 small interfering RNA reduced Akt protein expression (Fig. 4B) and significantly prevented 2- or 5-mediated protective effects against PM challenge (Fig. 4C). Taken together, these results indicated that collismycin C and pyrisulfoxin A induced Akt phosphorylation, which contributed to the attenuation of PM-induced MLMVEC barrier disruption.
Effects of collismycin C on PM2.5-stimulated ROS generation in MLMVECs Previous studies have shown that intracellular oxidative stress increases remarkably owing to mitochondrial dysregulation after PM exposure (Gualtieri et al., 2012; Wang et al., 2010; Xu et al., 2018; Zhao et al., 2009). In this study, compounds 2 and 5 abolished PM2.5-induced substantial ROS production, as evidenced by the results of DCFDA oxidation assay (Fig. 3). Thus, 2 and 5 acted as ROS scavengers in the endothelial cells, preventing PM-induced ROS accumulation and significantly inhibiting PM-mediated endothelial cell barrier disruption. Effects of collismycin C and pyrisulfoxin A on Akt phosphorylation and endothelial cell barrier function Phosphatidylinositol 3-phosphate (PI3K)/Akt signaling is one of the most critical pathways in the regulation of cellular survival, proliferation, and metabolism in mammals (Bellacosa et al., 1998; Koundouros 5
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 4. Effects of collismycin C (2) and pyrisulfoxin A (5) on Akt activation. (A) Mouse lung microvascular endothelial cells (MLMVECs) were pretreated with 2 or 5 (500 μM, 6 h) and challenged with PM2.5 (1 mg/ml) for 6 h. Phospho-Akt MAPK and total Akt MAPK expression were determined in cell lysates by using western blot analysis. Representative blots are shown. Values represent the means ± SD of three independent experiments. (B) Akt MAPK and β-actin expression was measured using western blot analysis in control siRNA (siCNTL, 100 μM)- and Akt1 siRNA (siAkt1, 100 μM)-treated MLMVECs. (C) MLMVECs were treated with LY294002 (LY, 10 μM, 1 h), Akt1 siRNA, or control siRNA (100 μM, 3 h). A permeability assay was performed after treatment with 2 or 5 (500 μM, 6 h) and PM2.5 (1 mg/ml). Values represent the mean ± SD of three independent experiments. *p< 0.05. NS, not significant.
Effects of collismycin C and pyrisulfoxin A on PM2.5-induced pulmonary inflammation
several vascular disorders (de Kok et al., 2005; Sun et al., 2008; Wu et al., 2014). The vascular endothelium acts as a dynamic barrier that selectively restricts the passage of plasma and cells from the blood into the adjacent tissues. Reversible alterations in barrier function typically occur during inflammatory responses, during which inflammatory mediators cause a transient increase in vascular permeability (Komarova et al., 2007; Mehta et al., 2014). Further, inflammatory mediators irreversibly disrupt vascular integrity and cause excessive loss of fluids from the circulation in inflammatory diseases (Komarova et al., 2007; Mehta et al., 2014). Therefore, the prevention of vascular disruptive responses could result in improved survival of patients with inflammatory diseases. The present study showed that collismycin C and pyrisulfoxin A inhibited PM2.5-induced endothelial barrier disruption and attenuated PM2.5-induced lung vascular leakage via the p38 MAPK pathway, indicating their potential barrier protective functions in guarding endothelial integrity against PM challenge. Growing epidemiological evidence supports the deleterious cardiopulmonary health effects, mortality, and morbidity associated with the exposure to ambient pollutant particles (Dominici et al., 2006; Peng et al., 2008). Various mechanisms have been proposed to explain the cardiopulmonary health effects of PM, including pulmonary and systemic oxidative stress and inflammation (Schicker et al., 2009), coagulation pathway activation (Mutlu et al., 2007), and cardiac autonomic function alteration (Baccarelli et al., 2008). Recent in vivo and in vitro studies revealed that PM triggered significant lung inflammation and vascular hyperpermeability responses, including endothelial barrier disruption, vascular protein leakage, neutrophil infiltration, and proinflammatory cytokine release (Wang et al., 2010, 2008; Zhao et al., 2009). These pathophysiological alterations in pulmonary cells and tissues lead to systemic inflammation, which is one of the primary
Since collismycin C and pyrisulfoxin A suppressed PM2.5-induced vascular barrier disruptive responses in vivo (Fig. 2), we determined their effects on PM-induced pulmonary inflammatory responses and injury in vivo. PM2.5 caused an increase in inflammatory leukocyte infiltration and leukocyte count in BAL, which was significantly prevented by 2 and 5 (Fig. 5A). Furthermore, 2 and 5 attenuated PM2.5stimulated release of inflammatory cytokines, IL-6 (Fig. 5B) and TNF-α (Fig. 5C), into the BAL fluid. Histological studies confirmed 2- and 5mediated reduction of PM2.5-induced remarkable increase in leukocyte infiltration in murine lung tissue (Fig. 5D). These data indicated that collismycin C and pyrisulfoxin A attenuated PM-induced pulmonary vascular hyperpermeability, inflammatory leukocyte infiltration, and proinflammatory cytokine release, thereby protecting against PMmediated pulmonary inflammatory injury. Discussion Endothelial integrity disruption and vascular hyperpermeability are prominent features of vascular inflammatory diseases, including severe inflammatory response syndrome, sepsis, pulmonary diseases, and atherosclerosis (Curry and Adamson, 2013; Komarova et al., 2007). Therefore, the prevention of endothelial barrier disruption has obvious therapeutic applications. Airborne particulates containing high concentrations of transition metals and polycyclic aromatic hydrocarbons stimulate the excessive generation of ROS in lung tissues, particularly in the vascular endothelium, which is readily susceptible to PM-induced oxidative stress and plays a critical role in the pathophysiology of 6
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Fig. 5. Effects of collismycin C (2) and pyrisulfoxin A (5) on particulate matter-induced pulmonary inflammation and injury. After the peroral administration of 2 or 5 for 10 days, mice were intratracheally challenged with PM2.5 (1 mg/kg in 100 μl of saline, 10 days). (A−C) Total leukocyte counts (A) as well as IL-6 (B) and TNF-α (C) concentrations in bronchoalveolar lavage (BAL) fluids were measured. (D) Lung histology was examined using hematoxylin and eosin staining. Representative images from each group are shown (n = 5). Scale bar, 200 μm. Arrows indicate leukocyte infiltration. Values represent the mean ± SD of three independent experiments. *p < 0.05 vs. PM2.5 challenge.
aggravators of existing cardiopulmonary conditions such as asthma, chronic obstructive pulmonary disease, cardiac arrhythmias, and congestive heart failure. In this study, collismycin C and pyrisulfoxin A scavenged PM2.5-induced ROS; inhibited oxidative stress-induced p38 MAPK and Akt activation; and reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the BAL fluid of PM-treated mice. Therefore, 2 and 5 might attenuate ROSmediated pulmonary inflammation caused by PM air pollution. Among the six tested 2,2′-bipyridines tested, compounds 2–5 showed does-dependent inhibition of PM2.5-mediated vascular barrier disruption and PM2.5-stimulated ROS generation, whereas pyrisulfoxin B (1) and SF2738D (6), which possess 6-nitrile groups, did not. Previously, the aldoxime moiety of caerulomycins was speculated to be the pharmacophore (Fu et al., 2011), and the bioactive 2,2′-bipyridines, including pyrisulfoxins A and B as well as collismycins A and B, have the 6-aldoxime group (Gomi et al., 1994; Tsuge et al., 1999). However, collismycin C (2) possesses 6-hydroxymethyl rather than 6-aldoxime. Therefore, the 6-aldoxime unit does not seem to be critical for the attenuation of airborne PM-induced ROS-dependent pulmonary inflammation by the 2,2′-bipyridines. The influences of other components such as 4-O-methyl, 5-S-methyl, and 5-methyl sulfoxide on the activities of 2,2′-bipyridines are not yet clear. The hydroxyl radical is a harmful byproduct of oxidative metabolism and can cause molecular damage in living systems; it plays a critical role in initiating and catalyzing various radical reactions (Sun et al., 2008). ROS formation exceeds the detoxification capacity of the antioxidant defense system, thereby producing a change in the cell redox status and causing a cascade of events that is closely involved in the inflammatory responses (Garcon et al., 2006; Pozzi et al., 2003). In addition, ROS are thought to play an active role in inducing airway inflammation and hyper-responsiveness (Hulsmann et al., 1994). Our findings suggest that collismycin C might exhibit beneficial effects in the respiratory system against PM-induced ROS formation. In addition, inhaled PM, particularly fine and ultrafine particles with diameters less than 2.5 mm, was shown to move from the alveolar space into the pulmonary circulation and to trigger amplified endothelial dysfunction (Karoly et al., 2007; Kreyling et al., 2002). Moreover, PM challenge results in the migration of activated neutrophils from the blood into the lung interstitium, which can lead to the production of significant amounts of ROS, thereby exacerbating inflammation (Walters et al., 2001). In this study, we showed that PM induced significant ROS
production in the pulmonary endothelial cells, thereby activating p38 MAPK. Activated p38 MAPK is involved in actin filament reorganization in response to oxidative stress, to facilitate stress actin fiber synthesis and paracellular gap formation, which are essential markers of endothelial cell integrity disruption (Wang et al., 2010), as was evident by the disruption of vascular barrier integrity in this study (Fig. 2). Collismycin C inhibited PM2.5-induced endothelial barrier disruption and attenuated PM2.5-induced lung vascular leakage via the p38 MAPK pathway; therefore, it could act as an endogenous ROS scavenger and maintain endothelial integrity during oxidative stress. Our findings showed that collismycin C and pyrisulfoxin A prevented PM-induced endothelial integrity disruption and vascular hyperpermeability via the dual actions of ROS scavenging and Akt pathway activation. Thus, they might attenuate PM air pollution-induced ROS-mediated pulmonary inflammation. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [Grant nos. 2018R1A5A2025272 and 2017R1A2B4006110]. Conflict of interest The authors declare no conflicts of interest. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2019.152939. References Baccarelli, A., Cassano, P.A., Litonjua, A., Park, S.K., Suh, H., Sparrow, D., Vokonas, P., Schwartz, J., 2008. Cardiac autonomic dysfunction: effects from particulate air pollution and protection by dietary methyl nutrients and metabolic polymorphisms. Circulation 117, 1802–1809. Bellacosa, A., Chan, T.O., Ahmed, N.N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J.N., Tsichlis, P., 1998. Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17, 313–325. Curry, F.R., Adamson, R.H., 2013. Tonic regulation of vascular permeability. Acta Physiol. (Oxf) 207, 628–649. de Kok, T.M., Engels, L.G., Moonen, E.J., Kleinjans, J.C., 2005. Inflammatory bowel disease stimulates formation of carcinogenic N-nitroso compounds. Gut 54, 731.
7
Phytomedicine 62 (2019) 152939
H. Choi, et al.
Peng, R.D., Chang, H.H., Bell, M.L., McDermott, A., Zeger, S.L., Samet, J.M., Dominici, F., 2008. Coarse particulate matter air pollution and hospital admissions for cardiovascular and respiratory diseases among Medicare patients. JAMA 299, 2172–2179. Piao, M.J., Ahn, M.J., Kang, K.A., Ryu, Y.S., Hyun, Y.J., Shilnikova, K., Zhen, A.X., Jeong, J.W., Choi, Y.H., Kang, H.K., Koh, Y.S., Hyun, J.W., 2018. Particulate matter 2.5 damages skin cells by inducing oxidative stress, subcellular organelle dysfunction, and apoptosis. Arch. Toxicol. 92, 2077–2091. Pozzi, R., De Berardis, B., Paoletti, L., Guastadisegni, C., 2003. Inflammatory mediators induced by coarse (PM2.5-10) and fine (PM2.5) urban air particles in RAW 264.7 cells. Toxicology 183, 243–254. Qin, Y.H., Dai, S.M., Tang, G.S., Zhang, J., Ren, D., Wang, Z.W., Shen, Q., 2009. HMGB1 enhances the proinflammatory activity of lipopolysaccharide by promoting the phosphorylation of MAPK p38 through receptor for advanced glycation end products. J. Immunol. 183, 6244–6250. Schade, A.E., Powers, J.J., Wlodarski, M.W., Maciejewski, J.P., 2006. Phosphatidylinositol-3-phosphate kinase pathway activation protects leukemic large granular lymphocytes from undergoing homeostatic apoptosis. Blood 107, 4834–4840. Schicker, B., Kuhn, M., Fehr, R., Asmis, L.M., Karagiannidis, C., Reinhart, W.H., 2009. Particulate matter inhalation during hay storing activity induces systemic inflammation and platelet aggregation. Eur. J. Appl. Physiol. 105, 771–778. Shindo, K., Yamagishi, Y., Okada, Y., Kawai, H., 1994. Collismycins A and B, novel nonsteroidal inhibitors of dexamethasone-glucocorticoid receptor binding. J. Antibiot. (Tokyo). 47, 1072–1074. Shiojima, I., Walsh, K., 2002. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ. Res. 90, 1243–1250. Singleton, P.A., Chatchavalvanich, S., Fu, P., Xing, J., Birukova, A.A., Fortune, J.A., Klibanov, A.M., Garcia, J.G., Birukov, K.G., 2009. Akt-mediated transactivation of the S1P1 receptor in caveolin-enriched microdomains regulates endothelial barrier enhancement by oxidized phospholipids. Circ. Res. 104, 978–986. Sun, C., Liang, C., Ren, Y., Zhen, Y., He, Z., Wang, H., Tan, H., Pan, X., Wu, Z., 2009. Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways. Basic Res. Cardiol. 104, 42–49. Sun, Y., Yin, Y., Zhang, J., Yu, H., Wang, X., Wu, J., Xue, Y., 2008. Hydroxyl radical generation and oxidative stress in Carassius auratus liver, exposed to pyrene. Ecotoxicol. Environ. Saf. 71, 446–453. Tsuge, N., Furihata, K., Shin-Ya, K., Hayakawa, Y., Seto, H., 1999. Novel antibiotics pyrisulfoxin A and B produced by Streptomyces californicus. J. Antibiot. (Tokyo). 52, 505–507. Walters, D.M., Breysse, P.N., Wills-Karp, M., 2001. Ambient urban Baltimore particulateinduced airway hyperresponsiveness and inflammation in mice. Am. J. Respir. Crit. Care Med. 164, 1438–1443. Wang, H., Song, L., Ju, W., Wang, X., Dong, L., Zhang, Y., Ya, P., Yang, C., Li, F., 2017a. The acute airway inflammation induced by PM2.5 exposure and the treatment of essential oils in Balb/c mice. Sci. Rep. 7, 44256. Wang, T., Chiang, E.T., Moreno-Vinasco, L., Lang, G.D., Pendyala, S., Samet, J.M., Geyh, A.S., Breysse, P.N., Chillrud, S.N., Natarajan, V., Garcia, J.G., 2010. Particulate matter disrupts human lung endothelial barrier integrity via ROS- and p38 MAPKdependent pathways. Am. J. Respir. Cell Mol. Biol. 42, 442–449. Wang, T., Moreno-Vinasco, L., Huang, Y., Lang, G.D., Linares, J.D., Goonewardena, S.N., Grabavoy, A., Samet, J.M., Geyh, A.S., Breysse, P.N., Lussier, Y.A., Natarajan, V., Garcia, J.G., 2008. Murine lung responses to ambient particulate matter: genomic analysis and influence on airway hyperresponsiveness. Environ. Health Perspect. 116, 1500–1508. Wang, T., Shimizu, Y., Wu, X., Kelly, G.T., Xu, X., Wang, L., Qian, Z., Chen, Y., Garcia, J.G.N., 2017b. Particulate matter disrupts human lung endothelial cell barrier integrity via Rho-dependent pathways. Pulm. Circ. 7, 617–623. Wu, S., Deng, F., Hao, Y., Wang, X., Zheng, C., Lv, H., Lu, X., Wei, H., Huang, J., Qin, Y., Shima, M., Guo, X., 2014. Fine particulate matter, temperature, and lung function in healthy adults: findings from the HVNR study. Chemosphere 108, 168–174. Xing, Y.F., Xu, Y.H., Shi, M.H., Lian, Y.X., 2016. The impact of PM2.5 on the human respiratory system. J. Thorac. Dis. 8, E69–E74. Xu, C., Shi, Q., Zhang, L., Zhao, H., 2018. High molecular weight hyaluronan attenuates fine particulate matter-induced acute lung injury through inhibition of ROS-ASK1p38/JNK-mediated epithelial apoptosis. Environ. Toxicol. Pharmacol. 59, 190–198. Zhang, L., Wang, M.C., 2018. Growth inhibitory effect of mangiferin on thyroid cancer cell line TPC1. Biotechnol. Bioprocess Eng. 23, 649–654. Zhao, Y., Usatyuk, P.V., Gorshkova, I.A., He, D., Wang, T., Moreno-Vinasco, L., Geyh, A.S., Breysse, P.N., Samet, J.M., Spannhake, E.W., Garcia, J.G., Natarajan, V., 2009. Regulation of COX-2 expression and IL-6 release by particulate matter in airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 40, 19–30.
Dominici, F., Peng, R.D., Bell, M.L., Pham, L., McDermott, A., Zeger, S.L., Samet, J.M., 2006. Fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases. JAMA 295, 1127–1134. Falcon-Rodriguez, C.I., Osornio-Vargas, A.R., Sada-Ovalle, I., Segura-Medina, P., 2016. Aeroparticles, composition, and lung diseases. Front. Immunol. 7, 3. Fu, P., Wang, S., Hong, K., Li, X., Liu, P., Wang, Y., Zhu, W., 2011. Cytotoxic bipyridines from the marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6. J. Nat. Prod. 74, 1751–1756. Funk, A., Divekar, P.V., 1959. Caerulomycin, a new antibiotic from Streptomyces caeruleus Baldacci. I. Production, isolation, assay, and biological properties. Can. J. Microbiol. 5, 317–321. Garcon, G., Dagher, Z., Zerimech, F., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., Shirali, P., 2006. Dunkerque city air pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells (L132) in culture. Toxicol. In Vitro 20, 519–528. Gomi, S., Amano, S., Sato, E., Miyadoh, S., Kodama, Y., 1994. Novel antibiotics SF2738A, SF2738B and SF2738C, and their analogs produced by Streptomyces sp. J. Antibiot. (Tokyo). 47, 1385–1394. Gualtieri, M., Longhin, E., Mattioli, M., Mantecca, P., Tinaglia, V., Mangano, E., Proverbio, M.C., Bestetti, G., Camatini, M., Battaglia, C., 2012. Gene expression profiling of A549 cells exposed to Milan PM2.5. Toxicol. Lett. 209, 136–145. Hulsmann, A.R., Raatgeep, H.R., den Hollander, J.C., Stijnen, T., Saxena, P.R., Kerrebijn, K.F., de Jongste, J.C., 1994. Oxidative epithelial damage produces hyperresponsiveness of human peripheral airways. Am. J. Respir. Crit. Care Med. 149, 519–525. Jang, M.H., Kang, N.H., Mukherjee, S., Yun, J.W., 2018. Theobromine, a methylxanthine in cocoa bean, stimulates thermogenesis by inducing white fat browning and activating brown adipocytes. Biotechnol. Bioprocess Eng. 23, 617–626. Karoly, E.D., Li, Z., Dailey, L.A., Hyseni, X., Huang, Y.C., 2007. Up-regulation of tissue factor in human pulmonary artery endothelial cells after ultrafine particle exposure. Environ. Health Perspect. 115, 535–540. Kim, J.E., Lee, W., Yang, S., Cho, S.H., Baek, M.C., Song, G.Y., Bae, J.S., 2019. Suppressive effects of rare ginsenosides, Rk1 and Rg5, on HMGB1-mediated septic responses. Food Chem. Toxicol. 124, 45–53. Komarova, Y.A., Mehta, D., Malik, A.B., 2007. Dual regulation of endothelial junctional permeability. Sci. STKE 2007, re8. Koundouros, N., Poulogiannis, G., 2018. Phosphoinositide 3-kinase/Akt signaling and redox metabolism in cancer. Front. Oncol. 8, 160. Kovacs-Kasa, A., Varn, M.N., Verin, A.D., Gonzales, J.N., 2017. Method for the culture of mouse pulmonary microvascular endothelial cells. Sci. Pages Pulmonol. 1, 7–18. Kreyling, W.G., Semmler, M., Erbe, F., Mayer, P., Takenaka, S., Schulz, H., Oberdorster, G., Ziesenis, A., 2002. Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low. J. Toxicol. Environ. Health A 65, 1513–1530. Kujur, W., Gurram, R.K., Haleem, N., Maurya, S.K., Agrewala, J.N., 2015. Caerulomycin A inhibits Th2 cell activity: a possible role in the management of asthma. Sci. Rep. 5, 15396. Kujur, W., Gurram, R.K., Maurya, S.K., Nadeem, S., Chodisetti, S.B., Khan, N., Agrewala, J.N., 2017. Caerulomycin A suppresses the differentiation of naive T cells and alleviates the symptoms of experimental autoimmune encephalomyelitis. Autoimmunity 50, 317–328. Lee, I.C., Bae, J.S., 2019. Pelargonidin protects against renal injury in a mouse model of sepsis. J. Med. Food 22, 57–61. Lee, J.H., Kim, E., Choi, H., Lee, J., 2017. Collismycin C from the Micronesian Marine Bacterium Streptomyces sp. MC025 Inhibits Staphylococcus aureus Biofilm Formation. Mar. Drugs 15 (12), 387. Lee, W., Cho, S.H., Kim, J.E., Lee, C., Lee, J.H., Baek, M.C., Song, G.Y., Bae, J.S., 2019. Suppressive effects of Ginsenoside Rh1 on HMGB1-mediated septic responses. Am. J. Chin. Med. 47, 119–133. Liu, Q., Xu, C., Ji, G., Liu, H., Shao, W., Zhang, C., Gu, A., Zhao, P., 2017. Effect of exposure to ambient PM2.5 pollution on the risk of respiratory tract diseases: a metaanalysis of cohort studies. J. Biomed. Res. 31, 130–142. Manna, P., Jain, S.K., 2015. Phosphatidylinositol-3,4,5-triphosphate and cellular signaling: implications for obesity and diabetes. Cell. Physiol. Biochem. 35, 1253–1275. Mehta, D., Ravindran, K., Kuebler, W.M., 2014. Novel regulators of endothelial barrier function. Am. J. Physiol. Lung Cell Mol. Physiol. 307, L924–L935. Mutlu, G.M., Green, D., Bellmeyer, A., Baker, C.M., Burgess, Z., Rajamannan, N., Christman, J.W., Foiles, N., Kamp, D.W., Ghio, A.J., Chandel, N.S., Dean, D.A., Sznajder, J.I., Budinger, G.R., 2007. Ambient particulate matter accelerates coagulation via an IL-6-dependent pathway. J. Clin. Invest. 117, 2952–2961. Ozdulger, A., Cinel, I., Koksel, O., Cinel, L., Avlan, D., Unlu, A., Okcu, H., Dikmengil, M., Oral, U., 2003. The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and puncture-induced sepsis model. Shock 19, 366–372.
8