Involvement of EGF receptor signaling and NLRP12 inflammasome in fine particulate matter‐induced lung inflammation in mice

Epidemiological studies have shown that exposure to ambient fine particulate matter (PM_2.5) is associated with respiratory diseases. Lung inflammation is a central feature of many pulmonary diseases, which can be induced by PM_2.5 exposure. However, the mechanisms underlying PM_2.5‐induced lung inflammation remain unclear. To characterize the role of epidermal growth factor receptor (EGFR) and inflammasome in PM_2.5‐induced lung inflammation in mice, 30 BALB/c mice were intrabroncheally instilled with saline and PM_2.5 suspension (4.0 mg/kg b.w.) for 5 consecutive days, respectively. Bronchoalveolar lavage (BAL) was conducted and BAL fluid (BALF) was collected. The levels of reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), epidermal growth factor (EGF), CXCL1, interleukin (IL)?1β, and IL‐18 in BALF were determined using ELISA. mRNA levels of IL‐6, IL‐1β, IL‐18, CXCL1, IL‐10, NLRP3, Caspase‐1, and NLRP12 in lung tissues were determined by RT‐PCR. Phospho‐EGFR (Tyr1068) and phospho‐Akt (Thr308) in lung tissues were examined using immunohistochemical staining and Western blotting, respectively. Protein levels of Caspase‐1, NLRP3, NF‐κB‐p52/p100, and NF‐κB‐p65 in bronchial epithelium were examined using immunohistochemical staining. It was shown that PM_2.5 exposure induced lung inflammation. Levels of total protein, ROS, iNOS, EGF, and CXCL1 and cell number in the BALF of mice exposed to PM_2.5 were markedly elevated relative to the control. mRNA levels of CXCL1, IL‐1β, and IL‐18 in lung tissues of PM_2.5‐exposed mice were increased in comparison with the control. However, level of NLRP12 mRNA in lung tissues of PM_2.5‐exposed mice was reduced. Phospho‐EGFR (Tyr1068) and phospho‐Akt (Thr308) levels in the lungs of PM_2.5‐instilled mice were higher than those in the lungs of the control. The protein levels of NF‐κB‐p52/p100 and NF‐κB‐p65 in bronchial epithelium of PM_2.5‐exposed mice were also increased compared with the control. This study suggests that EGF‐EGFR‐Akt‐NF‐κB signaling and NLRP12 inflammasome may be associated with PM_2.5‐induced lung inflammation in mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1121–1134, 2017.     

Resveratrol relieves particulate matter (mean diameter < 2.5 μm)‐induced oxidative injury of lung cells through attenuation of autophagy deregulation

Oxidative stress and inflammation are critically implicated in ambient fine particulate matter (mean diameter < 2.5 μm; PM_2.5)‐induced lung injury. Autophagy, playing a crucial role in various physiopathological conditions, modulates cellular homeostasis and stress adaptation. Resveratrol is a phytoalexin that exerts potent antioxidant effects on cardiopulmonary diseases. To date, the mechanisms by which resveratrol protects against PM_2.5 remain to be elucidated. In the present study, we investigated the effect of resveratrol on PM_2.5‐induced oxidative injury. The potential role of nuclear factor erythroid‐2‐related factor 2 and autophagy in this progress was explored. Human bronchial epithelial cells were treated with PM_2.5 and the cytotoxicity and oxidative stress markers were determined. The results showed that PM_2.5 decreased cell viability and elevated the level of lactate dehydrogenase. The levels of malondialdehyde and reactive oxygen species were increased by PM_2.5 exposure. PM_2.5 also induced a significant increase of the inflammatory cytokines including interleukin (IL)‐6, IL‐8, IL‐1β and tumor necrosis factor α. Meanwhile, PM_2.5 triggered autophagy formation and alteration of the nuclear factor erythroid‐2‐related factor 2 pathway. Furthermore, human bronchial epithelial cells were co‐treated with PM_2.5 and resveratrol in the presence or absence of 3‐methylamphetamine, an inhibitor of autophagic formation. It was revealed that resveratrol intervention abolished PM_2.5‐induced oxidative injury partially through the suppression of autophagy deregulation. Findings from this study could provide new insights into the molecular mechanisms of pulmonary intervention during PM_2.5 exposure.     

Inflammatory response and endothelial dysfunction in the hearts of mice co‐exposed to SO2, NO2, and PM2.5

SO₂, NO₂, and PM_2.5 are typical air pollutants produced during the combustion of coal. Increasing evidence indicates that air pollution has contributed to the development and progression of heart‐related diseases over the past decades. However, little experimental data and few studies of SO₂, NO₂, and PM_2.5 co‐exposure in animals exist; therefore, the relevant mechanisms underlying this phenomenon are unclear. An important characteristic of air pollution is that co‐exposure persists at a low concentration throughout a lifetime. In the present study, we treated adult mice with SO₂, NO₂, and PM_2.5 at various concentrations (0.5 mg/m³ SO₂, 0.2 mg/m³ NO₂ 6 h/d, with intranasal instillation of 1 mg/kg PM_2.5 every other day during these exposures; or 3.5 mg/m³ SO₂, 2 mg/m³ NO₂ 6 h/d, and 10 mg/kg PM_2.5 for 28 d). Blood pressure (BP), heart rate (HR), histopathological damage, and inflammatory and endothelial cytokines in the heart were assessed. The results indicate that co‐exposure caused endothelial dysfunction by elevating endothelin‐1 (ET‐1) expression and repressing the endothelial nitric oxide synthase (eNOS) level as well as stimulating the inflammatory response by increasing the levels of cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS), tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). Additionally, these alterations were confirmed by histological staining. Furthermore, we observed decreased BP and increased HR after co‐exposure. Our results indicate that co‐exposure to SO₂, NO₂, and PM_2.5 may be a major risk factor for cardiac disease and may induce injury to the hearts of mammals and contribute to heart disease. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1996–2005, 2016.     

Glyburide attenuates ozone‐induced pulmonary inflammation and injury by blocking the NLRP3 inflammasome

Glyburide is a classic antidiabetic drug that is dominant in inflammation regulation, but its specific role in ozone‐induced lung inflammation and injury remains unclear. In order to investigate whether glyburide prevents ozone‐induced pulmonary inflammation and its mechanism, C57BL/6 mice were intratracheally pre‐instilled with glyburide or the vehicle 1 hour before ozone (1 ppm, 3 hours) or filtered air exposure. After 24 hours, the total inflammatory cells and total protein in bronchoalveolar lavage fluid (BALF) were detected. The pathological alternations in lung tissues were evaluated by HE staining. The expression of NLRP3, interleukin‐1β (IL‐1β), and IL‐18 protein in lung tissues was detected by immunohistochemistry. Western blotting was used to examine the levels of caspase‐1 p10 and active IL‐1β protein. Levels of IL‐1β and IL‐18 in BALF were measured using ELISA kits. Glyburide treatment decreased the total cells in BALF, the inflammatory score, and the mean linear intercept induced by ozone in lung tissues. In addition, glyburide inhibited the expression of NLRP3, IL‐18, and IL‐1β protein in lung tissues, and also suppressed NLRP3 inflammasome activation, including caspase‐1 p10, active IL‐1β protein in lung tissues, IL‐1β, and IL‐18 in BALF. These results demonstrate that glyburide effectively attenuates ozone‐induced pulmonary inflammation and injury via blocking the NLRP3 inflammasome.     

Ambient air PM2.5 exposure induces heart injury and cardiac hypertrophy in rats through regulation of miR-208a/b, α/β-MHC,and GATA4

Ambient air fine particulate matter (PM_2.5) may increase cardiovascular disease risks. In this study, we investigated the miR-208/GATA4/myosin heavy chain (MHC) regulation mechanisms on cardiac injury in rats after PM_2.5 exposure via an animal inhalation device. The results showed that PM_2.5 exposure for 2 months caused pathological heart injury, reduced nucleus-cytoplasm ratio, and increased the levels of CK-MB and cTnI, showing cardiac hypertrophy. Oxidative stress and inflammatory responses were also observed in rats’ hearts exposed to PM_2.5. Of note, PM_2.5 exposure for 2-month significantly elevated GATA4 and β-MHC mRNA and protein expression compared with the corresponding controls, along with the high-expression of miR-208b. The ratios of β-MHC/α-MHC expression induced by PM_2.5 were remarkably raised in comparison to their controls. It suggested that the up-regulation of miR-208b/β-MHC and GATA4 and the conversion from α-MHC to β-MHC may be the important causes of cardiac hypertrophy in rats incurred by PM_2.5.     

MiR‐146a regulates PM1‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells

Exposure to particulate matter (PM) leads to kinds of cardiopulmonary diseases, such as asthma, COPD, arrhythmias, lung cancer, etc., which are related to PM‐induced inflammation. We have found that PM_2.5 (aerodynamics diameter <2.5 µm) exposure induces inflammatory response both in vivo and in vitro. Since the toxicity of PM is tightly associated with its size and components, PM₁ (aerodynamics diameter <1.0 µm) is supposed to be more toxic than PM_2.5. However, the mechanism of PM₁‐induced inflammation is not clear. Recently, emerging evidences prove that microRNAs play a vital role in regulating inflammation. Therefore, we studied the regulation of miR‐146a in PM₁‐induced inflammation in human lung bronchial epithelial BEAS‐2B cells. The results show that PM₁ induces the increase of IL‐6 and IL‐8 in BEAS‐2B cells and up‐regulates the miR‐146a expression by activating NF‐κB signaling pathway. Overexpressed miR‐146a prevents the nuclear translocation of p65 through inhibiting the IRAK1/TRAF6 expression, and downregulates the expression of IL‐6 and IL‐8. Taken together, these results demonstrate that miR‐146a can negatively feedback regulate PM₁‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells.     

PM2.5 upregulates rat mesenteric arteries 5‐HT2A receptor via inflammatory‐mediated mitogen‐activated protein kinases signaling pathway

Fine particulate matter (PM_2.5) is an important environmental risk factor for cardiovascular diseases. However, little is known about the effects of PM_2.5 on arteries. The present study investigated whether PM_2.5 alters 5‐hydroxytryptamine (5‐HT) receptor expression and inflammatory mediators on rat mesenteric arteries, and examined the underlying mechanisms. Isolated rat mesenteric arteries segments were cultured with PM_2.5 in the presence or absence of ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was monitored by a sensitive myograph. The expression of 5‐HT_2A/1B receptors and inflammatory mediators were studied by a real‐time polymerase chain reaction and/or by immunohistochemistry. The phosphorylation of mitogen‐activated protein kinases (MAPK) pathway was detected by Western blot. Compared with the fresh or culture alone groups, 1.0 μg/mL PM_2.5 cultured for 16 hours significantly enhanced contractile response induced by 5‐HT and increased 5‐HT_2A receptor mRNA and protein expressions, indicating PM_2.5 upregulates 5‐HT_2A receptor. SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly decreased PM_2.5‐induced elevated contraction and mRNA and protein expression of 5‐HT_2A receptor. Cultured with PM_2.5 significantly increased the mRNA expression of inflammatory mediators (NOS2, IL‐1β, and TNF‐α), while SB203580 decreased mRNA expression level of NOS2, IL‐1β, and TNF‐α. SP600125 (JNK inhibitor) decreased mRNA expression level of TNF‐α and IL‐1β. After PM_2.5 exposure, the phosphorylation of p38 and ERK1/2 protein were increased. SB203580 and U0126 inhibited the PM_2.5 caused increased phosphorylation protein of p38 and ERK1/2. In conclusion, PM_2.5 induces inflammatory‐mediated MAPK pathway in artery which subsequently results in enhanced vascular contraction responding to 5‐HT via the upregulated 5‐HT_2A receptors.     

Cardiomyopathy confers susceptibility to particulate matter-induced oxidative stress,vagal dominance,arrhythmia and pulmonary inflammation in heart failure-prone rats

Abstract

Acute exposure to ambient fine particulate matter (PM_2.5) is tied to cardiovascular morbidity and mortality, especially among those with prior cardiac injury. The mechanisms and pathophysiological events precipitating these outcomes remain poorly understood but may involve inflammation, oxidative stress, arrhythmia and autonomic nervous system imbalance. Cardiomyopathy results from cardiac injury, is the leading cause of heart failure, and can be induced in heart failure-prone rats through sub-chronic infusion of isoproterenol (ISO). To test whether cardiomyopathy confers susceptibility to inhaled PM_2.5 and can elucidate potential mechanisms, we investigated the cardiophysiologic, ventilatory, inflammatory and oxidative effects of a single nose-only inhalation of a metal-rich PM_2.5 (580?µg/m³, 4?h) in ISO-pretreated (35 days?×?1.0?mg/kg/day sc) rats. During the 5 days post-treatment, ISO-treated rats had decreased HR and BP and increased pre-ejection period (PEP, an inverse correlate of contractility) relative to saline-treated rats. Before inhalation exposure, ISO-pretreated rats had increased PR and ventricular repolarization time (QT) and heterogeneity (Tp-Te). Relative to clean air, PM_2.5 further prolonged PR-interval and decreased systolic BP during inhalation exposure; increased tidal volume, expiratory time, heart rate variability (HRV) parameters of parasympathetic tone and atrioventricular block arrhythmias over the hours post-exposure; increased pulmonary neutrophils, macrophages and total antioxidant status one day post-exposure; and decreased pulmonary glutathione peroxidase 8 weeks after exposure, with all effects occurring exclusively in ISO-pretreated rats but not saline-pretreated rats. Ultimately, our findings indicate that cardiomyopathy confers susceptibility to the oxidative, inflammatory, ventilatory, autonomic and arrhythmogenic effects of acute PM_2.5 inhalation.     

Activated iRhom2 drives prolonged PM2.5 exposure-triggered renal injury in Nrf2-defective mice

Abstract

Research suggests that particulate matter (PM_2.5) is a predisposing factor for metabolic syndrome-related systemic inflammation and oxidative stress injury. TNF-α as a major pro-inflammatory cytokine was confirmed to participate in various diseases. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for shedding of TNF-α in immune cells. Importantly, kidney-resident macrophages are critical to inflammation-associated chronic renal injury. Podocyte injury can be induced by stimulants and give rise to nephritis, but how iRhom2 contributes to PM_2.5-induced renal injury is unclear. Thus, we studied whether PM_2.5 causes renal injury and characterized iRhom2 with respect to TNF-α release in mice macrophages and renal tissues in long-term PM_2.5-exposed mouse models. After long-term PM_2.5 exposures, renal injury was confirmed via inflammatory cytokine, chemokine expression, and reduced antioxidant activity. Patients with kidney-related diseases had increased TNF-α, which may contribute to renal injury. We observed up-regulation of serum creatinine, serum urea nitrogen, kidney injury molecule 1, uric acid, TNF-α, MDA, H₂O_2, and O₂^– in PM_2.5-treated mice, which was greater than that found in Nrf2^?/? mice. Meanwhile, increases in metabolic disorder-associated indicators were involved in PM_2.5-induced nephritis. In vitro, kidney-resident macrophages were observed to be critical to renal inflammatory infiltration and function loss via regulation of iRhom2/TACE/TNF-α signaling, and suppression of Nrf2-associated anti-oxidant response. PM_2.5 exposure led to renal injury partly by inflammation-mediated podocyte injury. Reduced SOD1, SOD2, Nrf2 activation, and increased XO, NF-κB activity, TACE, iNOS, IL-1β, TNF-α, IL-6, MIP-1α, Emr-1, MCP-1, and Cxcr4, were also noted. Long-term PM_2.5 exposure causes chronic renal injury by up-regulation of iRhom2/TACE/TNF-α axis in kidney-resident macrophages. Overexpression of TNF-α derived from macrophages causes podocyte injury and kidney function loss. Thus, PM_2.5 toxicities are related to exposure duration and iRhom2 may be a potential therapeutic renal target.     

Epidermal growth factor receptor (EGFR)—MAPK—nuclear factor(NF)‐κB—IL8: A possible mechanism of particulate matter(PM) 2.5‐induced lung toxicity

Airway inflammation plays a central role in the pathophysiology of diverse pulmonary diseases. In this study, we investigated whether exposure to particulate matter (PM) 2.5, a PM with an aerodynamic diameter of less than 2.5 µm, enhances inflammation‐related toxicity in the human respiratory system through activation of the epidermal growth factor receptor (EGFR) signaling pathway. Through cytokine antibody array analysis of two extracts of PM_2.5 [water (W‐PM_2.5) and organic (O‐PM_2.5) soluble extracts] exposed to A549 (human alveolar epithelial cell), we identified eight cytokines changed their expression with W‐PM_2.5 and three cytokines with O‐PM_2.5. Among them, epidermal growth factor (EGF) was commonly up‐regulated by W‐PM_2.5 and O‐PM_2.5. Then, in both groups, we can identify the increase in EGF receptor protein levels. Likewise, increases in the phosphorylation of ERK1/2 MAP kinase and acetylation of nuclear factor(NF)‐κB were detected. We also detected an increase in IL‐8 that was related to inflammatory response. And using the erlotinib as an inhibitor of EGFR, we identified the erlotinib impaired the phosphorylation of EGFR, ERK1/2, acetylation of NF‐κB proteins and decreased IL‐8. Furthermore, at in vivo model, we were able to identify similar patterns. These results suggest that PM_2.5 may contribute to an abnormality in the human respiratory system through EGFR, MAP kinase, NF‐κB, and IL‐8 induced toxicity signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1628–1636, 2017.     

Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells

Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM_2.5) and cardiovascular disorders, but how PM_2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen‐activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM_2.5. H9c2 cells were incubated with PM_2.5 at 100–800 µg ml^–1 to evaluate the effects of PM_2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis‐related proteins as well as activation of MAPKs. PM_2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration‐dependent manner. PM_2.5 decreased the Bcl‐2/Bax ratio and increased cleaved caspase‐3 levels. A Western blots study showed up‐regulation of phosphorylated MAPKs including extracellular signal‐regulated protein kinases (ERKs), c‐Jun NH₂‐terminal kinases (JNKs) and p38 MAPK in the PM_2.5‐treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM_2.5 on apoptosis and the expression of related proteins. In conclusion, PM_2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM_2.5‐induced cardiac injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of ozone and fine particulate matter (PM2.5) on rat system inflammation and cardiac function

In order to understand the toxic mechanisms of cardiovascular system injuries induced by ambient PM_2.5 and/or ozone, a subacute toxicological animal experiment was designed with exposure twice a week for 3 continuous weeks. Wistar rats were randomly categorized into 8 groups (n = 6): 1 control group, 3 groups exposed to fine particulate matters (PM_2.5) alone at 3 doses (0.2, 0.8, or 3.2 mg/rat), 1 group to ozone (0.81 ppm) alone and 3 groups to ozone plus PM_2.5 at 3 doses (0.2, 0.8, or 3.2 mg/rat). Heart rate (HR) and electrocardiogram (ECG) was monitored at approximately 24-h both after the 3rd exposure and the last (6th) exposure, and systolic blood pressure (SBP) was monitored at approximately 24-h after the 6th exposure. Biomarkers of systemic inflammation and injuries (CRP, IL-6, LDH, CK), heart oxidative stress (MDA, SOD) and endothelial function (ET-1, VEGF) were analyzed after the 6th exposure. Additionally, myocardial ultrastructural alterations were observed under transmission electron microscopy (TEM) for histopathological analyses. Results showed that PM_2.5 alone exposure could trigger the significant increase of CRP, MDA, CK, ET-1 and SBP and decrease of heart rate variability (HRV), a marker of cardiac autonomic nervous system (ANS) function. Ozone alone exposure in rats did not show significant alterations in any indicators. Ozone plus PM_2.5 exposure, however, induced CRP, IL-6, CK, LDH and MDA increase, SOD and HRV decrease significantly in a dose–response way. Meanwhile, abnormal ECG types were monitored in rats exposed to PM_2.5 with and without ozone and obvious myocardial ultrastructural changes were observed by TEM. In conclusion, PM_2.5 alone exposure could cause inflammation, endothelial function and ANS injuries, and ozone potentiated these effects induced by PM_2.5.     

Rat lung response to ozone and fine particulate matter (PM2.5) exposures

Exposure to different ambient pollutants maybe more toxic to lung than exposure to a single pollutant. In this study, we discussed the inflammation and oxidative stress responses of rat lung caused by ozone and PM_2.5 versus that of rats exposed to saline, ozone, or single PM_2.5. Wistar rats inhaled 0.8 ppm ozone or air for 4 h and then placed in air for 3 h following intratracheal instillation with 0, 0.2 (low dose), 0.8 (medium dose), 3.2 (high dose) mg/rat PM_2.5 dissolved in sterile saline (0.25 mL/rat), repeated twice per week for 3 weeks, the cumulative doses of PM_2.5 in animals were 1.2, 4.8, and 19.2 mg. Rats were sacrificed 24 h after the last (sixth) exposure. The collected bronchoalveolar lavage fluid (BALF) was analyzed for inflammatory cells and cytokines. Lung tissues were processed for light microscopic and transmission electron microscopic (TEM) examinations. Results showed that total cell number in BALF of PM_2.5‐exposed groups were higher than control (p < 0.05). PM_2.5 instillation caused dose‐trend increase in tumor necrosis factor alpha (TNF‐α), interleukin‐6, lactate dehydrogenase, and total protein of BALF. Exposure to ozone alone only caused TNF‐α significant change in above‐mentioned indicators of lung injury. On the other hand, ozone could enhance PM_2.5‐induced inflammatory changes and pathological characters in rat lungs. SOD and GSH‐Px activities in lung were reduced in PM_2.5‐exposed rats with and without prior ozone exposure compared to control. To determine whether the PM_2.5 and ozone affect endothelium system, iNOS, eNOS, and ICAM‐1 mRNA levels in lung were analyzed by real‐time PCR. These data demonstrated that inflammation and oxidative stress were involved in toxicology mechanisms of PM_2.5 in rat lung and ozone potentiated these effects induced by PM_2.5. These results have implications for understanding the pulmonary effects induced by ozone and PM_2.5. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 343–356, 2015.     

Comparison of how ambient PMc and PM2.5 influence the inflammatory potential

Abstract

Airborne particulate matter (PM) is one of the six criteria air pollutants currently regulated by the U.S. Environmental Protection Agency (EPA), with existing ambient standards for PM_2.5 and PM₁₀. Currently there are no health-based regulations for the size fraction between 2.5 and 10?µm, commonly known as the coarse fraction (PM_c). The present study investigates current gaps in knowledge for PM_c including exposure toxicity and PM ratios (PM_c:PM_2.5) in PM₁₀. Throughout the world, all the three PM size fractions have been shown to be associated with adverse impacts. Recent studies have shown that PM_c can be more detrimental to susceptible populations when directly compared to PM_2.5, and that the PM_c fraction in PM₁₀ can account for the majority of the inflammatory response from PM₁₀ exposure. In our studies we utilized a bone marrow-derived mouse macrophage in vitro system to compare the inflammatory potential of PM_c, PM_2.5 and mixtures of the two. The result was a linear increase in interleukin(IL) -1β with increasing levels of exposure to winter and summer PM_c, as compared to PM_2.5, which exhibited logarithmic growth. Also, exposure to PM₁₀ as a function of PM_2.5 and PM_c mass ratios showed that IL-1β and TNF-α levels increased synergistically with a greater burden of PM_c. Endotoxin content in the PM did not correlate with these results, suggesting that other activators in PM_c are likely responsible for activating the NF-κB pathway and the inflammasome.     

GRK2/β‐arrestin mediates arginine vasopressin‐induced cardiac fibroblast proliferation

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl‐tetrazolium assay and 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α‐smooth muscle actin (α‐SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK_1/2 were determined by western blotting. AVP, in a time‐ and concentration‐dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein‐coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2‐Ct or knock‐down of GRK2 suppressed AVP‐induced BrdU incorporation and the expression of MMP2 and α‐SMA in NRCFs. Moreover, shRNA‐mediated silencing of β‐arrestin1 or β‐arrestin 2 abolished AVP‐induced BrdU incorporation and MMP2 expression. AVP‐induced NRCF proliferation depended on the phosphorylation of ERK_1/2, and inhibition of GRK2 or silencing of β‐arrestins blocked AVP‐induced ERK_1/2 phosphorylation. The effects of AVP on NRCF proliferation and α‐SMA expression were blocked by SR45059, a vasopressin receptor type1A (V_1AR) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V_1AR‐mediated GRK2/β‐arrestin/ERK_1/2 signalling.     

The mechanisms for lung cancer risk of PM2.5: Induction of epithelial‐mesenchymal transition and cancer stem cell properties in human non‐small cell lung cancer cells

Fine particulate matter (PM_2.5) is a major component of air pollutions that are closely associated with increased risk of lung cancer. However, the role of PM_2.5 in the etiology of lung cancer is largely unknown. In this study, we performed acute (24 hours) and chronic (five passages) exposure models to investigate the carcinogenetic mechanisms of PM_2.5 by targeting the induction of epithelial‐mesenchymal transition (EMT) and cancer stem cells (CSC) properties in human non‐small cell lung cancer cell line A549. We found that both acute and chronic PM_2.5 exposure enhanced cell migration and invasion, decreased mRNA expression of epithelial markers and increased mRNA expression of mesenchymal markers. Chronic PM_2.5 exposure further induced notable EMT morphology and CSC properties, indicating the developing process of cell malignant behaviors from acute to chronic PM_2.5 exposure. CSC properties induced by chronic PM_2.5 exposure characterized with increased cell‐surface markers (CD44, ABCG2), self‐renewal genes (SOX2 and OCT4), side population cells and neoplastic capacity. Furthermore, the levels of three stemness‐associated microRNAs, Let‐7a, miR‐16 and miR‐34a, were found to be significantly downregulated by chronic PM_2.5 exposure, with microarray data analysis from TCGA database showing their lower expression in human lung adenocarcinoma tissues than that in the adjacent normal lung tissues. These data revealed that the induction of EMT and CSC properties were involved in the lung cancer risk of PM_2.5, and implicated CSC properties and related microRNAs as possible biomarkers for carcinogenicity prediction of PM_2.5.     

Mitochondrial dysfunction in endothelial cells induced by airborne fine particulate matter (<2.5 μm)

Exposure to ambient fine particulate matter (<2.5 μm; PM_2.5) increases the risk of the physiopathology of vascular diseases. However, the underlying mechanism, particularly the mitochondrial damage mechanism, of PM_2.5‐induced vascular dysfunction is still unclear. In this study, we examined PM_2.5‐induced alterations of mitochondrial morphology, and further demonstrated the adverse effects on mitochondrial dynamics and function in vascular endothelial cells. Consequently, cultured EA.hy926 cells were subjected to PM_2.5 collected from Beijing. A Cell Counting Assay Kit‐8 demonstrated that PM_2.5 exposure decreased the proliferation of EA.hy926 cells in a dose‐dependent manner. The exposure caused an increment of abnormal mitochondria coupled with the decrease of fusion protein MFN2 and the increase of fission protein FIS1, suggesting that PM_2.5 inhibits mitochondrial fusion. Further analyses revealed PM_2.5 decreased the mitochondrial membrane potential (ΔΨm) and increased the mitochondrial permeability transport pore opening, eventually resulting in impairments in adenosine triphosphate synthesis. Therefore, it is clearly shown that PM_2.5 triggered endothelial toxicity through mitochondria as the target, including the damage of mitochondrial homeostasis.