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1,25-Vitamin D3 protects against cooking oil fumes-derived PM2.5-induced cell damage through its anti-inflammatory effects in cardiomyocytes
Ecotoxicology and Environmental Safety 179 (2019) 249–256
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1,25-Vitamin D3 protects against cooking oil fumes-derived PM2.5-induced cell damage through its anti-inflammatory effects in cardiomyocytes
T
Chun-Miao Luoa,b, Jun Fengb, Jing Zhangb, Chao Gaob, Ji-Yu Caoc,d, Gao-Liang Zhoub, Yong-Jing Jiangb, Xiao-Qing Jinb, Meng-Si Yangb, Jian-Yuan Panb, Ai-Ling Wanga,∗ a
Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, HeFei, Anhui, China Department of Cardiology, The Second People Hospital of Hefei, HeFei, Anhui, China c Department of Occupational and Environmental Health, School of Public Health, Anhui Medical University, HeFei, Anhui, China d The Teaching Center for Preventive Medicine, School of Public Health, Anhui Medical University, HeFei, Anhui, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: COFs-derived PM2.5 Cardiomyocytes Cell injury Inflammation 1,25-Vitamin D3 JAK/STAT pathway NF-κB pathway
The functional role of 1,25-vitamin D3 in cooking oil fumes (COFs)-derived PM2.5-induced cell damage is largely unexplored. The present study investigated the protective role of 1,25-vitamin D3 against cell injury by possible involvement of JAK/STAT and NF-κB signaling pathways in cardiomyocytes. Cell viability was measured using CCK-8 assay, and cell apoptosis was analyzed by flow cytometry, qRT-PCR and Western blot in cultured rat neonatal cardiomyocytes treated with 1,25-vitamin D3 and COFs-derived PM2.5. Expressions of JAK/STAT and NF-κB signaling pathway were measured by Western blot. The results suggested that treatment with COFs-derived PM2.5 significantly decreased cell viability and increased apoptosis and oxidative stress in cultured rat neonatal cardiomyocytes. 1,25-vitamin D3 pretreatment alleviated the cell injury by increasing cell viability and decreasing apoptosis in the cardiomyocytes. 1,25-vitamin D3 pretreatment also decreased the ROS level and inflammation in the cardiomyocytes. Furthermore, 1,25-vitamin D3 pretreatment alleviated COFsderived PM2.5-evoked elevation of JAK/STAT and NF-κB signaling pathways. Our study showed that 1,25vitamin D3 pretreatment protected cardiomyocytes from COFs-derived PM2.5-induced injury by decreasing ROS, apoptosis and inflammation level via activations of the JAK/STAT and NF-κB signaling pathways.
1. Introduction
2017). These processes are accompanied by the down-regulation of bcl2 and up-regulation of bax, which are important regulators of cellular apoptosis (D'Orsi et al., 2017). Data show that a decreased bcl-2/bax ratio increases the apoptosis risk in myocardial cells (Zhang and Yu, 2017). It has been confirmed that cardiomyocyte apoptosis is one of the most common pathophysiological processes in injury resulted from oxidative stress (Wang et al., 2017). Hypovitaminosis D is increasingly recognized as an independent predictor for primary cardiovascular events and related diseases, such as CVD (Al and Quyyumi, 2017; Zhang et al., 2017a). In addition, vitamin D deficiency is highly prevalent in patients with heart failure being a significant indicator of reduced survival, whereas supplementation was associated with an improved outcome (Witte et al., 2016). The National Health and Nutritional Examination Surveys (NHANES) (1988–1994, 2000–2004) conducted in the US have been designed to explore the association between vitamin D status and CVD and researchers found that individuals included in NHANES 1988–1994 with vitamin D deficiency (vitamin D < 20 ng/mL) had higher
Epidemiological investigations have showed a strong relationship between atmospheric particulate matters, especially an aerodynamic diameter less than 2.5 μm (PM2.5) and increased incidence, mortality and morbidity of cardiovascular disorders (CVD), including atherosclerosis, ischemic cardiovascular events, coronary artery disease and hypertension (Marcellin et al., 2016; Kim et al., 2017; Pope et al., 2015). One of the possible mechanisms by which PM2.5 exposure can contribute to an increased risk for adverse cardiovascular events is dysfunction of cardiac cells (Pope et al., 2016). PM2.5 can be absorbed and taken in cells, not only adhering to cell surfaces, and induce detrimental effects (Stone et al., 2017). Key indicators of cellular dysfunction include the loss of mitochondrial membrane potential, intracellular overproduction of ROS, the decrease of cell viability, and so on (Yang et al., 2017; Park et al., 2017). Intracellular overproduction of ROS is known to decrease the mitochondrial membrane potential, activate pro-caspases and results in apoptotic cell death (Crobeddu et al.,
∗
Corresponding author. E-mail addresses: [email protected], [email protected] (A.-L. Wang).
https://doi.org/10.1016/j.ecoenv.2019.04.064 Received 27 November 2018; Received in revised form 16 April 2019; Accepted 22 April 2019 Available online 01 May 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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were approved by the Ethics Committee of Anhui Medical University and the first Affiliated Hospital of Anhui Medical University and carried out under the institutional guidelines for ethical animal use. Briefly, ventricular cardiomyocytes were isolated from 1 to 2 days of old neonatal Sprague-Dawley rats, and then chopped and digested several times in collagenase II at 37 °C. To prepare almost pure cardiomyocytes, cells were incubated in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, antibiotics, L-glutamine. After 30 min, the plate was washed briefly and gently with the cell solution in the dish and plated again with fresh medium and repeat until most myocytes are washed off to avoid the non-myocyte cells. Further myocytes were plated in collagen precoated dishes or in fibronectin-precoated glass coverslips at a density of 160,000/cm2 in 10% fetal bovine serum DMEM supplemented with 1 mmol/l l-glutamine, antibiotic/antimycotic solution, 0.1 M AraC (1 μL/ml) and 100 μmol/l 5-bromo-2-deoxyuridine (BrdU). Under these high density conditions, the myocytes form cell-cell contacts and display spontaneous contractile activity within 24 h of plating. The cells were maintained at 37 °C with 100% relatively humidity in a CO2 incubator containing 5% CO2. Cardiac myocyte cell suspensions were transferred into 6-well gelatin-coated plates at a density of 1–5 × 106 cells/well for protein and mRNA extraction. Cells were plated 1 × 105 cells/well for cell viability, namely LDH leakage and CCK-8 assay.
prevalence of self-reported angina, heart failure, and myocardial infarction compared to the individuals with higher levels of vitamin D (Rautiainen et al., 2010). A few in vitro and in vivo studies have evaluated the role of vitamin D on cardiac tissue directly, especially in response to injury. It has been demonstrated that matrix metalloproteinases (MMP) proteins, which contribute to aberrant cardiomyocyte remodeling in response to injury and atherosclerosis, were upregulated in vitamin D receptor (VDR) knockout mice (Rahman et al., 2007). It has been also shown that VDR knockout mice have impaired cardiac relaxation and contractility and develop left ventricular hypertrophy (Rahman et al., 2007). A study in HL-1 murine cardiac myocytes showed that 1,25-vitamin D3 significantly decreased cell proliferation, increased cell size, and resulted in hypoplasia, slight hypertrophy, and altered morphology of dividing cardiomyocytes, demonstrating that 1,25-vitamin D3 is involved in maintaining structure and function of cardiac cell (Nibbelink et al., 2007a). In addition, Morán-Auth et al. demonstrated that 1,25-vitamin D3 promoted gene transcription in immune cells, such as 25-vitamin-Dhydroxylase (CYP2R1) and 25-hydroxyvitamin-D3-1a-hydroxylase (CYP27B1), suggesting that 1,25-vitamin D3 may also have a protective effect on the stress response to injury (Morán-Auth et al., 2013). Even though most evidences indicate a protective effect of vitamin D on inflammation, the role of 1,25-vitamin D3 on cardiac cell responding to COFs-derived PM2.5 remains poorly understood. In this study, we employed cultured rat neonatal cardiomyocytes, derived from neonatal rat heart, which have been extensively used as an in vitro model for cardiac function to examine the effect and mechanism of 1,25-vitamin D3 on myocardial injury induced by COFs-derived PM2.5 and attempt to explore the role of JAK/STAT and NF-κB signaling pathways in 1,25-vitamin D3 -mediated cardioprotective actions.
2.4. Quantitative real-time PCR analysis Total RNA was isolated from cells using TRIzol reagent (Invitrogen) as per manufacturer's instructions. DNaseI (Promega) was added to the isolated RNA to remove DNA impurities. Reverse transcription was performed by MultiScribe RT kit (Applied Biosystems) using random hexamers or oligo (dT). The PCR conditions for the experiments were 10 min at 25 °C, 30 min at 48 °C, and 5 min at 95 °C. All primers were designed using the Primer Premier 5.0 software and synthesized by Shenggong Co. Ltd. Specific primers were designed as follows: Bax. Forward: 5′-GGA GAC ACC TGA GCT GAC CT-3′ Reverse: 5′-GAC GAC TCC AGC CAC AAA GA-3′ Bcl-2. Forward: 5′-AAG CTG TCA CAG AGG GGC TC-3′ Reverse: 5′-CAG ATG CCG GTT CAG GTA CT-3′ GAPDH. Forward: 5′-GGC CTT CCG TGT TCC TAC C-3′ Reverse: 5′-CGG CAT GTC AGA TCC ACA AC-3′
2. Material and methods 2.1. 1,25-Vitamin D3 preparation 10−3 mol/L 1,25-vitamin D3 solution was prepared by dissolving 1 mg 1,25-vitamin D3 (molecular weight: 416.64, Sigma, USA) in 2.4 mL 95% ethanol, and stored at −20 °C in the dark until needed for used. 1 μL 10−3 mol/L 1,25-vitamin D3 solution was diluted with 99 μL DMEM medium with 10% fetal bovine serum (10−5 mol/L 1,25-vitamin D3 solution was prepared), 1 μL 10−5 mol/L 1,25-vitamin D3 solution was diluted with 999 μL DMEM medium (10−8 mol/L 1,25-vitamin D3 solution was prepared). 2.2. Preparation of COFs-derived PM2.5
2.5. Cell counting kit-8 assay
The collection and extraction of PM2.5 were in accordance with the procedures followed by Cao et al. methods, which were modified by ourselves (Cao et al., 2013). Peanut oil (200 ml) was poured into an iron pot and heated with an electric heater. The temperature was maintained at the smoke point (280 ± 10 °C). The fumes generated from the heating oil were collected, with filter paper placed 50 cm upon the oil surface, at a flow rate of 12 L/min, which connected to a vacuum pump. Each filter paper was renewed once after 2 h. The experiments were independently repeated three times. The gathered condensates were separately extracted with 50 ml of acetone using a Soxhlet extractor for 24 h. The extracts were then dried by rotary evaporation at 40 °C. The eluant was evaporated to a yellow viscous solution and diluted to 200 mg/mL with dimethylsulfoxide (DMSO). The samples were sealed up in brown glass vials and preserved at −80 °C until use.
Cell viability was assessed by a cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD). Cells were seeded in a 96well plate with 5000 cells/well. After stimulation, the CCK-8 solution was added to the culture medium, and the cultures were incubated for 1 h at 37 °C in humidified 95% air and 5% CO2. The absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA). 2.6. Annexin V/propidium iodide (PI) double staining assay Cell apoptosis analysis was performed using PI and fluorescein isothiocyanate (FITC)-conjugated Annexin V staining. Briefly, cells were washed in phosphate buffered saline (PBS) and fixed in 70% ethanol. Fixed cells were then washed twice in PBS and stained in PI/ FITC-Annexin V in the presence of 50 μg/mL RNase A (Sigma-Aldrich), and then incubated for 1 h at room temperature in the dark. Flow cytometry analysis was done using a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA) to differentiate apoptotic cells (Annexin-V positive and PI-negative) from necrotic cells (Annexin-V and PI-positive). The data were analyzed using FlowJo software.
2.3. Cell culture and treatment Ventricular myocytes were isolated from the hearts of neonatal rats (SD rats) that were up to 2 days old and were isolated and cultured as described previously (Rutering et al., 2015). All the animal experiments 250
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Fig. 1. VitD3 pretreatment prevented COFs-derived PM2.5-induced cell damage in cultured rat neonatal cardiomyocytes. (A) CCK-8 levels in cells incubated with different doses of COFs-derived PM2.5. (B) LDH levels in cells incubated with different doses of COFs-derived PM2.5. (C) CCK-8 levels in cells incubated with different doses VitD3. (D) LDH levels in cells incubated with different doses of VitD3. (E) CCK-8 levels in cells incubated with COFs-derived PM2.5 in absence or presence of VitD3 (10−8 mol/L). (F) LDH levels in cells incubated with COFs-derived PM2.5 in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
2.7. ROS assay
521 nm emission).
The total ROS was measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) (Nanjing Jiancheng, Nanjing, China). The cells seeded in a 6-well plate, were washed twice with PBS post treatment and co-incubated with serum-free culture medium containing 10 μM DCFH-DA (20 min, 37 °C, in dark). All the samples were centrifuged and the supernatants were resuspended in 500 μL PBS. The fluorescent intensities were measured using a fluorospectrophotometer (488 nm excitation,
2.8. Cellular antioxidant defense measurements To verify cellular antioxidant defenses, superoxide dismutase (SOD) activities and GSH levels were assessed. Then, 24 h after treatment, cells were washed twice with PBS and scraped in cell lysis buffer (10 mM Tris-HCl, 1% Triton X-100, pH 7.4). Cellular lysate was obtained after sonication for 1 min (5 s on, 5 s off, 30% amplitude) on ice 251
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3. Results
and centrifugation at 14,000 rpm for 10 min at 4 °C. Protein concentrations were determined by the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The supernatants were used to evaluate SOD activities and GSH levels. To determine SOD activity, 930 μL of buffer (200 mM Tris HCl, 2 mM EDTA, pH 8.2) containing supernatant aliquots (50 μg/mL of protein) was mixed with 70 μL of pyrogallol (15 mM) (Nanjing Jiancheng, Nanjing, China). After 2 min of incubation at room temperature, the absorbance was assessed at 420 nm. The amount of SOD that inhibited 50% of the pyrogallol oxidation compared to the negative control was considered one unit of SOD activity, and the results were expressed as unit of SOD/μg protein. The amount of yellow color produced by 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) directly reflected the content of GSH reacting for 25 min at 25 °C, according to the assay kit (Nanjing Jiancheng, Nanjing, China). It was measured by the absorbance at 412 nm and the results were expressed as unit of SOD/μg protein.
3.1. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced cell damage in cultured rat neonatal cardiomyocytes LDH leakage and cell viability generally served as indexes of cardiomyocyte injury, which were measured at 0, 12.5, 25, 50, and 100 μg/mL of the COFs-derived PM2.5. As shown in Fig. 1A and Fig. 1B, a dose-dependent increase of LDH leakage and a dose-dependent decrease of CCK-8 levels were observed in treated cells within different treated doses. A significant injury was found in treated cells with 50 μg/ mL COFs-derived PM2.5, and the dose was used in further experiments. In order to determine the cellular compatibility of 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes, the effects of 1,25-vitamin D3 with dose ranged from 1 to 100 nM were measured using CCK-8 kitbased viability assay and LDH assay after incubating the cells for 24 h (Fig. 1C and D). It was observed that dose ranged of 1,25-vitamin D3 showed non-significant toxic effect. 10 nM of 1,25-vitamin D3 was selected for further experiments. To determine the effects of pretreatment with 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes treated with COFs-derived PM2.5, CCK-8 assay was performed on the cells that were pretreated with 1,25-vitamin D3 for 24 h before undergoing COFsderived PM2.5-induced injury (Fig. 1E). The CCK-8 assay results showed that pretreatment with 1,25-vitamin D3 significantly suppressed the decrease of viability in a dose-dependent manner, indicating that 1,25-vitamin D3 increased cell viability and protected cardiomyocytes against COFs-derived PM2.5-induced injury. To further determine the protective effect of 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes undergoing COFs-derived PM2.5 treatment, the activities of LDH in the supernatant, which could indirectly reflect the degree of injury, were analyzed (Fig. 1F). Pretreatment with 1,25vitamin D3 also significantly suppressed increased LDH activity (P < 0.05 vs treated group).
2.9. Western blot assay The proteins used for western blotting were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Guangzhou, China). The proteins were quantified using BCA™ Protein Assay Kit. Western blot system was established using Bio-Rad Bis-Tris Gel system according to the manufacturer's instructions. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1000. Primary antibody was incubated with the membrane at 4 °C overnight, followed by wash and incubation with secondary antibody marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride membranecarried blots and antibodies were transferred into Bio-Rad ChemiDoc™ XRS system, and then 200 μL Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, China).
3.2. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced oxidative stress in cultured rat neonatal cardiomyocytes It is generally accepted that mitochondrial generation of reactive oxygen species plays a role in cell death. Levels of intracellular reactive oxygen species were reflected by DCF fluorescence intensity (Fig. 2A). The significant increase of the intracellular reactive oxygen species peak indicated that reactive oxygen species generation was significantly increased in the COFs-derived PM2.5-treated group (P < 0.05 vs control group). However, in the 1,25-vitamin D3 and COFs-derived PM2.5 group, the reactive oxygen species peak level significantly decreased (P < 0.05 vs treated group), indicating that 1,25-vitamin D3 pretreatment decreased reactive oxygen species generation. GSH assay and total SOD assay were also performed to determine the protective effect of 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes against COFs-derived PM2.5 treatment and the data were consistent with above results (Fig. 2 B and C).
2.10. ELISA assay Cells were incubated in culture medium containing a designated concentrations of COFs-derived PM2.5 and VitD3 for 24 h. Then IL-6 and TNF-α levels in the cell culture medium were determined according to the manufacturer's instructions (Boster Biological Technology, Wuhan, China). Briefly, 96-well immunoplates were coated with capture antibody overnight at 4 °C, washed with PBS containing 0.05% tween-20 (PBST) and blocked with assay diluent for 1 h at RT to avoid nonspecific binding. Samples and standard antibodies were added to each well and incubated for 2 h at RT. The reactants were incubated serially with detection antibody (1 h), horseradish peroxidase-streptavidin solution (30 min), and substrates (15 min). Finally, absorbance was measured at 450 nm using a multiplate reader, and the cytokine levels in each sample were calculated using the corresponding standard curve.
3.3. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced apoptosis in cultured rat neonatal cardiomyocytes Previous studies have shown that urban dust exposure can result in apoptosis and cardiac dysfunction, and destruction of heart tissue. In this study, we examined whether 1,25-vitamin D3 could protect cells against COFs-derived PM2.5-induced apoptosis. Cultured rat neonatal cardiomyocytes were harvested and subjected to annexin V/PI double staining, then analyzed by flow cytometry (Fig. 3A). Cell apoptosis of cells in the COFs-derived PM2.5 group was significantly increased (P < 0.05 vs control group). However, in cells that were pretreated with 1,25-vitamin D3, apoptosis of cardiomyocytes was decreased (P > 0.05 vs treated group). We also found that the levels of GRP78, caspase-1, and cleaved caspase-3 were increased in COFs-derived
2.11. Statistical analysis The statistical analysis was performed using SPSS 16.0 statistical software. All the experiments were replicated at least thrice and the results were expressed as mean ± SD. One-way analysis of variance and the Student-Newman-Keuls post hoc test were used to determine differences among different groups. P values of < 0.05 was considered to indicate a statistically significant result.
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Fig. 2. VitD3 pretreatment prevented COFs-derived PM2.5-induced oxidative stress in cultured rat neonatal cardiomyocytes. (A) ROS fluorescence intensities in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). (B) SOD activity in cells incubated with COFs-derived PM2.5 (50 μg/ mL) in absence or presence of VitD3 (10−8 mol/L). (C) GSH level in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/ L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
results in activities of caspases. To determine whether 1,25-vitamin D3 protects against COFs-derived PM2.5 induced apoptosis by modulating the bcl-2 family and activities of caspases in cultured rat neonatal cardiomyocytes, RT-qPCR and Western blot were performed to evaluate
PM2.5 group, whereas there was a decrease of caspase-3 activities in 1,25-vitamin D3 pretreated group (P > 0.05 vs treated group) (Fig. 3B). Furthermore, previous studies have shown that mitochondria integrate death signals through the bcl-2 family which eventually
Fig. 3. VitD3 pretreatment prevented COFs-derived PM2.5-induced apoptosis in cultured rat neonatal cardiomyocytes. (A) Apoptosis levels measured by flow cytometry of cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). (B) Expression of apoptosis biomarkers in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L) using Western blot. (C) Bax/Bcl-2 ratio detected with qRT-PCR in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group. 253
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Fig. 4. VitD3 pretreatment prevented COFs-derived PM2.5-induced inflammation in cultured rat neonatal cardiomyocytes. Elisa assay and quantitative analysis of IL6 (A) and TNF-α (B) expression. Cells were incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
2017; Pilz et al., 2016). Air pollution, especially urban PM2.5 is associated with a high prevalence of vitamin D deficiency, and low levels of vitamin D are in turn correlated with adverse clinical outcomes in this patient population (Mulligan et al., 2014; Rosser et al., 2014). Low vitamin D levels are associated with elevated blood pressure, increased hospitalization rates, and higher incidence of all-cause mortality. Oxidative stress-induced cell injury plays a critical role in the pathophysiology of many diseases. Oxidative stress especially increases excessive production of ROS, which may contribute to a number of cardiovascular diseases, such as atherosclerosis, hypertension and myocardial ischemic reperfusion injury (Niemann et al., 2017). The cellular sources of ROS are accumulated from mitochondrial electron transport chain, lipoxygenase and auto-oxidation of catecholamines (Belaidi et al., 2016). Continuous persistence of this oxidative insult can lead to programmed cell death, causing the loss of functional cardiomyocytes. Despite significant improvement in the treatment strategies to reduce cardiovascular risks, the disease still remains challenging in the developing world. The present study was undertaken to explore the effects of 1,25vitamin D3 on COFs-derived PM2.5-induced cell injury in cultured rat neonatal cardiomyocytes and further to investigate the mechanism of this phenomenon. COFs-derived PM2.5-induced oxidative stress, increased apoptosis, and inflammation in cultured rat neonatal cardiomyocytes and resulted in decreased cell viability. Pretreatment of 1,25vitamin D3 significantly increased cell viability, and decreased apoptosis, inflammation, and ROS level in cultured rat neonatal cardiomyocytes. The mechanistic study revealed that pretreatment of 1,25vitamin D3 deactivated the JAK/STAT and NF-κB signaling pathways, which were in accordance with previously published evidences. Studies have portrayed 1,25-vitamin D3 as a biomarker and modulator of heart failure in dilated cardiomyopathy, and vitamin D levels were significantly decreased in mice models with acute myocardial infarction (MI) compared to non-MI controls (Zhang et al., 2018; Kolaszko et al., 2018). An in vivo study was conducted to examine the functional role of pretreatment of 1,25-vitamin D3 on cardiomyocyte survival in acute MI (El-Gohary and Allam, 2017). The infarct size and the ratio of infarct area to area at risk 24 h post MI was decreased within 1,25-vitamin D3 pretreatment when compared to the wild-type rat model. The percentage of apoptotic cells was also significantly reduced in the infarcted left ventricle of 1,25-vitamin D3 pretreatment rat vs. control. Pretreatment of 1,25-vitamin D3 protected HL-1 cells from staurosporineinduced apoptosis by blocking cytochrome C release and caspase 3 cleavage (Nibbelink et al., 2007b). These findings are consistent with
the expression of bcl-2/bax and caspase-3, respectively. RT-qPCR analysis showed that COFs-derived PM2.5 treatment significantly downregulated bcl-2 expression and upregulated Bax expression (P < 0.05 vs control group). In addition, pretreatment with 1,25-vitamin D3 significantly upregulated bcl-2 expression and downregulated bax expression (P < 0.05 vs treated group) (Fig. 3C). 3.4. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced inflammation in cultured rat neonatal cardiomyocytes Inflammation played a major role in cell damage caused by COFsderived PM2.5. IL-6 and TNF-α were detected to determine the effect of 1,25-vitamin D3 pretreatment on inflammation resulted from COFsderived PM2.5 treatment (Fig. 4). Our results showed that IL-6 and TNF-α levels in the treated group were increased (P < 0.05 vs control group), which were attenuated by 1,25-vitamin D3 pretreatment (P < 0.05 vs treated group). 3.5. 1,25-Vitamin D3 inhibited COFs-derived PM2.5-induced activation of JAK/STAT and NF-κB signaling pathways in cultured rat neonatal cardiomyocytes The effects of 1,25-vitamin D3 on expression of JAK/STAT and NFκB signaling pathways in COFs-derived PM2.5-treated rat neonatal cardiomyocytes were analyzed. As expected, the levels of p-JAK and pSTAT3 were significantly elevated after COFs-derived PM2.5 treatment and decreased under COFs-derived PM2.5 plus vitamin D application. Interestingly, 1,25-vitamin D3 alone moderately inhibited phosphorylation of JAK and STAT3 in cultured rat neonatal cardiomyocytes (Fig. 5). Moreover, 1,25-vitamin D3 inhibited COFs-derived PM2.5-induced activation of JAK/STAT pathway in cultured rat neonatal cardiomyocytes. We also analyzed expression of NF-κB signaling pathway with 1,25-vitamin D3 pretreatment under COFs-derived PM2.5 exposure, and observed a similar result which was consistent with previously published article (Fig. 5). 4. Discussion Evidences linking vitamin D to cardiovascular health have accumulated in recent years, and numerous epidemiological studies report vitamin D deficiency as a significant cardiovascular disease (CVD) risk factor, as well as vitamin D levels are inversely correlated with known CVD risk factors and incidence of overt CVD (Afzal and Nordestgaard, 254
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Fig. 5. VitD3 pretreatment inhibited COFs-derived PM2.5-evoked activation of NF-κB pathway and JAK/STAT pathway in cultured rat neonatal cardiomyocytes. Cells were incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
further confirmed the essential role of JAK1 and STAT3 in COFs-derived PM2.5-induced oxidative stress, inflammation and apoptosis in cultured rat neonatal cardiomyocytes. We identified that 1,25-vitamin D3 may protect rat neonatal cardiomyocytes from COFs-derived PM2.5 induced injury through antiapoptotic and anti-oxidant properties, possibly via the involvement of JAK/STAT and NF-κB signaling pathways. Based on the 1,25-vitamin D3-protective effects observed in pretreated cells, the regulation of 1,25-vitamin D3 can be a fascinating approach to prevent/treat myocardial injury in response to pathological stress. Additional studies investigating the potential effects of 1,25-vitamin D3 in more transgenic models are certainly required to extrapolate the therapeutic efficacy of 1,25-vitamin D3.
the results observed in the present study. The JAK/STAT signaling pathway is important in inflammation factors and is activated when stimulated by upstream inflammatory factors such as interleukins and growth factors (Boengler et al., 2008). The activated JAK receptor tyrosine kinase is phosphorylated to phosphorylate STATs to form dimers, which are then translocated to the nucleus to bind with nuclear DNA to regulate the transcription and translation of downstream inflammatory genes (Welsch et al., 2017). The JAK family has four kinases JAK1, JAK2, JAK3 and Tyk2. All of them have an active domain of tyrosine kinase, respectively. STATs are the target proteins of JAKs, including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, and STAT3 is a key effector of JAK/STAT downstream signal pathway. Many cytokines and growth factors, such as EGF, IL-5, IL-6, bind to their corresponding receptors respectively and phosphorylate tyrosine 705 of STAT3 through receptor-associated kinases. As a transcription factor, STAT3 regulates a number of genes expression following the cell stimuli and consequently impacts many cellular behaviors such as growth, migration, apoptosis and autophagy. STAT3 persistent phosphorylation has been found in 22%∼65% tumors and this aberrant constitutive activation is associated with cancer cell proliferation, resistance to chemotherapy and poor prognosis (Zhang et al., 2017b). Reyes-Zárate reported that atmospheric particulate matter (PM10) phosphorylated STAT3 and further induced cell death in lung cells (Reyes-Zárate et al., 2016). In present work, the data suggested that COFs-derived PM2.5 caused STAT3 phosphorylation and further damaged cell viability in cultured rat neonatal cardiomyocytes. However, 1,25-vitamin D3 pretreatment in cardiomyocytes inhibited JAK1 and STAT3 phosphorylation, which attenuated cell death and
Conflicts of interest The authors declare that they have no competing interests. Author contributions L. CM and W. AL designed and conducted the experiments, analyzed the data, and wrote the manuscript; F. J, Z. J, G. C, J. XQ, Y. MS, and P. JY performed the experiments and acquired the data; C. JY, Z. GL, and J. YJ provided key reagents and critical comments. Acknowledgements This work was supported by the Science Research Fund of Anhui 255
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Province (090413265X).
Niemann, B., et al., 2017. Oxidative stress and cardiovascular risk: obesity, diabetes, smoking, and pollution: Part 3 of a 3-Part Series. J. Am. Coll. Cardiol. 70, 230–251. Park, J., et al., 2017. Ambient fine particulate matters induce cell death and inflammatory response by influencing mitochondria function in human corneal epithelial cells. Environ. Res. 159, 595–605. Pilz, S., Verheyen, N., Grübler, M.R., Tomaschitz, A., März, W., 2016. Vitamin D and cardiovascular disease prevention. Nat. Rev. Cardiol. 13, 404–417. Pope, A., et al., 2015. Relationships between fine particulate air pollution, cardiometabolic disorders, and cardiovascular mortality. Circ. Res. 116, 108–115. Pope, A., et al., 2016. Exposure to fine particulate air pollution is associated with endothelial injury and systemic inflammation. Circ. Res. 119, 1204–1214. Rahman, A., Hershey, S., Ahmed, S., Nibbelink, K., Simpson, U., 2007. Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice. J. Steroid Biochem. Mol. Biol. 103, 416–419. Rautiainen, S., et al., 2010. Multivitamin use and the risk of myocardial infarction: a population-based cohort of Swedish women. Am. J. Clin. Nutr. 92, 1251–1256. Reyes-Zárate, E., et al., 2016. Atmospheric particulate matter (PM10) exposure-induced cell cycle arrest and apoptosis evasion through STAT3 activation via PKCζ and Src kinases in lung cells. Environ. Pollut. 214, 646–656. Rosser, F., et al., 2014. Proximity to a major road, vitamin D insufficiency, and severe asthma exacerbations in Puerto Rican children. Am. J. Respir. Crit. Care Med. 190, 1190–1193. Rutering, J., et al., 2015. Improved method for isolation of neonatal rat cardiomyocytes with increased yield of C-Kit+ cardiac progenitor cells. J. Stem Cell Res. Ther. 5, 1–8. Stone, V., et al., 2017. Nanomaterials versus ambient ultrafine particles: an opportunity to exchange toxicology knowledge. Environ. Health Perspect. 125, 106002. Wang, S., Zhu, X., Xiong, L., Ren, J., 2017. Ablation of Akt2 prevents paraquat-induced myocardial mitochondrial injury and contractile dysfunction: role of Nrf2. Toxicol. Lett. 269, 1–14. Welsch, K., Holstein, J., Laurence, A., Ghoreschi, K., 2017. Targeting JAK/STAT signalling in inflammatory skin diseases with small molecule inhibitors. Eur. J. Immunol. 47, 1096–1107. Witte, K., et al., 2016. Effects of vitamin D on cardiac function in patients with chronic HF: the VINDICATE study. J. Am. Coll. Cardiol. 67, 2593–2603. Yang, J., et al., 2017. Oxidative stress and cell cycle arrest induced by short-term exposure to dustfall PM2.5 in A549 cells. Environ. Sci. Pollut. Res. Int. https://doi.org/ 10.1007/s11356-017-0430-3. Zhang, R., Yu, L., 2017. Effect of NF-κB signaling pathway mediated by miR-711 on the apoptosis of H9c2 cardiomyocytes in myocardial ischemia reperfusion. Eur. Rev. Med. Pharmacol. Sci. 21, 5781–5788. Zhang, R., et al., 2017a. Serum 25-hydroxyvitamin D and the risk of cardiovascular disease: dose-response meta-analysis of prospective studies. Am. J. Clin. Nutr. 105, 810–819. Zhang, L., et al., 2017b. The relationship between microRNAs and the STAT3-related signaling pathway in cancer. Tumour Biol. 39 1010428317719869. Zhang, L., et al., 2018. Vitamin D attenuates pressure overload-induced cardiac remodeling and dysfunction in mice. J. Steroid Biochem. Mol. Biol. 178, 293–302.
References Afzal, S., Nordestgaard, B.G., 2017. Vitamin D, hypertension, and ischemic stroke in 116 655 individuals from the general population: a genetic study. Hypertension. https:// doi.org/10.1161/HYPERTENSIONAHA.117.09411. Al, I., Quyyumi, A., 2017. Vitamin D and cardiovascular disease: controversy unresolved. J. Am. Coll. Cardiol. 70, 89–100. Belaidi, E., Morand, J., Gras, E., Pépin, J.L., Godin-Ribuot, D., 2016. Targeting the ROSHIF-1-endothelin axis as a therapeutic approach for the treatment of obstructive sleep apnea-related cardiovascular complications. Pharmacol. Ther. 168, 1–11. Boengler, K., Hilfiker-Kleiner, D., Drexler, H., Heusch, G., Schulz, R., 2008. The myocardial JAK/STAT pathway: from protection to failure. Pharmacol. Ther. 120, 172–185. Cao, J., et al., 2013. Toxic effect of cooking oil fumes in primary fetal pulmonary type IIlike epithelial cells. Environ. Toxicol. Pharmacol. 36, 320–331. Crobeddu, B., Aragao-Santiago, L., Bui, L.C., Boland, S., Baeza, S.A., 2017. Oxidative potential of particulate matter 2.5 as predictive indicator of cellular stress. Environ. Pollut. 230, 125–133. D'Orsi, B., Mateyka, J., JHM, P., 2017. Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok. Neurochem. Int. 109, 162–170. El-Gohary, A., Allam, M., 2017. Effect of vitamin D on isoprenaline-induced myocardial infarction in rats: possible role of peroxisome proliferator-activated receptor-γ. Can. J. Physiol. Pharmacol. 95, 641–646. Kim, H., et al., 2017. Cardiovascular effects of long-term exposure to air pollution: a population-based study with 900 845 person-years of follow-up. J. Am. Heart Assoc. 6. https://doi.org/10.1161/JAHA.117.007170. Kolaszko, A., Nowalany-Kozielska, E., Ceranowicz, P., Morawiec, B., Kubiak, G., 2018. The role of parathyroid hormone and vitamin D serum concentrations in patients with cardiovascular diseases. Dis. Markers 2018, 5287573. Marcellin, P., et al., 2016. Combination of tenofovir disoproxil fumarate and peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B. Gastroenterology 150, 134–144. Morán-Auth, Y., Penna-Martinez, M., Shoghi, F., Ramos-Lopez, E., Badenhoop, K., 2013. Vitamin D status and gene transcription in immune cells. J. Steroid Biochem. Mol. Biol. 136, 83–85. Mulligan, J.K., et al., 2014. Cigarette smoke exposure is associated with vitamin D3 deficiencies in patients with chronic rhinosinusitis. J. Allergy Clin. Immunol. 134, 342–349. Nibbelink, A., Tishkoff, X., Hershey, D., Rahman, A., Simpson, U., 2007a. 1,25(OH)2vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte. J. Steroid Biochem. Mol. Biol. 10, 533–537. Nibbelink, A., Tishkoff, X., Hershey, D., Rahman, A., Simpson, U., 2007b. 1,25(OH)2vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte. J. Steroid Biochem. Mol. Biol. 103, 533–537.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
1,25-Vitamin D3 protects against cooking oil fumes-derived PM2.5-induced cell damage through its anti-inflammatory effects in cardiomyocytes
T
Chun-Miao Luoa,b, Jun Fengb, Jing Zhangb, Chao Gaob, Ji-Yu Caoc,d, Gao-Liang Zhoub, Yong-Jing Jiangb, Xiao-Qing Jinb, Meng-Si Yangb, Jian-Yuan Panb, Ai-Ling Wanga,∗ a
Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, HeFei, Anhui, China Department of Cardiology, The Second People Hospital of Hefei, HeFei, Anhui, China c Department of Occupational and Environmental Health, School of Public Health, Anhui Medical University, HeFei, Anhui, China d The Teaching Center for Preventive Medicine, School of Public Health, Anhui Medical University, HeFei, Anhui, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: COFs-derived PM2.5 Cardiomyocytes Cell injury Inflammation 1,25-Vitamin D3 JAK/STAT pathway NF-κB pathway
The functional role of 1,25-vitamin D3 in cooking oil fumes (COFs)-derived PM2.5-induced cell damage is largely unexplored. The present study investigated the protective role of 1,25-vitamin D3 against cell injury by possible involvement of JAK/STAT and NF-κB signaling pathways in cardiomyocytes. Cell viability was measured using CCK-8 assay, and cell apoptosis was analyzed by flow cytometry, qRT-PCR and Western blot in cultured rat neonatal cardiomyocytes treated with 1,25-vitamin D3 and COFs-derived PM2.5. Expressions of JAK/STAT and NF-κB signaling pathway were measured by Western blot. The results suggested that treatment with COFs-derived PM2.5 significantly decreased cell viability and increased apoptosis and oxidative stress in cultured rat neonatal cardiomyocytes. 1,25-vitamin D3 pretreatment alleviated the cell injury by increasing cell viability and decreasing apoptosis in the cardiomyocytes. 1,25-vitamin D3 pretreatment also decreased the ROS level and inflammation in the cardiomyocytes. Furthermore, 1,25-vitamin D3 pretreatment alleviated COFsderived PM2.5-evoked elevation of JAK/STAT and NF-κB signaling pathways. Our study showed that 1,25vitamin D3 pretreatment protected cardiomyocytes from COFs-derived PM2.5-induced injury by decreasing ROS, apoptosis and inflammation level via activations of the JAK/STAT and NF-κB signaling pathways.
1. Introduction
2017). These processes are accompanied by the down-regulation of bcl2 and up-regulation of bax, which are important regulators of cellular apoptosis (D'Orsi et al., 2017). Data show that a decreased bcl-2/bax ratio increases the apoptosis risk in myocardial cells (Zhang and Yu, 2017). It has been confirmed that cardiomyocyte apoptosis is one of the most common pathophysiological processes in injury resulted from oxidative stress (Wang et al., 2017). Hypovitaminosis D is increasingly recognized as an independent predictor for primary cardiovascular events and related diseases, such as CVD (Al and Quyyumi, 2017; Zhang et al., 2017a). In addition, vitamin D deficiency is highly prevalent in patients with heart failure being a significant indicator of reduced survival, whereas supplementation was associated with an improved outcome (Witte et al., 2016). The National Health and Nutritional Examination Surveys (NHANES) (1988–1994, 2000–2004) conducted in the US have been designed to explore the association between vitamin D status and CVD and researchers found that individuals included in NHANES 1988–1994 with vitamin D deficiency (vitamin D < 20 ng/mL) had higher
Epidemiological investigations have showed a strong relationship between atmospheric particulate matters, especially an aerodynamic diameter less than 2.5 μm (PM2.5) and increased incidence, mortality and morbidity of cardiovascular disorders (CVD), including atherosclerosis, ischemic cardiovascular events, coronary artery disease and hypertension (Marcellin et al., 2016; Kim et al., 2017; Pope et al., 2015). One of the possible mechanisms by which PM2.5 exposure can contribute to an increased risk for adverse cardiovascular events is dysfunction of cardiac cells (Pope et al., 2016). PM2.5 can be absorbed and taken in cells, not only adhering to cell surfaces, and induce detrimental effects (Stone et al., 2017). Key indicators of cellular dysfunction include the loss of mitochondrial membrane potential, intracellular overproduction of ROS, the decrease of cell viability, and so on (Yang et al., 2017; Park et al., 2017). Intracellular overproduction of ROS is known to decrease the mitochondrial membrane potential, activate pro-caspases and results in apoptotic cell death (Crobeddu et al.,
∗
Corresponding author. E-mail addresses: [email protected], [email protected] (A.-L. Wang).
https://doi.org/10.1016/j.ecoenv.2019.04.064 Received 27 November 2018; Received in revised form 16 April 2019; Accepted 22 April 2019 Available online 01 May 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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were approved by the Ethics Committee of Anhui Medical University and the first Affiliated Hospital of Anhui Medical University and carried out under the institutional guidelines for ethical animal use. Briefly, ventricular cardiomyocytes were isolated from 1 to 2 days of old neonatal Sprague-Dawley rats, and then chopped and digested several times in collagenase II at 37 °C. To prepare almost pure cardiomyocytes, cells were incubated in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, antibiotics, L-glutamine. After 30 min, the plate was washed briefly and gently with the cell solution in the dish and plated again with fresh medium and repeat until most myocytes are washed off to avoid the non-myocyte cells. Further myocytes were plated in collagen precoated dishes or in fibronectin-precoated glass coverslips at a density of 160,000/cm2 in 10% fetal bovine serum DMEM supplemented with 1 mmol/l l-glutamine, antibiotic/antimycotic solution, 0.1 M AraC (1 μL/ml) and 100 μmol/l 5-bromo-2-deoxyuridine (BrdU). Under these high density conditions, the myocytes form cell-cell contacts and display spontaneous contractile activity within 24 h of plating. The cells were maintained at 37 °C with 100% relatively humidity in a CO2 incubator containing 5% CO2. Cardiac myocyte cell suspensions were transferred into 6-well gelatin-coated plates at a density of 1–5 × 106 cells/well for protein and mRNA extraction. Cells were plated 1 × 105 cells/well for cell viability, namely LDH leakage and CCK-8 assay.
prevalence of self-reported angina, heart failure, and myocardial infarction compared to the individuals with higher levels of vitamin D (Rautiainen et al., 2010). A few in vitro and in vivo studies have evaluated the role of vitamin D on cardiac tissue directly, especially in response to injury. It has been demonstrated that matrix metalloproteinases (MMP) proteins, which contribute to aberrant cardiomyocyte remodeling in response to injury and atherosclerosis, were upregulated in vitamin D receptor (VDR) knockout mice (Rahman et al., 2007). It has been also shown that VDR knockout mice have impaired cardiac relaxation and contractility and develop left ventricular hypertrophy (Rahman et al., 2007). A study in HL-1 murine cardiac myocytes showed that 1,25-vitamin D3 significantly decreased cell proliferation, increased cell size, and resulted in hypoplasia, slight hypertrophy, and altered morphology of dividing cardiomyocytes, demonstrating that 1,25-vitamin D3 is involved in maintaining structure and function of cardiac cell (Nibbelink et al., 2007a). In addition, Morán-Auth et al. demonstrated that 1,25-vitamin D3 promoted gene transcription in immune cells, such as 25-vitamin-Dhydroxylase (CYP2R1) and 25-hydroxyvitamin-D3-1a-hydroxylase (CYP27B1), suggesting that 1,25-vitamin D3 may also have a protective effect on the stress response to injury (Morán-Auth et al., 2013). Even though most evidences indicate a protective effect of vitamin D on inflammation, the role of 1,25-vitamin D3 on cardiac cell responding to COFs-derived PM2.5 remains poorly understood. In this study, we employed cultured rat neonatal cardiomyocytes, derived from neonatal rat heart, which have been extensively used as an in vitro model for cardiac function to examine the effect and mechanism of 1,25-vitamin D3 on myocardial injury induced by COFs-derived PM2.5 and attempt to explore the role of JAK/STAT and NF-κB signaling pathways in 1,25-vitamin D3 -mediated cardioprotective actions.
2.4. Quantitative real-time PCR analysis Total RNA was isolated from cells using TRIzol reagent (Invitrogen) as per manufacturer's instructions. DNaseI (Promega) was added to the isolated RNA to remove DNA impurities. Reverse transcription was performed by MultiScribe RT kit (Applied Biosystems) using random hexamers or oligo (dT). The PCR conditions for the experiments were 10 min at 25 °C, 30 min at 48 °C, and 5 min at 95 °C. All primers were designed using the Primer Premier 5.0 software and synthesized by Shenggong Co. Ltd. Specific primers were designed as follows: Bax. Forward: 5′-GGA GAC ACC TGA GCT GAC CT-3′ Reverse: 5′-GAC GAC TCC AGC CAC AAA GA-3′ Bcl-2. Forward: 5′-AAG CTG TCA CAG AGG GGC TC-3′ Reverse: 5′-CAG ATG CCG GTT CAG GTA CT-3′ GAPDH. Forward: 5′-GGC CTT CCG TGT TCC TAC C-3′ Reverse: 5′-CGG CAT GTC AGA TCC ACA AC-3′
2. Material and methods 2.1. 1,25-Vitamin D3 preparation 10−3 mol/L 1,25-vitamin D3 solution was prepared by dissolving 1 mg 1,25-vitamin D3 (molecular weight: 416.64, Sigma, USA) in 2.4 mL 95% ethanol, and stored at −20 °C in the dark until needed for used. 1 μL 10−3 mol/L 1,25-vitamin D3 solution was diluted with 99 μL DMEM medium with 10% fetal bovine serum (10−5 mol/L 1,25-vitamin D3 solution was prepared), 1 μL 10−5 mol/L 1,25-vitamin D3 solution was diluted with 999 μL DMEM medium (10−8 mol/L 1,25-vitamin D3 solution was prepared). 2.2. Preparation of COFs-derived PM2.5
2.5. Cell counting kit-8 assay
The collection and extraction of PM2.5 were in accordance with the procedures followed by Cao et al. methods, which were modified by ourselves (Cao et al., 2013). Peanut oil (200 ml) was poured into an iron pot and heated with an electric heater. The temperature was maintained at the smoke point (280 ± 10 °C). The fumes generated from the heating oil were collected, with filter paper placed 50 cm upon the oil surface, at a flow rate of 12 L/min, which connected to a vacuum pump. Each filter paper was renewed once after 2 h. The experiments were independently repeated three times. The gathered condensates were separately extracted with 50 ml of acetone using a Soxhlet extractor for 24 h. The extracts were then dried by rotary evaporation at 40 °C. The eluant was evaporated to a yellow viscous solution and diluted to 200 mg/mL with dimethylsulfoxide (DMSO). The samples were sealed up in brown glass vials and preserved at −80 °C until use.
Cell viability was assessed by a cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD). Cells were seeded in a 96well plate with 5000 cells/well. After stimulation, the CCK-8 solution was added to the culture medium, and the cultures were incubated for 1 h at 37 °C in humidified 95% air and 5% CO2. The absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA). 2.6. Annexin V/propidium iodide (PI) double staining assay Cell apoptosis analysis was performed using PI and fluorescein isothiocyanate (FITC)-conjugated Annexin V staining. Briefly, cells were washed in phosphate buffered saline (PBS) and fixed in 70% ethanol. Fixed cells were then washed twice in PBS and stained in PI/ FITC-Annexin V in the presence of 50 μg/mL RNase A (Sigma-Aldrich), and then incubated for 1 h at room temperature in the dark. Flow cytometry analysis was done using a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA) to differentiate apoptotic cells (Annexin-V positive and PI-negative) from necrotic cells (Annexin-V and PI-positive). The data were analyzed using FlowJo software.
2.3. Cell culture and treatment Ventricular myocytes were isolated from the hearts of neonatal rats (SD rats) that were up to 2 days old and were isolated and cultured as described previously (Rutering et al., 2015). All the animal experiments 250
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Fig. 1. VitD3 pretreatment prevented COFs-derived PM2.5-induced cell damage in cultured rat neonatal cardiomyocytes. (A) CCK-8 levels in cells incubated with different doses of COFs-derived PM2.5. (B) LDH levels in cells incubated with different doses of COFs-derived PM2.5. (C) CCK-8 levels in cells incubated with different doses VitD3. (D) LDH levels in cells incubated with different doses of VitD3. (E) CCK-8 levels in cells incubated with COFs-derived PM2.5 in absence or presence of VitD3 (10−8 mol/L). (F) LDH levels in cells incubated with COFs-derived PM2.5 in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
2.7. ROS assay
521 nm emission).
The total ROS was measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) (Nanjing Jiancheng, Nanjing, China). The cells seeded in a 6-well plate, were washed twice with PBS post treatment and co-incubated with serum-free culture medium containing 10 μM DCFH-DA (20 min, 37 °C, in dark). All the samples were centrifuged and the supernatants were resuspended in 500 μL PBS. The fluorescent intensities were measured using a fluorospectrophotometer (488 nm excitation,
2.8. Cellular antioxidant defense measurements To verify cellular antioxidant defenses, superoxide dismutase (SOD) activities and GSH levels were assessed. Then, 24 h after treatment, cells were washed twice with PBS and scraped in cell lysis buffer (10 mM Tris-HCl, 1% Triton X-100, pH 7.4). Cellular lysate was obtained after sonication for 1 min (5 s on, 5 s off, 30% amplitude) on ice 251
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3. Results
and centrifugation at 14,000 rpm for 10 min at 4 °C. Protein concentrations were determined by the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The supernatants were used to evaluate SOD activities and GSH levels. To determine SOD activity, 930 μL of buffer (200 mM Tris HCl, 2 mM EDTA, pH 8.2) containing supernatant aliquots (50 μg/mL of protein) was mixed with 70 μL of pyrogallol (15 mM) (Nanjing Jiancheng, Nanjing, China). After 2 min of incubation at room temperature, the absorbance was assessed at 420 nm. The amount of SOD that inhibited 50% of the pyrogallol oxidation compared to the negative control was considered one unit of SOD activity, and the results were expressed as unit of SOD/μg protein. The amount of yellow color produced by 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) directly reflected the content of GSH reacting for 25 min at 25 °C, according to the assay kit (Nanjing Jiancheng, Nanjing, China). It was measured by the absorbance at 412 nm and the results were expressed as unit of SOD/μg protein.
3.1. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced cell damage in cultured rat neonatal cardiomyocytes LDH leakage and cell viability generally served as indexes of cardiomyocyte injury, which were measured at 0, 12.5, 25, 50, and 100 μg/mL of the COFs-derived PM2.5. As shown in Fig. 1A and Fig. 1B, a dose-dependent increase of LDH leakage and a dose-dependent decrease of CCK-8 levels were observed in treated cells within different treated doses. A significant injury was found in treated cells with 50 μg/ mL COFs-derived PM2.5, and the dose was used in further experiments. In order to determine the cellular compatibility of 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes, the effects of 1,25-vitamin D3 with dose ranged from 1 to 100 nM were measured using CCK-8 kitbased viability assay and LDH assay after incubating the cells for 24 h (Fig. 1C and D). It was observed that dose ranged of 1,25-vitamin D3 showed non-significant toxic effect. 10 nM of 1,25-vitamin D3 was selected for further experiments. To determine the effects of pretreatment with 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes treated with COFs-derived PM2.5, CCK-8 assay was performed on the cells that were pretreated with 1,25-vitamin D3 for 24 h before undergoing COFsderived PM2.5-induced injury (Fig. 1E). The CCK-8 assay results showed that pretreatment with 1,25-vitamin D3 significantly suppressed the decrease of viability in a dose-dependent manner, indicating that 1,25-vitamin D3 increased cell viability and protected cardiomyocytes against COFs-derived PM2.5-induced injury. To further determine the protective effect of 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes undergoing COFs-derived PM2.5 treatment, the activities of LDH in the supernatant, which could indirectly reflect the degree of injury, were analyzed (Fig. 1F). Pretreatment with 1,25vitamin D3 also significantly suppressed increased LDH activity (P < 0.05 vs treated group).
2.9. Western blot assay The proteins used for western blotting were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Guangzhou, China). The proteins were quantified using BCA™ Protein Assay Kit. Western blot system was established using Bio-Rad Bis-Tris Gel system according to the manufacturer's instructions. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1000. Primary antibody was incubated with the membrane at 4 °C overnight, followed by wash and incubation with secondary antibody marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride membranecarried blots and antibodies were transferred into Bio-Rad ChemiDoc™ XRS system, and then 200 μL Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, China).
3.2. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced oxidative stress in cultured rat neonatal cardiomyocytes It is generally accepted that mitochondrial generation of reactive oxygen species plays a role in cell death. Levels of intracellular reactive oxygen species were reflected by DCF fluorescence intensity (Fig. 2A). The significant increase of the intracellular reactive oxygen species peak indicated that reactive oxygen species generation was significantly increased in the COFs-derived PM2.5-treated group (P < 0.05 vs control group). However, in the 1,25-vitamin D3 and COFs-derived PM2.5 group, the reactive oxygen species peak level significantly decreased (P < 0.05 vs treated group), indicating that 1,25-vitamin D3 pretreatment decreased reactive oxygen species generation. GSH assay and total SOD assay were also performed to determine the protective effect of 1,25-vitamin D3 in cultured rat neonatal cardiomyocytes against COFs-derived PM2.5 treatment and the data were consistent with above results (Fig. 2 B and C).
2.10. ELISA assay Cells were incubated in culture medium containing a designated concentrations of COFs-derived PM2.5 and VitD3 for 24 h. Then IL-6 and TNF-α levels in the cell culture medium were determined according to the manufacturer's instructions (Boster Biological Technology, Wuhan, China). Briefly, 96-well immunoplates were coated with capture antibody overnight at 4 °C, washed with PBS containing 0.05% tween-20 (PBST) and blocked with assay diluent for 1 h at RT to avoid nonspecific binding. Samples and standard antibodies were added to each well and incubated for 2 h at RT. The reactants were incubated serially with detection antibody (1 h), horseradish peroxidase-streptavidin solution (30 min), and substrates (15 min). Finally, absorbance was measured at 450 nm using a multiplate reader, and the cytokine levels in each sample were calculated using the corresponding standard curve.
3.3. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced apoptosis in cultured rat neonatal cardiomyocytes Previous studies have shown that urban dust exposure can result in apoptosis and cardiac dysfunction, and destruction of heart tissue. In this study, we examined whether 1,25-vitamin D3 could protect cells against COFs-derived PM2.5-induced apoptosis. Cultured rat neonatal cardiomyocytes were harvested and subjected to annexin V/PI double staining, then analyzed by flow cytometry (Fig. 3A). Cell apoptosis of cells in the COFs-derived PM2.5 group was significantly increased (P < 0.05 vs control group). However, in cells that were pretreated with 1,25-vitamin D3, apoptosis of cardiomyocytes was decreased (P > 0.05 vs treated group). We also found that the levels of GRP78, caspase-1, and cleaved caspase-3 were increased in COFs-derived
2.11. Statistical analysis The statistical analysis was performed using SPSS 16.0 statistical software. All the experiments were replicated at least thrice and the results were expressed as mean ± SD. One-way analysis of variance and the Student-Newman-Keuls post hoc test were used to determine differences among different groups. P values of < 0.05 was considered to indicate a statistically significant result.
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Fig. 2. VitD3 pretreatment prevented COFs-derived PM2.5-induced oxidative stress in cultured rat neonatal cardiomyocytes. (A) ROS fluorescence intensities in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). (B) SOD activity in cells incubated with COFs-derived PM2.5 (50 μg/ mL) in absence or presence of VitD3 (10−8 mol/L). (C) GSH level in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/ L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
results in activities of caspases. To determine whether 1,25-vitamin D3 protects against COFs-derived PM2.5 induced apoptosis by modulating the bcl-2 family and activities of caspases in cultured rat neonatal cardiomyocytes, RT-qPCR and Western blot were performed to evaluate
PM2.5 group, whereas there was a decrease of caspase-3 activities in 1,25-vitamin D3 pretreated group (P > 0.05 vs treated group) (Fig. 3B). Furthermore, previous studies have shown that mitochondria integrate death signals through the bcl-2 family which eventually
Fig. 3. VitD3 pretreatment prevented COFs-derived PM2.5-induced apoptosis in cultured rat neonatal cardiomyocytes. (A) Apoptosis levels measured by flow cytometry of cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). (B) Expression of apoptosis biomarkers in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L) using Western blot. (C) Bax/Bcl-2 ratio detected with qRT-PCR in cells incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group. 253
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Fig. 4. VitD3 pretreatment prevented COFs-derived PM2.5-induced inflammation in cultured rat neonatal cardiomyocytes. Elisa assay and quantitative analysis of IL6 (A) and TNF-α (B) expression. Cells were incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
2017; Pilz et al., 2016). Air pollution, especially urban PM2.5 is associated with a high prevalence of vitamin D deficiency, and low levels of vitamin D are in turn correlated with adverse clinical outcomes in this patient population (Mulligan et al., 2014; Rosser et al., 2014). Low vitamin D levels are associated with elevated blood pressure, increased hospitalization rates, and higher incidence of all-cause mortality. Oxidative stress-induced cell injury plays a critical role in the pathophysiology of many diseases. Oxidative stress especially increases excessive production of ROS, which may contribute to a number of cardiovascular diseases, such as atherosclerosis, hypertension and myocardial ischemic reperfusion injury (Niemann et al., 2017). The cellular sources of ROS are accumulated from mitochondrial electron transport chain, lipoxygenase and auto-oxidation of catecholamines (Belaidi et al., 2016). Continuous persistence of this oxidative insult can lead to programmed cell death, causing the loss of functional cardiomyocytes. Despite significant improvement in the treatment strategies to reduce cardiovascular risks, the disease still remains challenging in the developing world. The present study was undertaken to explore the effects of 1,25vitamin D3 on COFs-derived PM2.5-induced cell injury in cultured rat neonatal cardiomyocytes and further to investigate the mechanism of this phenomenon. COFs-derived PM2.5-induced oxidative stress, increased apoptosis, and inflammation in cultured rat neonatal cardiomyocytes and resulted in decreased cell viability. Pretreatment of 1,25vitamin D3 significantly increased cell viability, and decreased apoptosis, inflammation, and ROS level in cultured rat neonatal cardiomyocytes. The mechanistic study revealed that pretreatment of 1,25vitamin D3 deactivated the JAK/STAT and NF-κB signaling pathways, which were in accordance with previously published evidences. Studies have portrayed 1,25-vitamin D3 as a biomarker and modulator of heart failure in dilated cardiomyopathy, and vitamin D levels were significantly decreased in mice models with acute myocardial infarction (MI) compared to non-MI controls (Zhang et al., 2018; Kolaszko et al., 2018). An in vivo study was conducted to examine the functional role of pretreatment of 1,25-vitamin D3 on cardiomyocyte survival in acute MI (El-Gohary and Allam, 2017). The infarct size and the ratio of infarct area to area at risk 24 h post MI was decreased within 1,25-vitamin D3 pretreatment when compared to the wild-type rat model. The percentage of apoptotic cells was also significantly reduced in the infarcted left ventricle of 1,25-vitamin D3 pretreatment rat vs. control. Pretreatment of 1,25-vitamin D3 protected HL-1 cells from staurosporineinduced apoptosis by blocking cytochrome C release and caspase 3 cleavage (Nibbelink et al., 2007b). These findings are consistent with
the expression of bcl-2/bax and caspase-3, respectively. RT-qPCR analysis showed that COFs-derived PM2.5 treatment significantly downregulated bcl-2 expression and upregulated Bax expression (P < 0.05 vs control group). In addition, pretreatment with 1,25-vitamin D3 significantly upregulated bcl-2 expression and downregulated bax expression (P < 0.05 vs treated group) (Fig. 3C). 3.4. 1,25-Vitamin D3 alleviated COFs-derived PM2.5-induced inflammation in cultured rat neonatal cardiomyocytes Inflammation played a major role in cell damage caused by COFsderived PM2.5. IL-6 and TNF-α were detected to determine the effect of 1,25-vitamin D3 pretreatment on inflammation resulted from COFsderived PM2.5 treatment (Fig. 4). Our results showed that IL-6 and TNF-α levels in the treated group were increased (P < 0.05 vs control group), which were attenuated by 1,25-vitamin D3 pretreatment (P < 0.05 vs treated group). 3.5. 1,25-Vitamin D3 inhibited COFs-derived PM2.5-induced activation of JAK/STAT and NF-κB signaling pathways in cultured rat neonatal cardiomyocytes The effects of 1,25-vitamin D3 on expression of JAK/STAT and NFκB signaling pathways in COFs-derived PM2.5-treated rat neonatal cardiomyocytes were analyzed. As expected, the levels of p-JAK and pSTAT3 were significantly elevated after COFs-derived PM2.5 treatment and decreased under COFs-derived PM2.5 plus vitamin D application. Interestingly, 1,25-vitamin D3 alone moderately inhibited phosphorylation of JAK and STAT3 in cultured rat neonatal cardiomyocytes (Fig. 5). Moreover, 1,25-vitamin D3 inhibited COFs-derived PM2.5-induced activation of JAK/STAT pathway in cultured rat neonatal cardiomyocytes. We also analyzed expression of NF-κB signaling pathway with 1,25-vitamin D3 pretreatment under COFs-derived PM2.5 exposure, and observed a similar result which was consistent with previously published article (Fig. 5). 4. Discussion Evidences linking vitamin D to cardiovascular health have accumulated in recent years, and numerous epidemiological studies report vitamin D deficiency as a significant cardiovascular disease (CVD) risk factor, as well as vitamin D levels are inversely correlated with known CVD risk factors and incidence of overt CVD (Afzal and Nordestgaard, 254
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Fig. 5. VitD3 pretreatment inhibited COFs-derived PM2.5-evoked activation of NF-κB pathway and JAK/STAT pathway in cultured rat neonatal cardiomyocytes. Cells were incubated with COFs-derived PM2.5 (50 μg/mL) in absence or presence of VitD3 (10−8 mol/L). Data are presented as mean ± SD of three individual experiments performed in triplicate. *P < 0.05 versus the untreated control group and #P < 0 05 versus the treated group.
further confirmed the essential role of JAK1 and STAT3 in COFs-derived PM2.5-induced oxidative stress, inflammation and apoptosis in cultured rat neonatal cardiomyocytes. We identified that 1,25-vitamin D3 may protect rat neonatal cardiomyocytes from COFs-derived PM2.5 induced injury through antiapoptotic and anti-oxidant properties, possibly via the involvement of JAK/STAT and NF-κB signaling pathways. Based on the 1,25-vitamin D3-protective effects observed in pretreated cells, the regulation of 1,25-vitamin D3 can be a fascinating approach to prevent/treat myocardial injury in response to pathological stress. Additional studies investigating the potential effects of 1,25-vitamin D3 in more transgenic models are certainly required to extrapolate the therapeutic efficacy of 1,25-vitamin D3.
the results observed in the present study. The JAK/STAT signaling pathway is important in inflammation factors and is activated when stimulated by upstream inflammatory factors such as interleukins and growth factors (Boengler et al., 2008). The activated JAK receptor tyrosine kinase is phosphorylated to phosphorylate STATs to form dimers, which are then translocated to the nucleus to bind with nuclear DNA to regulate the transcription and translation of downstream inflammatory genes (Welsch et al., 2017). The JAK family has four kinases JAK1, JAK2, JAK3 and Tyk2. All of them have an active domain of tyrosine kinase, respectively. STATs are the target proteins of JAKs, including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, and STAT3 is a key effector of JAK/STAT downstream signal pathway. Many cytokines and growth factors, such as EGF, IL-5, IL-6, bind to their corresponding receptors respectively and phosphorylate tyrosine 705 of STAT3 through receptor-associated kinases. As a transcription factor, STAT3 regulates a number of genes expression following the cell stimuli and consequently impacts many cellular behaviors such as growth, migration, apoptosis and autophagy. STAT3 persistent phosphorylation has been found in 22%∼65% tumors and this aberrant constitutive activation is associated with cancer cell proliferation, resistance to chemotherapy and poor prognosis (Zhang et al., 2017b). Reyes-Zárate reported that atmospheric particulate matter (PM10) phosphorylated STAT3 and further induced cell death in lung cells (Reyes-Zárate et al., 2016). In present work, the data suggested that COFs-derived PM2.5 caused STAT3 phosphorylation and further damaged cell viability in cultured rat neonatal cardiomyocytes. However, 1,25-vitamin D3 pretreatment in cardiomyocytes inhibited JAK1 and STAT3 phosphorylation, which attenuated cell death and
Conflicts of interest The authors declare that they have no competing interests. Author contributions L. CM and W. AL designed and conducted the experiments, analyzed the data, and wrote the manuscript; F. J, Z. J, G. C, J. XQ, Y. MS, and P. JY performed the experiments and acquired the data; C. JY, Z. GL, and J. YJ provided key reagents and critical comments. Acknowledgements This work was supported by the Science Research Fund of Anhui 255
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Province (090413265X).
Niemann, B., et al., 2017. Oxidative stress and cardiovascular risk: obesity, diabetes, smoking, and pollution: Part 3 of a 3-Part Series. J. Am. Coll. Cardiol. 70, 230–251. Park, J., et al., 2017. Ambient fine particulate matters induce cell death and inflammatory response by influencing mitochondria function in human corneal epithelial cells. Environ. Res. 159, 595–605. Pilz, S., Verheyen, N., Grübler, M.R., Tomaschitz, A., März, W., 2016. Vitamin D and cardiovascular disease prevention. Nat. Rev. Cardiol. 13, 404–417. Pope, A., et al., 2015. Relationships between fine particulate air pollution, cardiometabolic disorders, and cardiovascular mortality. Circ. Res. 116, 108–115. Pope, A., et al., 2016. Exposure to fine particulate air pollution is associated with endothelial injury and systemic inflammation. Circ. Res. 119, 1204–1214. Rahman, A., Hershey, S., Ahmed, S., Nibbelink, K., Simpson, U., 2007. Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice. J. Steroid Biochem. Mol. Biol. 103, 416–419. Rautiainen, S., et al., 2010. Multivitamin use and the risk of myocardial infarction: a population-based cohort of Swedish women. Am. J. Clin. Nutr. 92, 1251–1256. Reyes-Zárate, E., et al., 2016. Atmospheric particulate matter (PM10) exposure-induced cell cycle arrest and apoptosis evasion through STAT3 activation via PKCζ and Src kinases in lung cells. Environ. Pollut. 214, 646–656. Rosser, F., et al., 2014. Proximity to a major road, vitamin D insufficiency, and severe asthma exacerbations in Puerto Rican children. Am. J. Respir. Crit. Care Med. 190, 1190–1193. Rutering, J., et al., 2015. Improved method for isolation of neonatal rat cardiomyocytes with increased yield of C-Kit+ cardiac progenitor cells. J. Stem Cell Res. Ther. 5, 1–8. Stone, V., et al., 2017. Nanomaterials versus ambient ultrafine particles: an opportunity to exchange toxicology knowledge. Environ. Health Perspect. 125, 106002. Wang, S., Zhu, X., Xiong, L., Ren, J., 2017. Ablation of Akt2 prevents paraquat-induced myocardial mitochondrial injury and contractile dysfunction: role of Nrf2. Toxicol. Lett. 269, 1–14. Welsch, K., Holstein, J., Laurence, A., Ghoreschi, K., 2017. Targeting JAK/STAT signalling in inflammatory skin diseases with small molecule inhibitors. Eur. J. Immunol. 47, 1096–1107. Witte, K., et al., 2016. Effects of vitamin D on cardiac function in patients with chronic HF: the VINDICATE study. J. Am. Coll. Cardiol. 67, 2593–2603. Yang, J., et al., 2017. Oxidative stress and cell cycle arrest induced by short-term exposure to dustfall PM2.5 in A549 cells. Environ. Sci. Pollut. Res. Int. https://doi.org/ 10.1007/s11356-017-0430-3. Zhang, R., Yu, L., 2017. Effect of NF-κB signaling pathway mediated by miR-711 on the apoptosis of H9c2 cardiomyocytes in myocardial ischemia reperfusion. Eur. Rev. Med. Pharmacol. Sci. 21, 5781–5788. Zhang, R., et al., 2017a. Serum 25-hydroxyvitamin D and the risk of cardiovascular disease: dose-response meta-analysis of prospective studies. Am. J. Clin. Nutr. 105, 810–819. Zhang, L., et al., 2017b. The relationship between microRNAs and the STAT3-related signaling pathway in cancer. Tumour Biol. 39 1010428317719869. Zhang, L., et al., 2018. Vitamin D attenuates pressure overload-induced cardiac remodeling and dysfunction in mice. J. Steroid Biochem. Mol. Biol. 178, 293–302.
References Afzal, S., Nordestgaard, B.G., 2017. Vitamin D, hypertension, and ischemic stroke in 116 655 individuals from the general population: a genetic study. Hypertension. https:// doi.org/10.1161/HYPERTENSIONAHA.117.09411. Al, I., Quyyumi, A., 2017. Vitamin D and cardiovascular disease: controversy unresolved. J. Am. Coll. Cardiol. 70, 89–100. Belaidi, E., Morand, J., Gras, E., Pépin, J.L., Godin-Ribuot, D., 2016. Targeting the ROSHIF-1-endothelin axis as a therapeutic approach for the treatment of obstructive sleep apnea-related cardiovascular complications. Pharmacol. Ther. 168, 1–11. Boengler, K., Hilfiker-Kleiner, D., Drexler, H., Heusch, G., Schulz, R., 2008. The myocardial JAK/STAT pathway: from protection to failure. Pharmacol. Ther. 120, 172–185. Cao, J., et al., 2013. Toxic effect of cooking oil fumes in primary fetal pulmonary type IIlike epithelial cells. Environ. Toxicol. Pharmacol. 36, 320–331. Crobeddu, B., Aragao-Santiago, L., Bui, L.C., Boland, S., Baeza, S.A., 2017. Oxidative potential of particulate matter 2.5 as predictive indicator of cellular stress. Environ. Pollut. 230, 125–133. D'Orsi, B., Mateyka, J., JHM, P., 2017. Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok. Neurochem. Int. 109, 162–170. El-Gohary, A., Allam, M., 2017. Effect of vitamin D on isoprenaline-induced myocardial infarction in rats: possible role of peroxisome proliferator-activated receptor-γ. Can. J. Physiol. Pharmacol. 95, 641–646. Kim, H., et al., 2017. Cardiovascular effects of long-term exposure to air pollution: a population-based study with 900 845 person-years of follow-up. J. Am. Heart Assoc. 6. https://doi.org/10.1161/JAHA.117.007170. Kolaszko, A., Nowalany-Kozielska, E., Ceranowicz, P., Morawiec, B., Kubiak, G., 2018. The role of parathyroid hormone and vitamin D serum concentrations in patients with cardiovascular diseases. Dis. Markers 2018, 5287573. Marcellin, P., et al., 2016. Combination of tenofovir disoproxil fumarate and peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B. Gastroenterology 150, 134–144. Morán-Auth, Y., Penna-Martinez, M., Shoghi, F., Ramos-Lopez, E., Badenhoop, K., 2013. Vitamin D status and gene transcription in immune cells. J. Steroid Biochem. Mol. Biol. 136, 83–85. Mulligan, J.K., et al., 2014. Cigarette smoke exposure is associated with vitamin D3 deficiencies in patients with chronic rhinosinusitis. J. Allergy Clin. Immunol. 134, 342–349. Nibbelink, A., Tishkoff, X., Hershey, D., Rahman, A., Simpson, U., 2007a. 1,25(OH)2vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte. J. Steroid Biochem. Mol. Biol. 10, 533–537. Nibbelink, A., Tishkoff, X., Hershey, D., Rahman, A., Simpson, U., 2007b. 1,25(OH)2vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte. J. Steroid Biochem. Mol. Biol. 103, 533–537.
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