Integrative analysis of mRNA and microRNA expression of a human alveolar epithelial cell(A549) exposed to water and organic‐soluble extract from particulate matter (PM)2.5

MicroRNA (miRNA) is now attracting attention as a powerful negative regulator of messenger RNA(mRNA) levels, and is implicated in the modulation of important mRNA networks involved in toxicity. In this study, we assessed the effects of particulate matter 2.5 (PM_2.5), one of the most significant air pollutants, on miRNA and target gene expression. We exposed human alveolar epithelial cell (A549) to two types of PM_2.5[water (W‐PM_2.5) and organic (O‐PM_2.5) soluble extracts] and performed miRNA microarray analysis. A total of 37 miRNAs and 62 miRNAs were altered 1.3‐fold in W‐PM_2.5 and O‐PM_2.5, respectively. Integrated analyses of miRNA and mRNA expression profiles identified negative correlations between miRNA and mRNA in both W‐PM_2.5 and O‐PM_2.5 exposure groups. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses showed that the 35 W‐PM_2.5 target genes are involved in responses to nutrients, positive regulation of biosynthetic processes, positive regulation of nucleobase, nucleoside, and nucleotide, and nucleic acid metabolic processes; while the 69 O‐PM_2.5 target genes are involved in DNA replication, cell cycle processes, the M phase, and the cell cycle check point. We suggest that these target genes may play important roles in PM_2.5‐induced respiratory toxicity by miRNA regulation. These results demonstrate an integrated miRNA‐mRNA approach for identifying molecular events induced by environmental pollutants in an in vitro human model. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 302–310, 2017.     

Effect of c-fos gene silence on PM2.5-induced miRNA alteration in human bronchial epithelial cells

Human bronchial epithelial (HBE) cells and c-fos-silenced HBE cells were first exposed to fine particulate matter (PM_2.5) and the resulting miRNA sequenced. Thereafter, a weighted gene co-expression network analysis was performed using Cytoscape software to visualize the interactions between identified hub miRNAs and their target genes. Nine differentially expressed miRNAs in hub miRNAs were identified in the different treatment groups, of which miR-25−3p, miR-215−5p, and miR-145−5p were selected for further study. Following qPCR validation, both miR-25−3p and miR-215−5p were found to be significantly up-regulated whilst, miR-145−5p was significantly down-regulated (p < 0.05) in the PM_2.5 group. Furthermore, miR-25−3p and miR-145−5p were also significantly down-regulated in the untreated group of c-fos silenced HBE cells. However, miR-215−5p was significantly down-regulated in both the untreated and PM_2.5-treated groups of c-fos silenced HBE cells. Subsequent analysis of their target genes also illustrated differential gene expression when comparing the treatment groups of the two cell types. The present data indicated that the c-fos gene has an important effect on the miRNA expression profiles and the related signaling pathways in PM_2.5-treated HBE cells. Therefore, each of miR-25−3p, miR-145−5p, and miR-215−5p may potentially provide future research information for additional exploration of a PM_2.5-induced carcinogenesis mechanism.     

Exposure to PM2.5 causes genetic changes in fetal rat cerebral cortex and hippocampus

PM_2.5 travels along the respiratory tract and enters systemic blood circulation. Studies have shown that PM_2.5 increases the incidence of various diseases not only in adults but also in newborn infants. It causes chronic inflammation in pregnant women and retards fetal development. In this study, pregnant rats were exposed to PM_2.5 for extended periods of time and it was found that PM_2.5 exposure increased immune cells in mother rats. In addition, cytokines and free radicals rapidly accumulated in the amniotic fluid and indirectly affected the fetuses. The authors collected cerebral cortex and hippocampus samples at E18 and analyzed changes of miRNA levels. Expression levels of cortical miR‐6315, miR‐3588, and miR‐466b‐5p were upregulated, and positively correlated with the genes Pkn2 (astrocyte migration), Gorab (neuritogenesis), and Mobp (allergic encephalomyelitis). In contrast, PM_2.5 decreased expression of miR‐338‐5p and let‐7e‐5p, both related to mental development. Further, PM_2.5 exposure increased miR‐3560 and let‐7b‐5p in the hippocampus, two proteins that regulate genes Oxct1 and Lin28b that control ketogenesis and glycosylation, and neural cell differentiation, respectively. miR‐99b‐5p, miR‐92b‐5p, and miR‐99a‐5p were decreased, leading to reduced expression of Kbtbd8 and Adam11 which reduced cell mitosis, migration, and differentiation, and inhibited learning abilities and motor coordination of the fetus. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1412–1425, 2017.     

p53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM2.5 sampled from air in Guangzhou,China

Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16–128?µg/ml) of PM_2.5 for 24?h to investigate the apoptosis induced by PM_2.5. The results showed that PM_2.5 exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM_2.5 in 16-HBE cells, whereas the expression of p21^Cip1/WAF1 decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM_2.5 may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM_2.5-induced apoptosis in 16-HBE cells.     

NLRP3 inflammasome activation is associated with PM2.5‐induced cardiac functional and pathological injury in mice

Growing evidences indicate that inflammation induced by PM_2.5 exposure has been considered as a major driving force for the development of cardiovascular diseases. However, the mechanisms underlying PM_2.5‐induced cardiac injury remain unclear. This study aims to investigate the role of NLRP3 inflammasome in PM_2.5‐induced cardiac functional and pathological injury in mice. In this study, BALB/c mice were intratracheally instilled with PM_2.5 suspension (4.0 mg/kg BW) for 5 days to set up a cardiac injury model, which was evaluated by electrocardiogram monitoring, HE and Masson staining. Then, the effects of PM_2.5 on the expression of α‐SMA, NLRP3, IL‐1β, and IL‐18 proteins and the activation of caspase‐1 and IL‐1β were investigated. The results showed that PM_2.5 exposure induced characteristic abnormal ECG changes such as the abnormality of heart rhythm, tachycardia, and T‐wave reduction. Inflammatory cell infiltration and fibrosis were observed in the heart tissues of PM_2.5‐exposed mice. Meanwhile, PM_2.5 exposure increased the expression of α‐SMA. And, NLRP3 activation‐associated proteins of NLRP3, IL‐1β, IL‐18, Cleaved caspase‐1 p10, and Cleaved IL‐1β were upregulated in heart tissue of PM_2.5‐induced mice. In summary, PM_2.5 exposure could induce cardiac functional and pathological injury, which may be associated with the activation of NLRP3 inflammasome.     

Changes in RANKL and osteoprotegerin expression after chronic exposure to indoor air pollution as a result of cooking with biomass fuel

The impact of indoor air pollution as a result of cooking with unprocessed biomass on membrane‐bound and serum receptor activator of nuclear factor‐kappa ligand 1 (RANKL), its soluble decoy receptor osteoprotegerin (OPG) and osteoclast precursor CD14⁺CD16⁺ monocytes was investigated. Seventy‐four pre‐menopausal women from eastern India using biomass and 65 control women who cooked with cleaner liquefied petroleum gas were enrolled. PM₁₀ and PM_2.5 levels in their indoor air were measured with real‐time aerosol monitors. The levels of membrane‐bound RANKL on leukocytes and percentage CD14⁺CD16⁺ monocytes in the subjects' blood were assayed by flow cytometry. Soluble RANKL and OPG in serum were measured by ELISA. The results showed that PM₁₀ and PM_2.5 levels were significantly higher in the indoor air of biomass‐using households. Compared with the control women, the levels of CD4⁺ and CD19⁺ lymphocytes and circulating granulocytes with elevated levels of membrane‐bound RANKL were higher in biomass users. The serum levels of RANKL were increased by 41% whereas serum OPG was reduced by 22% among biomass users. The absolute number of CD14⁺CD16⁺ monocytes was significantly increased in biomass users than the control women. After controlling for potential confounders, PM₁₀ and PM_2.5 levels were found to be positively associated with leukocyte and serum RANKL and CD14⁺CD16⁺ monocyte levels, but negatively with serum OPG. From these results, we can conclude that chronic exposure to biomass smoke increased membrane‐bound and soluble RANKL and circulating osteoclast precursors but decreased OPG, suggesting an increased risk of bone resorption and consequent osteoporosis in biomass‐exposed women of a child‐bearing age. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM_2.5) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM_2.5 in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM_2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM_2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24 h of exposure (p < 0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48 h of exposure (p < 0.01), while those genes expressions were significantly reduced after 72 h of exposure, at which time the expression of p21 was predominant (p < 0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM_2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM_2.5-induced damage to the trachea and lung tissue was time-dependent.     

Effects of ambient ozone exposure on circulating extracellular vehicle microRNA levels in coronary artery disease patients

ABSTRACT

Exposure to ambient air pollutants such as ozone (O₃) and particulate matter (PM) is associated with increased cardiovascular morbidity and rate of mortality, but the underlying biological mechanisms have yet to be described. Emerging evidence shows that extracellular vehicle (EV) microRNAs (miRNAs) may facilitate cell-to-cell and organ-to-organ communications and play a role in the air pollution-induced cardiovascular effects. This study aims to explore the association between air pollutant exposure and miRNA changes related to cardiovascular diseases. Using a panel study design, 14 participants with coronary artery diseases were enrolled in this study. Each participant had up to 10 clinical visits and their plasma samples were collected and measured for expression of miRNA-21 (miR-21), miR-126, miR-146, miR-150, and miR-155. Mixed effects models adjusted for temperature, humidity, and season were used to examine the association between miRNA levels and exposure to 8-hr O₃ or 24-hr PM_2.5 up to 4 days prior. Results demonstrated that miR-150 expression was positively associated with O₃ exposure at 1–4 days lag and 5day moving average while miR-155 expression tracked with O₃ exposure at lag 0. No significant association was found between miRNA expression and ambient PM_2.5 at any time point. β-blocker and diabetic medication usage significantly modified the correlation between O₃ exposure and miR-150 expression where the link was more prominent among non-users. In conclusion, evidence indicated an association between exposure to ambient O₃ and circulating levels of EV miR-150 and miR-155 was observed. These findings pointed to a future research direction involving miRNA-mediated mechanisms of O₃-induced cardiovascular effects.     

PM2.5 promotes replication of VSV by ubiquitination degradation of phospho-IRF3 in A549 cells

Both PM_2.5 and respiratory viruses are part of the atmospheric constituents. Respiratory viruses are often associated with PM_2.5 exposure, but the mechanism of toxicity remains to be explored. The vitro models that adequately reproduce healthy cells or diseased cells exposing to PM_2.5 and infecting VSV can provide a useful tool for studying innate immune mechanisms and investigating new therapeutic focus.In the environment of PM_2.5, an infection model in which VSV infected A549 cells was established, that mimics the state in which the antiviral innate immune pathways are activated after the respiratory system is infected with RNA viruses. Subsequently, the model was exposed to PM_2.5 for 24 h. PM_2.5 could be ingested by A549 cells and synergize with VSV to inhibit cell viability and promote apoptosis. The expression of VSV-G were more abundant after VSV-infected A549 cells were exposed to PM_2.5. Furthermore, PM_2.5 inhibits VSV-induced IFN-β expression in A549 cells. ISG15, CCL-5, and CXCL-10 had the same expression tendency with IFN-β mRNA, consistently. Interestingly, when MG132 was applied, the expression of p-IRF-3 and IFN-β proteins reduced by PM_2.5 were refreshed. Conversely, the expression of VSV-G proteins were decreased.PM_2.5 could degrade p-IRF-3 proteins by ubiquitination pathway to inhibit VSV-induced IFN-β expression in A549 cells. Therefore, replication of the VSV viruses was promoted.     

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.     

Proteomic characteristics of PM2.5 -induced differentially expressed proteins in human renal tubular epithelial cells

Human renal epithelial (HK-2) cells were treated with PM_2.5 (50 μg/mL) from Shenzhen and Taiyuan, proteomics and bioinformatics were used to screen the differentially expressed proteins (DEPs). A total of 577 DEPs were screened after HK-2 cells exposed to Shenzhen PM_2.5, of which 426 were up-regulated and 151 were down-regulated. A total of 1250 DEPs were screened in HK-2 cells after exposure to Taiyuan PM_2.5, of which 488 were up-regulated and 185 were down-regulated. The top 10 proteins with the highest number of nodes were screened using the interaction network map of DEPs. HK-2 cells exposed to Shenzhen PM_2.5 contained CYR61, CTGF, and THBS1 proteins, while HK-2 cells exposed to Taiyuan PM_2.5 contained ALB, FN1, and CYR61 proteins. Additionally, PM_2.5 components were detected, PM_2.5 samples from Shenzhen and Taiyuan induced obvious changes in DEPs expression, the difference in DEPs between the two cities was probably associated with the different PM_2.5 components.     

Acute kidney damage by PM2.5 exposure in a rat model

PM_2.5 exposure is associated with a glomerular filtration rate (GFR) reduction, and renal tissue damage. The goal of this study was demonstrate the acute effect of PM_2.5 on the kidney. Male rats were acutely exposed to PM_2.5 or filtered air. Blood pressure was mesure and early kidney biomarkers were evaluated in serum and urine samples, and also IL-1β, IL-6 and TNFα were determined. Oxidative biomarkers, angiotensin/bradykinin-related proteins, KIM-1, IL-6 and histology were determined. Blood pressure, GFR, and early kidney damage biomarkers increase together with oxidative biomarkers and angiotensin/bradykinin endocrine-related proteins increased after exposure to PM_2.5. Urinary IL-6 increased after exposure to PM_2.5, whereas in kidney cortex decreased. Histological changes were observed and accompanied by the induction of KIM-1. Acute exposure to PM_2.5 not decline kidney function. However, it can induce early kidney damage biomarkers, oxidative stress, inflammation and angiotensin mediators, which perhabs culminates in a lose of renal function.     

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.     

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.     

Dust fall PM2.5-induced lung inflammation in rats is associated with hypermethylation of the IFN-γ gene promoter via the PI3K-Akt-DNMT3b pathway

Inflammation is one of the major adverse effects of fine particulate matter (PM_2.5) on the lung system; however, its mechanisms remain unclear. Rats were exposed to different concentrations of PM_2.5 to investigate the mechanism of short-term exposure-induced lung inflammation. The regulation of PI3K-Akt and DNA methyltransferase 3b (DNMT3b) was assessed by using a PI3K inhibitor and a DNA methyltransferase inhibitor. We found that PM_2.5 could decrease interferon-γ (IFN-γ) levels and increase interleukin 4 (IL-4), IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF) to promote eosinophil infiltration and eventually lead to allergic pulmonary inflammation. Moreover, the CpG island methylation rate of the IFN-γ promoter and the protein expression of DNMT3b, PI3K and p-Akt were increased in lung tissues after PM_2.5 exposure. Both inhibitors reversed the CpG island hypermethylation of IFN-γ. In conclusion, in PM_2.5-induced lung injury, the activated PI3K-Akt pathway, via an increase in DNMT3b expression, is involved in CpG hypermethylation of the IFN-γ gene promoter.     

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.     

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.     

The injury of fine particulate matter from cooking oil fumes on umbilical cord blood vessels in vitro

Cooking oil fumes (COFs) derived PM_2.5 is the major source of indoor air pollution in Asia. For this, a pregnant rat model within different doses of cooking oil fumes (COFs) derived PM_2.5 was established in pregnancy in our research. Our previous studies have showed that exposure to COFs-derived PM_2.5 was related to adverse pregnancy outcomes. However, the mechanisms of signaling pathways remain unknown. Therefore, this study aimed to investigate the underlying mechanisms induced by COFs-derived PM2.5 injury on umbilical cord blood vessels (UCs) in vitro. Exposure to COFs-derived PM_2.5 resulted in changing the expression of eNOS, ET-1, ETRA, and ETRB. In additions, western blot analysis indicated that the HIF-1α/iNOS/NO signaling pathway and VEGF/VEGFR1/iNOS signaling pathway were involved in UCs injury triggered by COFs-derived PM_2.5. In conclusion, our data suggested that exposure to COFs-derived PM_2.5 resulted in increasing of oxidative stress and inflammation, as well as dysfunction of UCs.     

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.