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.     

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.     

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.     

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.     

Let‐7a modulates particulate matter (≤ 2.5 μm)‐induced oxidative stress and injury in human airway epithelial cells by targeting arginase 2

Epidemiological studies show that particulate matter (PM) with an aerodynamic diameter ≤ 2.5 μm (PM2.5) is associated with cardiorespiratory diseases via the induction of excessive oxidative stress. However, the precise mechanism underlying PM2.5‐mediated oxidative stress injury has not been fully elucidated. Accumulating evidence has indicated the microRNA let‐7 family might play a role in PM‐mediated pathological processes. In this study, we investigated the role of let‐7a in oxidative stress and cell injury in human bronchial epithelial BEAS2B (B2B) cells after PM2.5 exposure. The let‐7a level was the most significantly decreased in B2B cells after PM2.5 exposure. The overexpression of let‐7a suppressed intracellular reactive oxygen species levels and the percentage of apoptotic cells after PM2.5 exposure, while the let‐7a level decreased arginase 2 (ARG2) mRNA and protein levels in B2B cells by directly targeting the ARG2 3′‐untranslated region. ARG2 expression was upregulated in B2B cells during PM2.5 treatment, and ARG2 knockdown could remarkably reduce oxidative stress and cellular injury. Moreover, its restoration could abrogate the protective effects of let‐7a against PM2.5‐induced injury. In conclusion, let‐7a decreases and ARG2 increases resulting from PM2.5 exposure may exacerbate oxidative stress, cell injury and apoptosis of B2B cells. The let‐7a/ARG2 axis is a likely therapeutic target for PM2.5‐induced airway epithelial injury. Copyright © 2016 John Wiley & Sons, Ltd.     

Resveratrol protects human bronchial epithelial cells against nickel‐induced toxicity via suppressing p38 MAPK,NF‐κB signaling,and NLRP3 inflammasome activation

Nickel is a common environmental pollutant that can impair the lung, but the underlying mechanisms have not yet been fully elucidated. Furthermore, natural products are generally used to inhibit cell damage induced by heavy metal. Resveratrol possesses wide biological activities, including anti‐inflammation and antioxidative stress. This study was conducted to explore the toxicity of nickel on human bronchial epithelial (BEAS‐2B) cells and evaluate the protective effect of resveratrol. The results showed that nickel could induce cell apoptosis, increase oxidative stress, and promote the expression of pro‐inflammatory cytokines, including tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐6, IL‐8, C‐reaction protein. Western blot analysis showed that nickel activated p38 mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B, and nucleotide‐binding oligomerization domain‐like receptor pyrin‐domain‐containing protein 3 pathways, while resveratrol could reverse these effects. Our results suggested that resveratrol could protect BEAS‐2B cells from nickel‐induced cytotoxicity. Therefore, resveratrol is a potential chemopreventive agent against nickel‐induced lung disease.     

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.     

PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells

Exposure to higher levels of air pollution particulate matter (PM) with an aerodynamic diameter of less than 2.5 μm (PM_2.5) links with an increased risk of cardiovascular and respiratory deaths and hospital admission as well as lung cancer. Although the mechanism underlying the correlation between PM_2.5 exposure and adverse effects has not fully elucidated, PM_2.5-induced oxidative stress has been considered as an important molecular mechanism of PM_2.5-mediated toxicity. In this work, human lung epithelial A549 cells were used to further investigate the biological effects of PM_2.5 on autophagy. The cell viability showed both time- and concentration-dependent decrease when exposure to PM_2.5, which can be attributed to increase of the levels of extracellular lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) generation in A549 cells. Moreover, PM_2.5-induced oxidative damage in A549 cells was observed through the alteration of superoxide dismutase (SOD) and catalase (CAT) activities compared to the unexposed control cells. PM_2.5-induced autophagy was indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, and accumulation of LC3 in both time- and concentration-dependent manner. PM_2.5-induced mRNA expression of autophagy-related protein Atg5 and Beclin1 was also observed compared with those of the unexposed control cells. These results suggest the possibility that PM_2.5-induced oxidative stress probably plays a key role in autophagy in A549 cells, which may contribute to PM_2.5-induced impairment of pulmonary function.     

Oxidative stress and endocytosis are involved in upregulation of interleukin‐8 expression in airway cells exposed to PM2.5

Inhaled PM2.5 (particulate matter with an aerodynamic diameter of 2.5 μm or less) can induce lung inflammation through released inflammatory mediators from airway cells, such as interleukin‐8 (IL‐8) and tumor necrosis factor alpha (TNF‐α). However, the mechanisms underlying PM2.5‐induced IL‐8 gene expression have not been fully characterized. BEAS‐2B cells (a human bronchial epithelial cell line) and THP‐1 cells (a human macrophage‐like cell line) were used as the in vitro models to investigate the underlying mechanism in this study. IL‐8 expression was increased in the cells treated with PM2.5 in a dose‐dependent manner. The water‐soluble and insoluble fractions of PM2.5 suspension were both shown to induce IL‐8 expression. PM2.5 exposure could obviously induce ROS (reactive oxygen species) generation, indicative of oxidative stress. Pretreatment with the antioxidant N‐acetyl‐l ‐cysteine (NAC) potently inhibited PM2.5‐induced IL‐8 expression. Employment of the transition metal chelators including TPEN (N,N,N',N'‐tetrakis (2‐pyridylmethyl) ethylenediamine) or DFO (desferrioxamine) inhibited IL‐8 expression induced by PM2.5 by over 20% in BEAS‐2B cells, but had minimal effect in THP‐1 cells. Pretreatment with the endocytosis inhibitor CytD markedly blocked IL‐8 expression induced by PM2.5 in both BEAS‐2B and THP‐1 cells. In summary, exposure to PM2.5 induced IL‐8 gene expression through oxidative stress induction and endocytosis in airway cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1869–1878, 2016.     

Regulation of Eclipta prostrata L. components on cigarette smoking‐induced autophagy of bronchial epithelial cells via keap1‐Nrf2 pathway

Cigarette smoking extract (CSE)‐induced autophagic injury has been regarded as an important contributor to the pathogenesis of lung cancer. We previously found that Eclipta prostrata L. component (CCE) reduced CSE‐induced bronchial epithelial cells damage. However, the mechanism remains unknown. Human normal bronchial epithelial cells (NHBE) were exposed to CSE to establish stress model. Nrf2‐siRNA and Keap1‐siRNA transfection were performed. mRFP‐GFP‐LC3 dual fluorescence and transmission electron microscopy were used to observe the autophagic characteristics. CCE prevented CSE‐induced Nrf2 transfer into cytoplasm and up‐regulated Keap1 level of NHBE cells. Furthermore, CCE significantly increased p‐p16, p‐p21 and p‐p53 phosphorylation levels in Nrf2‐siRNA‐ or Keap1‐siRNA‐transfected cells. As demonstrated by transmission electron microscopy and mRFP‐GFP‐LC3 dual fluorescence assays, CCE mitigated autophagic injury, and also down‐regulated autophagy‐related Beclin‐1, LC3II/LC3I ratio, Atg5 and ATF4 levels. Our findings showed the attenuation of CCE on CSE‐induced NHBE cells injury was associated with Nrf‐2‐mediated oxidative signaling pathway.     

The toxic effects of indoor atmospheric fine particulate matter collected from allergic and non‐allergic families in Wuhan on mouse peritoneal macrophages

Recent studies have shown that fine particulate matter (PM_2.5) is associated with multiple adverse health outcomes and PM_2.5‐induced oxidative stress is now commonly known as a proposed mechanism of PM_2.5‐mediated toxicity. However, the association between allergic symptoms in children and exposure to PM_2.5 has not been fully elucidated, particularly the role of PM_2.5 on the indoor environment involved in allergy or non‐allergy is unknown. The aim of the present study was to explore whether indoor PM_2.5 from the homes of children with allergic symptoms had more increased risks of allergy than that of healthy ones and then compare the toxicity and inflammatory response of them. In this study, indoor PM_2.5 was collected from the homes of schoolchildren with allergic symptoms and those of healthy ones respectively, and components of PM_2.5 were analyzed. PM_2.5‐mediated oxidative damage and inflammatory response were further evaluated in mouse peritoneal macrophages based on its effects on the levels of reactive oxygen species accumulation, lipid peroxidation, DNA damage or cytokine production. It seems that oxidative stress may contribute to PM_2.5‐induced toxicity, and PM_2.5 from the allergic indoor environment produced more serious toxic effects and an inflammatory response on mouse peritoneal macrophages than that from a non‐allergic indoor environment. Copyright © 2015 John Wiley & Sons, Ltd.     

Airborne fine particulate matter causes murine bronchial hyperreactivity via MAPK pathway‐mediated M3 muscarinic receptor upregulation

Regarding the human health effects, airborne fine particulate matter 2.5 (PM_2.5) is an important environmental risk factor. However, the underlying molecular mechanisms are largely unknown. The present study examined the hypothesis that PM_2.5 causes bronchial hyperreactivity by upregulated muscarinic receptors via the mitogen‐activated protein kinase (MAPK) pathway. The isolated rat bronchi segments were cultured with different concentration of PM_2.5 for different time. The contractile response of the bronchi segments were recorded by a sensitive myograph. The mRNA and protein expression levels of M₃ muscarinic receptors were studied by quantitative real‐time PCR and immunohistochemistry, respectively. The muscarinic receptors agonist, carbachol induced a remarkable contractile response on fresh and DMSO cultured bronchial segments. Compared with the fresh or DMSO culture groups, 1.0 µg/mL of PM_2.5 cultured for 24 h significantly enhanced muscarinic receptor‐mediated contractile responses in bronchi with a markedly increased maximal contraction. In addition, the expression levels of mRNA and protein for M₃ muscarinic receptors in bronchi of PM_2.5 group were higher than that of fresh or DMSO culture groups. SB203580 (p38 inhibitor) and U0126 (MEK1/2 inhibitor) significantly inhibited the PM_2.5‐induced enhanced contraction and increased mRNA and protein expression of muscarinic receptors. However, JNK inhibitor SP600125 had no effect on PM_2.5‐induced muscarinic receptor upregulation and bronchial hyperreactivity. In conclusion, airborne PM_2.5 upregulates muscarinic receptors, which causes subsequently bronchial hyperreactivity shown as enhanced contractility in bronchi. This process may be mediated by p38 and MEK1/2 MAPK pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 371–381, 2017.     

Resveratrol protects Leydig cells from nicotine‐induced oxidative damage through enhanced autophagy

Some studies have revealed that nicotine can damage the male reproductive system through various means including oxidative stress, which is a primary factor in the pathogenesis of male infertility. The strong anti‐oxidative capacity of resveratrol has been demonstrated previously, but its role in the context of male reproduction remains inconclusive. To explore the biological role of resveratrol in protecting male reproductive function and the potential underlying mechanism, nicotine‐induced Leydig cells were used as a cell model of oxidative damage. The data showed that resveratrol treatment increased cell viability, SOD activity and anti‐apoptotic activity in nicotine‐stressed Leydig cells. This effect was accompanied by the upregulation of autophagy, which was illustrated by MDC‐LysoTracker red staining. Moreover, pretreating with 3‐methyladenine (3‐MA), an autophagy inhibitor, attenuated resveratrol‐induced Leydig cells autophagy and promoted apoptosis. Apart from this, resveratrol enhanced AMPK phosphorylation but reduced mTOR phosphorylation. Subsequently, upon inhibiting AMPK phosphorylation by AMPK inhibitors, Leydig cell autophagy induced by resveratrol was obviously abolished. In conclusion, resveratrol may exert its cytoprotective role against oxidative injury by the activation of autophagy via AMPK/mTOR pathway.     

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.     

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.     

Silymarin attenuates paraquat‐induced lung injury via Nrf2‐mediated pathway in vivo and in vitro

The present study aims to investigate the impacts and mechanisms of silymarin on paraquat (PQ)‐induced lung injury in vivo and in vitro. In in vivo experiments, a total of 32 male Sprague‐Dawley (SD) rats were randomly divided into four groups. The rats were killed on day 3. Histopathological changes in lung tissue were examined using HE and Masson's trichrome staining. Biomarkers of neutrophil activation, pulmonary oedema, pulmonary fibrosis, lung permeability and oxidative stress were detected. Several proinflammatory mediators and antioxidant related proteins were measured. In in vitro experiments, A549 cells were transfected with Nrf2 special siRNA to investigate the roles of Nrf2. The results show that silymarin administration abated PQ‐induced lung histopathologic changes, decreased inflammatory cell infiltration and lung wet weight/dry weight (W/D) ratio, suppressed myeloperoxidase (MPO) activity and nitric oxide (NO)/inducible nitric oxide synthases (iNOS) expression, downregulated hydroxyproline (HYP) levels, reduced total protein concentration and proinflammatory mediator release, and improved oxidative stress (malondialdehyde, MDA; superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GSH‐Px) in lung tissue and serum. Meanwhile, treatment with silymarin upregulated the levels of nuclear factor‐erythroid‐2‐related factor 2 (Nrf2), heme oxygenase‐1 (HO‐1) and NAD(P)H:quinone oxidoreductase‐1(NQO1). However, the addition of Nrf2 siRNA reduced the expression of Nrf2‐mediated antioxidant protein HO‐1 and thus reversed the protective effects of silymarin against oxidative stress and inflammatory response. These results suggest that silymarin may exert protective effects against PQ‐induced lung injury. Its mechanisms were associated with the Nrf2‐mediated pathway. Therefore, silymarin may be a potential therapeutic drug for lung injury.     

Ginsenoside Rg1 reduces toxicity of PM2.5 on human umbilical vein endothelial cells by upregulating intracellular antioxidative state

Ambient airborne particulate matter (PM) is an important environmental pollutant responsible for many human diseases. Oxidative stress is suggested to be involved in PM-induced cell injury. The present study is designed to study unsalutary effects of the organic extracts of PM with an aerodynamic diameter of less than 2.5 μm (PM_2.5) and protective effect of Ginsenoside Rg1 (Rg1) against PM_2.5 on human umbilical vein endothelial cells (HUVECs) in vitro. Cytotoxic effects of the organic extract PM_2.5 on HUVECs were measured by means of HUVEC cell viability and the generation of intracellular reactive oxygen species (ROS). Expression of heme oxygenase-1(HO-1) and Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Nrf2 cytoplasm–nucleus location were assayed. The present results showed that PM_2.5 (50–800 μg/ml) decreased HUVEC viability and increased intracellular generation of ROS and malondialdehyde (MDA) in a concentration dependent manner, but increased HO-1 expression without concentration dependence. Rg1 (10 and 40 μg/ml) diminished PM_2.5-induced HUVEC viability, decrease ROS and MDA generation, increased HO-1 and Nrf2 expression and promoted Nrf2 translocation to nucleus in a concentration dependent manner. These results suggested that organic extracts of PM_2.5 increase oxidative stress and decrease cell viability; Rg1 antagonize PM_2.5-induced excess oxidative stress; HO-1 expression increase and Nrf2 translocation to nucleus may be involved in the effects of both PM_2.5 and Rg1 on HUVECs.     

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.     

Di‐(2‐ethylhexyl) phthalate (DEHP)‐induced testicular toxicity through Nrf2‐mediated Notch1 signaling pathway in Sprague–Dawley rats

Di‐(2‐ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor widely used in China that is harmful to the male reproductive system. Many studies have shown that DEHP causes testicular toxicity through oxidative stress, but the specific mechanism is unknown. Because the Notch pathway is a key mechanism for regulating cell growth and proliferation, we investigated whether Notch is involved in DEHP‐induced testicular toxicity and whether vitamins E and C could rescue testicular impairment in Sprague–Dawley (SD) rats. Compared with the control group, we found that DEHP exposure induced testicular toxicity through oxidative stress injury, and it decreased the testosterone level (P < .01) and upregulated nuclear factor‐erythroid 2 related factor (Nrf2) expression (P < .01). Therefore, because oxidative stress might be the initiating factor of DEHP‐induced testicular toxicity, treatment with the antioxidant vitamins E and C activated the Notch1 signaling pathway in the testis and in Leydig cells. Treatment with vitamins E and C normalized the oxidative stress state after DEHP exposure and restored testicular development to be similar to the control group. In summary, antioxidant vitamins E and C may be used to treat DEHP‐induced testicular toxicity.