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Potent In Vitro Protection Against PM2.5-Caused ROS Generation and Vascular Permeability by Long-Term Pretreatment with Ganoderma tsugae
Abstract
Epidemiological studies show increased particulate matter (PM2.5) particles in ambient air are correlated with increased myocardial infarctions. Given the close association of capillaries and alveoli, the dysfunction is caused when inhaled PM2.5 particles come in close proximity to capillary endothelial cells. We previously suggested that the inhalation of PM2.5 diesel exhaust particles (DEP) induces oxidative stress and upregulates the Nrf2/HO-1 pathway, inducing vascular permeability factor VEGFA secretion, which results in cell-cell adherens junction disruption and PM2.5 transmigratation into circulation. Here, we minimized the level that PM2.5 traveled in the bloodstream by pre-supplementing with a traditional Chinese medicine (TCM) Ganoderma tsugae DMSO extract (GTDE) prior to PM2.5 exposure. Our results show that PM2.5 caused alterations in enzyme activities and cellular anti-oxidant balance. We found decreased glutathione levels, a reduced cellular redox ratio, increased ROS generation and cytotoxicity in the cellular fractions. The oxidative stress caused DNA damage and apoptosis, likely causing downstream molecular events that trigger vasculature permeabilization and, eventually, cardiovascular disorders. Our results show long-term GTDE treatment increased endogenous glutathione level, while PM2.5-reduced glutathione levels and the cellular redox ratio. GTDE was protective against the genotoxic and apoptotic effects initiated by PM2.5 oxidative stress. Vascular permeability revealed that PM2.5 only accumulated on the surface of cells after GTDE treatment; no penetration was detected. After two weeks of GTDE treatment, VEGFA secretion was significantly reduced in human umbilical vein endothelial cells (HUVEC) and endothelial cell migration was blocked. Our results suggest GTDE prevents PM2.5 transmigration into the bloodstream, and the resultant dysfunction, by inhibiting oxidative stress production and endothelial permeability.
... Dr. Hseu provided and authorized us to use the water extract for this study. The crude mixture contained 1.96% triterpenes and 3.93% polysaccharides [75]. The dried extract was further analyzed for the contents of total water-soluble polysaccharides, total triterpenoids, beta-Dglucans, and heavy metals. ...
Lingzhi has long been regarded as having life-prolonging effects. Research in recent years has also reported that Lingzhi possesses anti-tumor, anti-inflammatory, immunomodulatory, hepatoprotective, and anti-lipogenic effects. The D-galactose (D-gal, 100 mg/kg/day)-induced aging Long-Evans rats were simultaneously orally administered a DMSO extract of Ganoderma tsugae (GTDE, 200 μg/kg/day) for 25 weeks to investigate the effects of GTDE on oxidative stress and memory deficits in the D-galactose-induced aging rats. We found that GTDE significantly improved the locomotion and spatial memory and learning in the aging rats. GTDE alleviated the aging-induced reduction of dendritic branching in neurons of the hippocampus and cerebral cortex. Immunoblotting revealed a significant increase in the protein expression levels of the superoxide dismutase-1 (SOD-1) and catalase, and the brain-derived neurotrophic factor (BDNF) in rats that received GTDE. D-gal-induced increase in the lipid peroxidation product 4-hydroxynonenal (4-HNE) was significantly attenuated after the administration of GTDE, and pyrin domain-containing 3 protein (NLRP3) revealed a significant decrease in NLRP3 expression after GTDE administration. Lastly, GTDE significantly reduced the advanced glycosylation end products (AGEs). In conclusion, GTDE increases antioxidant capacity and BDNF expression of the brain, protects the dendritic structure of neurons, and reduces aging-induced neuronal damage, thereby attenuating cognitive impairment caused by aging.
... Finally, they identified a major active ingredient in GTE, namely F5-2, which a peptidoglycan-like compound [72]. In addition, it has been reported that long-term GTE therapy upregulates GSH, SOD-1 and HO-1; decreases vascular permeability; and alleviates the genotoxicity and apoptosis caused by PM 2.5 in endothelial cells [73]. Korean red ginseng (KRG) is a food and drug with various pharmacological activities. ...
Introduction
Recently, Nrf2/HO-1 has received extensive attention as the main regulatory pathway of intracellular defense against oxidative stress and is considered an ideal target for alleviating endothelial cell (EC) injury
Objectives
This paper aimed to summarized the natural monomers/extracts that potentially exert protective effects against oxidative stress in ECs
Methods
A literature search was carried out regarding our topic with the keywords of “atherosclerosis” or “Nrf2/HO-1” or “vascular endothelial cells” or “oxidative stress” or “Herbal medicine” or “natural products” or “natural extracts” or “natural compounds” or “traditional Chinese medicines” based on classic books of herbal medicine and scientific databases including Pubmed, SciFinder, Scopus, the Web of Science, GoogleScholar, BaiduScholar, and others. Then, we analyzed the possible molecular mechanisms for different types of natural compounds in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress. In addition, perspectives for possible future studies are discussed.
Results
These agents with protective effects against oxidative stress in ECs mainly include phenylpropanoids, flavonoids, terpenoids, and alkaloids. Most of these agents alleviate cell apoptosis in ECs due to oxidative stress, and the mechanisms are related to Nrf2/HO-1 signaling activation. However, despite continued progress in research on various aspects of natural agents exerting protective effects against EC injury by activating Nrf2/HO-1 signaling, the development of new drugs for the treatment of atherosclerosis (AS) and other CVDs based on these agents will require more detailed preclinical and clinical studies.
Conclusion
Our present paper provides updated information of natural agents with protective activities on ECs against oxidative stress by activating Nrf2/HO-1. We hope this review will provide some directions for the further development of novel candidate drugs from natural agents for the treatment of AS and other CVDs.
... Macrophages were reported to promote angiogenesis, vascular permeability, and inflammatory cell recruitment via the activation of VEGFA, inducing human atherosclerotic plaque progression and rupture [38]. Furthermore, the oxidative stress induced vascular permeability factor VEGFA secretion, which was related to increased myocardial infarctions [39]. In our study, we observed that VEGFA was upregulated in MI patients, which was also supported by the above studies. ...
Background:
Myocardial infarction (MI) is the most terrible appearance of cardiovascular disease. The incidence of heart failure, one of the complications of MI, has increased in the past few decades. Therefore, the identification of MI from angina patients and the determination of new diagnoses and therapies of MI are increasingly important. The present study was aimed at identifying differentially expressed genes and miRNAs as biomarkers for the clinical and prognosis factors of MI compared with angina using microarray data analysis.
Methods:
Differentially expressed miRNAs and genes were manifested by GEO2R. The biological function of differentially expressed genes (DEGs) was examined by GO and KEGG. The construction of a protein-protein network was explored by STRING. cytoHubba was utilized to screen hub genes. Analysis of miRNA-gene pairs was executed by the miRWalk 3.0 database. The miRNA-target pairs overlapped with hub genes were seen as key genes. Logistic regressive analysis was performed by SPSS.
Results:
A number of 779 DEGs were recorded. The biological function containing extracellular components, signaling pathways, and cell adhesion was enriched. Twenty-four hub genes and three differentially expressed miRNAs were noted. Eight key genes were demonstrated, and 6 out of these 8 key genes were significantly related to clinical and prognosis factors following MI.
Conclusions:
CALCA, CDK6, MDM2, NRXN1, SOCS3, VEGFA, SMAD4, NCAM1, and hsa-miR-127-5p were thought to be potential diagnosis biomarkers for MI. Meanwhile, CALCA, CDK6, NRXN1, SMAD4, SOCS3, and NCAM1 were further identified to be potential diagnosis and therapy targets for MI.
... Many studies have confirmed that PM2.5 could increase ROS production. ROS increased in peripheral blood supernatants, lung and heart tissues in mice exposed to PM2.5 [6][7][8][9]. Excessive ROS could cause a series of damage to airway epithelial cells and alveolar cells, including structural damage, activation of inflammatory-related transcription factors, and the release of inflammatory mediators [10][11][12]. Frossi et al. found that oxidative stress increased Th2-mediated inflammatory response, the severity of asthma, the bronchial hyperresponsiveness, and promoted airway remodeling [13]. ...
Bronchial asthma is the most common chronic respiratory disease. Chronic airway inflammation, airflow restriction and airway hyper-responsiveness are its main manifestations. In recent decades, the prevalence and mortality of asthma have been increasing all over the world, which seriously threatens public health. Research suggests that air pollution is associated with the increased incidence of asthma. PM2.5 is one of the most complex pollutants in the atmospheric environment and harmful to human health. It is related to the incidence of asthma. However, the molecular mechanism of PM2.5 in the development of asthma is still unclear. In this study, we established a mouse model of asthma using CRE to observe the effect of PM2.5 on the symptoms of asthmatic mice and its possible molecular mechanism. The results showed that PM2.5 could significantly increase airway resistance and pulmonary inflammation, increase the number of inflammatory cells, eosinophils, macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid in asthmatic mice. Moreover, PM2.5 could reduce the contents of antioxidant enzymes such as CAT, GSH, GSH-Px and T-SOD in lung tissue of mice, and increase the ROS level. PM2.5 can promote the expression of microRNA-206 in lung tissue of mice. miR-206 can target the 3'-UTR of SOD1 to inhibit SOD1 expression, which leads to the increase of ROS level and aggravates pulmonary inflammatory response and asthma symptoms in asthmatic mice. This study found the possible molecular mechanism of PM2.5 aggravating asthma, and miR-206 may be a potential target for asthma treatment.
... A second physiological property of Ganoderma is the protection of cardiac capillary permeability against penetration of small particles that can damage underlying cardiac muscle and similar protection of the gut lining against increased permeability [127,128]. Urothelial permeability can be easily evaluated in our in vivo and in vitro bladder models using a variety of methodologies [129][130][131]. Thus, the rabbit urinary bladder is an excellent model to study the beneficial effects of GL on the response of organ system to oxidative stress. ...
Oxidative stress plays an important role in specific disease pathophysiology and the aging process. In the history of human kind, many herbs were utilized for disease prevention and anti-aging treatment. However, there are few direct evidences provided by modern laboratory technology. The current study was designed to evaluate Ganoderma Lucidum's (GL) ability to reduce the damage from in vivo ischemia/reperfusion (I/R) using a rabbit model of I/R that has been effectively utilized to prove the effects of drugs and supplements to reduce oxidative stress. Urinary bladder dysfunction secondary to benign prostatic hyperplasia (BPH) is a major affliction of aging men. One of the major etiologies of obstructive bladder dysfunction (OBD) is oxidative stress induced by I/R. Pharmaceutical studies and clinical research have proven that GL is useful in helping to prevent certain types of pathology and also helpful in prolonging human life in part by acting as an antioxidant. Using an in vivo model of I/R, we have investigated the ability of GL to protect bladder function from oxidative damage mediated by I/R. Our studies demonstrated that ischemia followed by reperfusion resulted in a significant decrease in bladder compliance and decreases in the contractile responses to a variety of forms of contractile stimulation. Pretreatment of rabbits with Ganoderma Lucidum prior to subjecting the rabbits to I/R completely inhibited the negative effects of I/R on both the compliance and contractile responses. These results demonstrate that Ganoderma provides excellent protection of bladder function following I/R (oxidative stress).
Particulate matter (PM) has long been appreciated as a biologically significant component of air pollution that has major impacts on public health. Exposure to PM less than 2.5 µm in diameter (PM2.5) is associated with mortality, and increased incidence of lung diseases. While the effect that PM2.5 exposure has on many cell types, such as epithelial cells, is relatively well studied, the effect it has on mesenchymal cells, such as fibroblasts, is less understood. This study seeks to determine the effect that PM2.5 exposure has on fibroblast-to-myofibroblast differentiation, which is a critical step in pulmonary fibrosis, a disease that has been shown to be exacerbated by PM2.5 exposure. We assessed the effect that PM2.5 exposure has on fibroblast biology and myofibroblast differentiation through four research aims. In Aim 1, we delineated the conditions and determinants by which ambient PM2.5 affected fibroblast-to-myofibroblast differentiation and found that, interestingly, repeated low concentration PM2.5 exposures promoted increases in αSMA and collagen, markers of myofibroblast differentiation. Furthermore, follow-up studies utilizing pharmacological inhibitors showed that NF-κB is critical for this effect. In Aim 2, we sought to investigate the effect that PM2.5 exposure has on bone morphogenic protein (BMP)2 expression and secretion in fibroblasts. BMP2 is a cytokine that can promote or inhibit myofibroblast differentiation and can be activated by NF-κB. We found that PM2.5 exposure promoted a dose-dependent increase in BMP2 transcript and secreted protein. The role of BMP2 on PM2.5-induced increase in myofibroblast differentiation was less clear; however, as treatment with exogenous BMP2 promoted myofibroblast differentiation, while blocking endogenous BMP2 with an siRNA or inhibitory protein (noggin), also promoted myofibroblast differentiation. This implies that a certain level of BMP2 signaling is critical for fibroblasts to maintain homeostasis, but that too much signaling has deleterious effects as well. In Aim 3, we examine the effect that PM2.5 exposure has on cytokine secretion and DNA methylation in lung fibroblasts. We analyze fibroblasts exposed to PM2.5 on a DNA methylation array, as well as supernatants collected from similarly exposed fibroblasts on a Luminex cytokine kit. We found that low concentrations of PM2.5 promoted methylation changes in genes enriched in cell cycle whereas high concentrations promoted methylation changes in a completely different set of genes, including those enriched in adhesion, neuronogeneis, and cell signaling. Additionally, IL-12 was found to be significantly upregulated following low concentration exposure to PM2.5 in our Luminex experiments. We sought to determine if PM2.5 exposure in vivo increases susceptibility to pulmonary fibrosis in Aim 4. The data in our prior Aims suggest that PM2.5 exposure promotes myofibroblast differentiation, an important process in fibrogenesis. Here, we exposed mice to PM2.5 and assess their susceptibility to bleomycin-induced fibrosis. Despite promising results in a pilot experiment, where we demonstrated a trend toward higher collagen content, as measured by hydroxyproline, in the lungs of male mice exposed to PM2.5 prior to bleomycin treatment, we were unable to replicate the results in a larger study with female mice. This implies that sex may have significant effects on susceptibility to PM2.5 exposure. PM2.5 exposure results in a myriad of changes in fibroblast biology, changes that can often promote fibrosis. The exact effects of PM2.5 on fibroblasts vary depending on the duration, frequency, and concentration of exposure, demonstrating the public health significance of even low levels of air pollution.
Long term particulate matter (PM) exposure has been associated with an increased incidence of respiratory diseases. Here, an in vitro model was developed to study how long term diesel exhaust particle (DEP) exposure might predispose to the development of allergic reactions. Airway epithelial (16HBE) cells were exposed to low concentrations of diesel exhaust particle (DEP) for 4 days after which they were challenged with house dust mite (HDM) extract (24 h). Compared to acute exposure (24 h), 4 days DEP exposure to 16HBE cells further reduced the transepithelial electrical resistance (TEER) and increased CXCL-8 release. DEP pre-exposure aggravated HDM-induced loss of TEER, increased tracer flux across the barrier and reduced CLDN-3 expression in these 16HBE cells. HDM-induced cytokine (IL-6, CCL-22, IL-10 and CXCL-8) release was significantly increased after DEP pre-exposure. In the current study an in vitro model with long term PM exposure was presented, which might be helpful for further understanding the interplay between long term PM exposure and allergic responses.
Objective
Numerous epidemiological studies have investigated the effects of short-term and long-term exposure to ambient air pollution on hypertension and blood pressure among children and adolescents. However, the results were controversial. To provide researchers reliable evidence, this meta-analysis was performed.
Methods
We searched all published studies in four databases examining the effects of particulate matter (PM10, PM2.5 and PM1.0), nitrogen oxide (NO2), sulfur dioxide (SO2), ozone (O3) and carbon monoxide (CO) on hypertension and blood pressure in children and adolescents. Overall risk estimates associated with per 10 μg/m³ increase of air pollution were analyzed by a random-effect model for articles with significant heterogeneity, otherwise, a fixed-effect model was applied. Subgroup analysis was conducted for studies with significant heterogeneity.
Results
Of 3918 identified literatures, 154 were evaluated in-depth with 15 satisfying inclusion criteria. Increased risk of hypertension was associated with long-term PM10 exposure (OR = 1.17, 95% confidence interval [CI]:1.13, 1.21). For systolic blood pressure (SBP), significant results were found for short-term PM10 (β = 0.26, 95% CI: −0.00, 0.53) exposure, long-term PM2.5 (β = 1.80, 95% CI: 0.94, 2.65) and PM10 (β = 0.50, 95% CI: 0.19, 0.81) exposure. The corresponding estimates of diastolic blood pressure (DBP) were 0.32 mmHg (95% CI: 0.19, 0.45) for short-term PM10 exposure, 1.06 mmHg (95% CI: 0.32, 1.80), 0.34 mmHg (95% CI: 0.11, 0.57) and 0.44 mmHg (95% CI: 0.25, 0.63) for long-term PM2.5, PM10 and NO2 exposure, respectively. Stratified analyses showed stronger effects of PM10 on blood pressure among studies with ≥50% boys' percentage (0.57 mmHg [95% CI: 0.44, 0.70] for SBP, 0.44 mmHg, [95% CI: 0.34, 0.54] for DBP, respectively) and articles using models to estimate exposure (0.90 mmHg [95% CI: 0.20 1.59] for SBP).
Conclusion
Ambient air pollution was associated with higher hypertension prevalence and elevated blood pressure in children and adolescents.
Although in-vivo exposure of PM2.5 has been suggested to initiate a disorder on vascular permeability, the effects and related mechanism has not been well defined. In this work, an obvious increase on vascular permeability has been confirmed in vivo by vein injection of PM2.5 into Balb/c mouse. Human umbilical vein vascular endothelial cells and the consisted ex-vivo vascular endothelium were used as model to investigate the effects of PM2.5 on the vascular permeability and the underlying molecular mechanism. Upon PM2.5 exposure, the vascular endothelial growth factor receptor 2 on cell membrane phosphorylates and activates the downstream mitogen-activated protein kinase (MAPK)/ERK signaling. The adherens junction protein VE-cadherin sheds and the intercellular junction opens, damaging the integrity of vascular endothelium via paracellular pathway. Besides, PM2.5 induces the intracellular reactive oxygen species (ROS) production and triggers the oxidative stress including activity decrease of superoxide dismutase, lactate dehydrogenase release and permeability increase of cell membrane. Taken together, the paracellular and transcellular permeability enhancement jointly contributes to the significant increase of endothelium permeability and thus vascular permeability upon PM2.5 exposure. This work provides an insight into molecular mechanism of PM2.5 associated cardiovascular disease and offered a real-time screening method for the health risk of PM2.5.
The genus Ganoderma (Ganodermataceae) has a long history in traditional medicine to improve longevity and health in Asia. Ganoderma has been widely used in multiple therapeutic activities as well as dietary supplements to prevent and treat many diseases. Several classes of bioactive substances have been isolated and identified from Ganoderma, such as polysaccharides, triterpenoids, nucleosides, sterols, fatty acids, protein and alkaloids. There are numerous research publications, which report the abundance and variety of biological actions initiated by the metabolites of Ganoderma. Investigation on different metabolic activities of Ganoderma species has been performed both in vitro and in vivo. In many cases, however, it has been questioned whether Ganoderma is solely a nourishment supplement for wellbeing or merely a helpful "medication" for restorative purposes. There has been no any conclusive report of human trials using Ganoderma species as a direct control agent for diseases. In addition, there is no evidence supporting the usage of Ganoderma species (excluding G. lucidum) as potential supplements for cancer or other diseases in humans since no preclinical trials have been performed up to date. In this review, the beneficial medicinal properties of several species of Ganoderma (excluding G. lucidum) and their secondary metabolites are discussed. Ganoderma species can be used as a therapeutic drug, but more direct scientific evidence should be made available in the future. The efficiency of Ganoderma in clinical treatments should be substantiated with more biomedical research and their true impact assessed on human health with more standardized clinical evaluations so that the feasibility of biologically active extracts of Ganoderma species in alternative treatments can be recommended.
Substantial epidemiological evidence has consistently reported that fine particulate matter (PM2.5) is associated with an increased risk of cardiovascular outcomes. PM2.5 is a complex mixture of extremely small particles and liquid droplets composed of multiple components, and there has been high interest in identifying the specific health-relevant physical and/or chemical toxic constituents of PM2.5. In the present study, we analyzed 8 heavy metals (Cr, Ni, Cu, Cd, Pb, Zn, Mn and Co) in the PM2.5 collected during four different seasons in Taiyuan, a typical coal-burning city in northern China. Our results indicated that total concentrations of the 8 heavy metals differed among the seasons. Zn and Pb, which are primarily derived from the anthropogenic source, coal burning, were the dominant elements, and high concentrations of these two elements were observed during the spring and winter. To clarify whether these heavy metals in the locally collected PM2.5 were associated with health effects, we conducted health risk assessments using validated methods. Interestingly, Pb was responsible for greater potential health risks to children. Because cardiovascular disease (CVD) is a main contributor to the mortality associated with PM2.5 exposure, we performed experimental assays to evaluate the myocardial toxicity. Our in vitro experiments showed that the heavy metal-containing PM2.5 induced season-dependent apoptosis in rat H9C2 cells through a reactive oxygen species (ROS)-mediated inflammatory response. Our findings suggested that heavy metals bound to PM2.5 produced by coal burning play an important role in myocardial toxicity and contribute to season-dependent health risks.
Exposure to diesel exhaust particles (DEP) is associated with pulmonary and cardiovascu-lar diseases. Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane. This allows DEPs to penetrate into the cell and capillary lumen. In addition, pro-inflammatory cytokines are up-regulated and mediate vascular permeability in response to DEP. However, the mechanisms through which these DEP-induced pro-inflammatory cyto-kines increase vascular permeability remain unknown. Hence, we examined the ability of DEP to induce permeability of human umbilical vein endothelial cell tube cells to investigate these mechanisms. Furthermore, supplementation with NAC reduces ROS production following exposure to DEP. HUVEC tube cells contributed to a pro-inflammatory response to DEP-induced intracellular ROS generation. Endothelial oxidative stress induced the release of TNF-α and IL-6 from tube cells, subsequently stimulating the secretion of VEGF-A independent of HO-1. Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF-α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability. Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.
Hebei is one of the most air polluted provinces in China. According to the Ministry of Environmental Protection (MEP) for the severe fog-haze month of Jan. 2013, seven of the top ten most polluted cities in China are located in Hebei Province. In this study, the air pollution history and status of the Hebei Province are reviewed and discussed, using the governmental published Air Pollution Index (API), the academic observations by various scientific research groups and the long-term statistics of visibility and haze frequencies. It is found that within the Hebei Province, the air pollution in the southern cities is much more severe than the northern cities. Particulate matter (PM) is undoubtedly the major air pollutant, sulfur dioxide (SO2) and nitrogen oxides (NOX) pollutions are also unnegligible. Ozone (O3) pollution in larger cities, such as Shijiazhuang, is significant. Air pollution control history from 1998 is discussed as well. Although Hebei Province has made a great effort on air quality, the pollutant emissions, such as SO2 and fly ash, showed a notable increase in 2001 to 2006. However, after 2006 the emissions started to decrease due to the strict implementation of the national 11th Five Year Plan (FYP). In addition, regional jointly air pollution control and prevention strategies are expected in the future to substantially change the severe air pollution status in Hebei Province.
This study was to investigate antiallergic effects of triterpenoids (Gt-TRE) and polysaccharide (Gt-PS) extracts from Ganoderma tsugae, using mast cell line RBL-2H3, T cell line EL4, primary T cells, and transfected RAW264.7 macrophage cells. The results showed that histamine secreted from activated RBL-2H3 mast cells was significantly suppressed by Gt-TRE but not Gt-PS. Interleukin- (IL-) 4 secreted from activated EL4 cells was significantly suppressed by Gt-TRE but not Gt-PS. Further primary CD4+ T cells cultures also confirmed that Gt-TRE (
5
~
50
µg/mL) significantly suppressed Th2 cytokines IL-4 and IL-5 secretions but had no effect on Th1 cytokines IL-2 and interferon (IFN)-γ. Gt-PS did not affect IL-4 and IL-5 secretions until higher doses (400, 500 µg/mL) and significantly suppressed IFNγ secretions but enhanced IL-2 at these high doses. The reporter gene assay indicated that Gt-TRE inhibited but Gt-PS enhanced the transcriptional activity of NF-κB in activated transfected RAW264.7 cells and transfected EL4 cells. IL-4 secreted by this transfected EL-4 cells was also significantly decreased by Gt-TRE but not by Gt-PS, suggesting that these two fractions may exert different effects on NF-κB related cytokines expression. These data suggested that triterpenoids fraction of Ganoderma tsugae might be the main constituents to alleviate allergic asthma.
We have previously shown that underground railway particulate matter (PM) is rich in iron and other transition metals across coarse (PM10-2.5), fine (PM2.5), and quasi-ultrafine (PM0.18) fractions, and is able to generate reactive oxygen species (ROS). However, there is little knowledge of whether the metal-rich nature of such particles exerts toxic effects in mucus-covered airway epithelial cell cultures, or whether there is an increased risk posed by the ultrafine fraction. Monolayer and mucociliary air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) were exposed to size-fractionated underground railway PM (1.1-11.1 μg/cm(2)), and release of lactate dehydrogenase and IL-8 was assayed. ROS generation was measured, and the mechanism of generation studied using desferrioxamine (DFX) and N-acetylcysteine (NAC). Expression of haem oxygenase-1 (HO-1) was determined by RT-qPCR. Particle uptake was studied by transmission electron microscopy. Underground PM increased IL-8 release from PBECs, but this was diminished in mucus-secreting ALI cultures. Fine and ultrafine PM generated a greater level of ROS than coarse PM. ROS generation by ultrafine PM was ameliorated by DFX and NAC, suggesting an iron-dependent mechanism. Despite the presence of mucus, ALI cultures displayed increased HO-1 expression. Intracellular PM was observed within vesicles, mitochondria, and free in the cytosol. The results indicate that, although the mucous layer appears to confer some protection against underground PM, ALI PBECs nonetheless detect PM and mount an antioxidant response. The combination of increased ROS-generating ability of the metal-rich ultrafine fraction and ability of PM to penetrate the mucous layer merits further research.
© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.
Previous studies suggest a direct correlation between exposure to diesel exhaust particles (DEP) and the onset of vascular permeability, presumably through the disruption of the adherens junctions. This would lead to deleterious effects on vasculature, such as acute myocardial infarction and atherosclerosis. Although the mechanism remains unclear, we demonstrate DEP-induced mitochondrial reactive oxygen species generation, which may be a central cause of the above vascular disorders. In vitro capillary-like HUVEC tube cells are used in this study and show that acute DEP exposure stimulates ATP depletion, followed by depolarization of their actin cytoskeleton, which sequentially inhibits PI3K/Akt activity and induces endothelial apoptosis. These events are accompanied by induction of p53/Mdm2 feedback regulation at 10 µg/mL DEP and produce 20 % cell apoptosis. Nevertheless, 100 µg/mL DEP augments tube cell apoptosis up to 70 % but disrupts the p53 negative regulator Mdm2. Addition of N-acetylcysteine provides substantial protection against the cytotoxic effects of DEP. In summary, exposure to a low dose of DEP actin triggers cytoskeleton depolarization, reduces PI3K/Akt activity, and induces a p53/Mdm2 feedback loop, and a high dose causes apoptosis by depleting Mdm2.
A field study was carried out from 2003 to 2004 with the aim to develop the PM 2.5 emission source profiles from light-duty gasoline and heavy-duty diesel vehicles, as well as emission source profiles from waste incineration, wood burning, LP gas combustion, and meat broiling. Over 25 chemical species were quantified from the fine particles emitted by the different combustion sources investigated, including organic and elemental carbon, ions, and elements. The OC/TC ratio found in the different PM 2.5 profiles was dissimilar as well as the sulfate, nitrate, ammonium, soil species, and trace element content. Consequently, these combustion emission profiles could be used in source reconciliation studies for fine particles.
A major constituent of urban air pollution is diesel exhaust, a complex mixture of gases, chemicals, and particles. Recent evidence suggests that exposure to air pollution can increase the risk of a fatal stroke, cause cerebrovascular damage, and induce neuroinflammation and oxidative stress that may trigger neurodegenerative diseases, such as Parkinson's disease. The specific aim of this study was to determine whether ultrafine diesel exhaust particles (DEPs), the particle component of exhaust from diesel engines, can induce oxidative stress and effect dopamine metabolism in PC-12 cells. After 24h exposure to DEPs of 200nm or smaller, cell viability, ROS and nitric oxide (NO2) generation, and levels of dopamine (DA) and its metabolites, (dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)), were evaluated. Results indicated cell viability was not significantly changed by DEP exposure. However, ROS showed dramatic dose-dependent changes after DEP exposure (2.4 fold increase compared to control at 200μg/mL). NO2 levels were also dose-dependently increased after DEP exposure. Although not in a dose-dependent manner, upon DEP exposure, intracellular DA levels were increased while DOPAC and HVA levels decreased when compared to control. Results suggest that ultrafine DEPs lead to dopamine accumulation in the cytoplasm of PC-12 cells, possibly contributing to ROS formation. Further studies are warranted to elucidate this mechanism.
Clinical studies have now confirmed the link between short-term exposure to elevated levels of air pollution and increased cardiovascular mortality, but the mechanisms are complex and not completely elucidated. The present study was designed to investigate the hypothesis that activation of pulmonary sensory receptors and the sympathetic nervous system underlies the influence of pulmonary exposure to diesel exhaust particulate on blood pressure, and on the myocardial response to ischemia and reperfusion.Methods & Results: 6 h after intratracheal instillation of diesel exhaust particulate (0.5 mg), myocardial ischemia and reperfusion was performed in anesthetised rats. Blood pressure, duration of ventricular arrhythmia, arrhythmia-associated death, tissue edema and reperfusion injury were all increased by diesel exhaust particulate exposure. Reperfusion injury was also increased in buffer perfused hearts isolated from rats instilled in vivo, excluding an effect dependent on continuous neurohumoral activation or systemic inflammatory mediators. Myocardial oxidant radical production, tissue apoptosis and necrosis were increased prior to ischemia, in the absence of recruited inflammatory cells. Intratracheal application of an antagonist of the vanilloid receptor TRPV1 (AMG 9810, 30 mg/kg) prevented enhancement of systolic blood pressure and arrhythmia in vivo, as well as basal and reperfusion-induced myocardial injury ex vivo. Systemic beta1 adrenoreceptor antagonism with metoprolol (10 mg/kg) also blocked enhancement of myocardial oxidative stress and reperfusion injury.
Pulmonary diesel exhaust particulate increases blood pressure and has a profound adverse effect on the myocardium, resulting in tissue damage, but also increases vulnerability to ischemia-associated arrhythmia and reperfusion injury. These effects are mediated through activation of pulmonary TRPV1, the sympathetic nervous system and locally generated oxidative stress.
To evaluate the efficacy and safety of Shensong Yangxin (SSYX) in patients with bradycardia arrhythmias, a randomized, double-blind, and placebo-controlled study was conducted. Patients with bradycardia were randomly assigned to receive either SSYX (trial group, n = 115) or placebo (control group, n = 104) for 4 weeks. ECG, 24-hour continuous ECG recording, echocardiography, and hepatic and renal function were evaluated at baseline and after treatment. Results showed that the average heart rate, the fastest heart rate, and the lowest heart rate in the trial group were all significantly higher than those in the control group at the end of treatment (P < 0.05 or 0.01, resp.). Compared with pretreatment, the average heart rate, the fastest heart rate, and the lowest heart rate in the trial group all increased significantly after treatment (P < 0.05 or 0.01, resp.). Both the efficacy and the symptom scores in the trial group were significantly better than those in the control group after treatment (both having P < 0.01). No severe adverse effects were reported. In conclusion, SSYX treatment significantly increased the heart rate in patients with bradycardia without severe side effects. The exact mechanisms remain to be further explored.
The endothelium forms a selective semi-permeable barrier controlling bidirectional transfer between blood vessel and irrigated tissues. This crucial function relies on the dynamic architecture of endothelial cell-cell junctions, and in particular, VE-cadherin-mediated contacts. VE-cadherin indeed chiefly organizes the opening and closing of the endothelial barrier, and is central in permeability changes. In this review, the way VE-cadherin-based contacts are formed and maintained is first presented, including molecular traits of its expression, partners, and signaling. In a second part, the mechanisms by which VE-cadherin adhesion can be disrupted, leading to cell-cell junction weakening and endothelial permeability increase, are described. Overall, the molecular basis for VE-cadherin control of the endothelial barrier function is of high interest for biomedical research, as vascular leakage is observed in many pathological conditions and human diseases.
This study explores and characterizes cell cycle alterations induced by urban PM2.5 in the human epithelial cell line BEAS-2B, and elucidates possible mechanisms involved.
The cells were exposed to a low dose (7.5 mug/cm2) of Milan winter PM2.5 for different time points, and the cell cycle progression was analyzed by fluorescent microscopy and flow cytometry. Activation of proteins involved in cell cycle control was investigated by Western blotting and DNA damage by 32P-postlabelling, immunostaining and comet assay. The formation of reactive oxygen species (ROS) was quantified by flow cytometry. The role of PM organic fraction versus washed PM on the cell cycle alterations was also examined. Finally, the molecular pathways activated were further examined using specific inhibitors.
Winter PM2.5 induced marked cell cycle alteration already after 3 h of exposure, represented by an increased number of cells (transient arrest) in G2. This effect was associated with an increased phosphorylation of Chk2, while no changes in p53 phosphorylation were observed at this time point. The increase in G2 was followed by a transient arrest in the metaphase/anaphase transition point (10 h), which was associated with the presence of severe mitotic spindle aberrations. The metaphase/anaphase delay was apparently followed by mitotic slippage at 24 h, resulting in an increased number of tetraploid G1 cells and cells with micronuclei (MN), and by apoptosis at 40 h. Winter PM2.5 increased the level of ROS at 2 h and DNA damage (8-oxodG, single- and double stand breaks) was detected after 3 h of exposure. The PM organic fraction caused a similar G2/M arrest and augmented ROS formation, while washed PM had no such effects. DNA adducts were detected after 24 h. Both PM-induced DNA damage and G2 arrest were inhibited by the addition of antioxidants and alpha-naphthoflavone, suggesting the involvement of ROS and reactive electrophilic metabolites formed via a P450-dependent reaction.
Milan winter PM2.5 rapidly induces severe cell cycle alterations, resulting in increased frequency of cells with double nuclei and MN. This effect is related to the metabolic activation of PM2.5 organic chemicals, which cause damages to DNA and spindle apparatus.
Reactive oxygen or nitrogen species (ROS, RNS) and oxidative stress in the respiratory system increase the production of mediators of pulmonary inflammation and initiate or promote mechanisms of carcinogenesis. The lungs are exposed daily to oxidants generated either endogenously or exogenously (air pollutants, cigarette smoke, etc.). Cells in aerobic organisms are protected against oxidative damage by enzymatic and non-enzymatic antioxidant systems. Recent epidemiologic investigations have shown associations between increased incidence of respiratory diseases and lung cancer from exposure to low levels of various forms of respirable fibers and particulate matter (PM), at occupational or urban air polluting environments. Lung cancer increases substantially for tobacco smokers due to the synergistic effects in the generation of ROS, leading to oxidative stress and inflammation with high DNA damage potential. Physical and chemical characteristics of particles (size, transition metal content, speciation, stable free radicals, etc.) play an important role in oxidative stress. In turn, oxidative stress initiates the synthesis of mediators of pulmonary inflammation in lung epithelial cells and initiation of carcinogenic mechanisms. Inhalable quartz, metal powders, mineral asbestos fibers, ozone, soot from gasoline and diesel engines, tobacco smoke and PM from ambient air pollution (PM10 and PM2.5) are involved in various oxidative stress mechanisms. Pulmonary cancer initiation and promotion has been linked to a series of biochemical pathways of oxidative stress, DNA oxidative damage, macrophage stimulation, telomere shortening, modulation of gene expression and activation of transcription factors with important role in carcinogenesis. In this review we are presenting the role of ROS and oxidative stress in the production of mediators of pulmonary inflammation and mechanisms of carcinogenesis.
Epidemiological studies have shown that air pollutants, such as diesel exhaust particle (DEP), are implicated in the increased incidence of allergic airway disorders. In vitro studies of molecular mechanisms have focused on the role of reactive oxygen species generated directly and indirectly by the exposure to DEP. Antioxidants effectively reduce the allergic inflammatory effects induced by DEP both in vitro and in vivo. On the other hand, Nrf2 is a transcription factor essential for the inducible and/or constitutive expression of phase II and antioxidant enzymes. Disruption of Nrf2 enhances susceptibility to airway inflammatory responses and exacerbation of allergic inflammation induced by DEP in mice. Host responses to DEP are regulated by a balance between antioxidants and proinflammatory responses. Nrf2 may be an important protective factor against oxidative stresses induced by DEP in airway inflammation and allergic asthma and is expected to contribute to chemoprevention against DEP health effects in susceptible individuals.
Ganoderma, also known as Lingzhi or Reishi, has been used for medicinal purposes in Asian countries for centuries. It is a medicinal fungus with a variety of biological properties including immunomodulatory and antitumor activities. In this study, we investigated the molecular mechanisms by which Ganoderma tsugae (GT), one of the most common species of Ganoderma, inhibits the proliferation of HER2-overexpressing cancer cells. Here, we show that a quality assured extract of GT (GTE) inhibited the growth of HER2-overexpressing cancer cells in vitro and in vivo and enhanced the growth-inhibitory effect of antitumor drugs (e.g., taxol and cisplatin) in these cells. We also demonstrate that GTE induced cell cycle arrest by interfering with the HER2/PI3K/Akt signaling pathway. Furthermore, GTE curtailed the expression of the HER2 protein by modulating the transcriptional activity of the HER2 gene and the stability/degradation of the HER2 protein. In conclusion, this study suggests that GTE may be a useful adjuvant therapeutic agent in the treatment of cancer cells that highly express HER2.
With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.
The first suggestion that physical exercise results in free radical-mediated damage to tissues appeared in 1978, and the past three decades have resulted in a large growth of knowledge regarding exercise and oxidative stress. Although the sources of oxidant production during exercise continue to be debated, it is now well established that both resting and contracting skeletal muscles produce reactive oxygen species and reactive nitrogen species. Importantly, intense and prolonged exercise can result in oxidative damage to both proteins and lipids in the contracting myocytes. Furthermore, oxidants can modulate a number of cell signaling pathways and regulate the expression of multiple genes in eukaryotic cells. This oxidant-mediated change in gene expression involves changes at transcriptional, mRNA stability, and signal transduction levels. Furthermore, numerous products associated with oxidant-modulated genes have been identified and include antioxidant enzymes, stress proteins, DNA repair proteins, and mitochondrial electron transport proteins. Interestingly, low and physiological levels of reactive oxygen species are required for normal force production in skeletal muscle, but high levels of reactive oxygen species promote contractile dysfunction resulting in muscle weakness and fatigue. Ongoing research continues to probe the mechanisms by which oxidants influence skeletal muscle contractile properties and to explore interventions capable of protecting muscle from oxidant-mediated dysfunction.
Dimethylsulfoxide (DMSO) is a widely used solvent for the extraction of chlorophylls (Chls) from leaves of higher plants. The method is preferred because the time-consuming steps of grinding and centrifuging are not required and the extracts are stable for a long time period. However, the extraction efficiency of this solvent is not comparable among plant species, whereas the particular leaf anatomical characteristics responsible for this unevenness remain unknown. In order to examine the influence of leaf anatomy on the extraction efficiency of DMSO (i.e. the concentration of Chls extracted with DMSO as % of the concentration of Chls extracted with 80% acetone), leaves of 19 plant species with different anatomical characteristics were incubated for 40min in DMSO at 65 degrees C. Under these conditions, heterobaric leaves, which are characterized by the occurrence of bundle sheath extensions in the mesophyll, showed lower extraction efficiency of DMSO compared to homobaric leaves and conifer needles. Microscopical observations of DMSO incubated leaf tissues showed that bundle sheath extensions behave as anatomical barriers which prevent the diffusion of DMSO within heterobaric leaves, even after prolonged incubation with the solvent. The effect was stronger in heterobaric leaves possessing thick bundle sheath extensions. The extraction efficiency of DMSO in these leaves was improved by vacuum infiltration of the samples in the presence of warm (65 degrees C) solvent.
The determination of thiobarbituric acid reactants (TBARs) is a widely used method for investigating overall lipid peroxidation. An assay kit that could be used with plasma and lipid fractions would facilitate standardization of the method. The results reported here indicate that the malondialdehyde (MDA) kit manufactured by Sobioda (Grenoble, France) complies with criteria of good analytical practices. The detection limit was 0.11 mumol of MDA per liter of plasma. The within-run (CV = 1.8-3.3%) and between-run (CV = 3.3-4.4%) precisions were acceptable. The analytical recovery of MDA after supplementing human plasma samples with tetraethoxypropane standards varied from 88% to 100%. The mean (SD) lipoperoxide concentration determined in 32 healthy adults, ages 20-40 years, was 2.51 (0.25) mumol/L. No significant sex-related difference was noted: 2.57 (0.28) in men vs 2.44 (0.20) mumol/L in women. Applying the method to lipid fractions showed that lipoprotein fractions contain relatively little MDA: 0.07 (0.03) mumol/L of plasma for very-low-density lipoproteins and 0.19 (0.10) mumol/L for low-density lipoproteins.
We have shown that carbon monoxide (CO) generated by heme oxygenase-1 (HO-1) protects endothelial cells (EC) from tumor necrosis
α (TNF-α)-mediated apoptosis. This effect relies on the activation of p38 MAPK. We now demonstrate that HO-1/CO requires the
activation of the transcription factor NF-κB to exert this anti-apoptotic effect. Our data suggest that EC have basal levels
of NF-κB activity that sustain the expression of NF-κB-dependent anti-apoptotic genes required to support the anti-apoptotic
effect of HO-1/CO. Over-expression of the inhibitor of NF-κB α (IκBα) suppresses the anti-apoptotic action of HO-1/CO. Reconstitution
of NF-κB activity, by co-expression of IκBα with different members of the NF-κB family, i.e.p65/RelA or p65/RelA plus c-Rel, restores the anti-apoptotic effect of HO-1/CO. Expression of the NF-κB family members p65/RelA
or p65/RelA with p50 or c-Rel up-regulates the expression of the anti-apoptotic genes A1, A20, c-IAP2, and manganese superoxide dismutase (MnSOD). Inhibition of NF-κB activity by over-expression of IκBα suppresses the expression of some of these anti-apoptotic genes,
i.e.c-IAP2. Under inhibition of NF-κB, co-expression of some of these anti-apoptotic genes, i.e. c-IAP2 andA1, restores the anti-apoptotic action of HO-1/CO, whereas expression of A20 or MnSOD cannot. The ability of c-IAP2 and/or A1 to restore the anti-apoptotic action of HO-1/CO is abolished when p38 MAPK activation
is blocked by over-expression of a p38 MAPK dominant negative mutant. In conclusion, we demonstrate that HO-1/CO cooperates
with NF-κB-dependent anti-apoptotic genes, i.e. c-IAP2 and A1, to protect EC from TNF-α-mediated apoptosis. This effect is dependent on the ability of HO-1/CO to activate the p38 MAPK
signal transduction pathway.
Particulate air pollution is associated with cardiovascular morbidity and mortality. To investigate this association, we studied the effect of ultrafine (60 nm) polystyrene particles on thrombus formation in a hamster model after intravenous and intratracheal administration of unmodified, carboxylate-polystyrene, or amine-polystyrene particles. Unmodified particles had no effect on thrombosis up to 5 mg/kg. Carboxylate-polystyrene particles significantly inhibited thrombus formation at 500 and 100 microg/kg body weight but not at 50 microg/kg body weight. In contrast, amine-polystyrene particles significantly enhanced thrombosis at 500 and 50 microg/kg body weight but not at 5 microg/kg body weight. Similarly, the intratracheal instillation of 5,000 microg of amine-polystyrene particles significantly increased thrombus formation. The unmodified particles and carboxylate-polystyrene particles had no effect. During platelet aggregation in human platelet-rich plasma, induced with 1.25 microM ADP, unmodified particles had no effect up to 100 microg/ml, and carboxylate-polystyrene particles weakly enhanced platelet aggregation at 25 to 100 microg/ml. However, amine-polystyrene particles (50 and 100 microg/ml) induced platelet aggregation themselves and strongly increased the ADP-induced aggregation. We conclude that the presence of (ultrafine) particles in the circulation may affect hemostasis. The observed in vivo prothrombotic tendency results, at least in part, from platelet activation by positively charged amine-polystyrene particles.
The endothelium forms a selective semi-permeable barrier controlling bidirectional transfer between blood vessel and irrigated tissues. This crucial function relies on the dynamic architecture of endothelial cell-cell junctions, and in particular, VE-cadherin-mediated contacts. VE-cadherin indeed chiefly organizes the opening and closing of the endothelial barrier, and is central in permeability changes. In this review, the way VE-cadherin-based contacts are formed and maintained is first presented, including molecular traits of its expression, partners, and signaling. In a second part, the mechanisms by which VE-cadherin adhesion can be disrupted, leading to cell-cell junction weakening and endothelial permeability increase, are described. Overall, the molecular basis for VE-cadherin control of the endothelial barrier function is of high interest for biomedical research, as vascular leakage is observed in many pathological conditions and human diseases.
We compare the relative toxicity of various organic aerosol (OA) components identified by an Aerosol Mass Spectrometer (AMS) based on their ability to generate reactive oxygen species (ROS). Ambient fine aerosols were collected from urban (three in Atlanta, GA and one in Birmingham, AL) and rural (Yorkville, GA and Centerville, AL) sites in the Southeastern United States. The ROS generating capability of the water-soluble fraction of the particles was measured by the dithiothreitol (DTT) assay. Water-soluble PM extracts were further separated into the hydrophobic and hydrophilic fractions using a C-18 column, and both fractions were analyzed for DTT activity and water-soluble metals. Organic aerosol composition was measured at selected sites using a High-Resolution Time-of-Flight AMS. Positive matrix factorization of the AMS spectra resolved the organic aerosol into isoprene-derived OA (Isop_OA), hydrocarbon-like OA (HOA), less-oxidized oxygenated OA, (LO-OOA), more-oxidized OOA (MO-OOA), cooking OA (COA), and biomass burning OA (BBOA). The association of the DTT activity of water-soluble PM2.5 (WS_DTT) with these factors was investigated by linear regression techniques. BBOA and MO-OOA were most consistently linked with WS_DTT, with intrinsic water-soluble activities of 151±20 and 36±22 pmol/min/μg, respectively. Although less toxic, MO-OOA was most widespread, contributing to WS_DTT activity at all sites and during all seasons. WS_DTT activity was least associated with biogenic secondary organic aerosol. The OA components contributing to WS_DTT were humic-like substances (HULIS), which are abundantly emitted in biomass burning (BBOA) and include highly oxidized OA from multiple sources (MO-OOA). Overall, OA contributed approximately 60% to the WS_DTT activity, with the remaining probably from water-soluble metals, which were mostly associated with the hydrophilic WS_DTT fraction.
Ambient fine particulate matter (PM2.5) exposure increases the respiratory disease risk. Although previous mitochondrial researches provided new information about the PM toxicity on the lung, the exact mechanism of PM2.5 on structural and functional damage of lung mitochondria remains unclear so far. In this study the changes of lung mitochondrial morphology and expression of mitochondrial fission/fusion markers, lipid peroxidation and transport ATPase in SD rats exposed to ambient PM2.5 with different dosages were investigated. Also, the reactive oxygen species (ROS) release through respiratory burst in rat alveolar macrophages (AMs) exposed to PM2.5 was performed by luminol-dependent chemiluminescence (CL). The results showed that (1) PM2.5 deposited in the lung and induced lung pathological damage, especially causing abnormal alterations of mitochondrial structure including mitochondrial swell and cristae disorder, or even fragmentation in the presence of higher dosage of PM2.5; (2) PM2.5 significantly affected the expression of specific mitochondrial fission/fusion markers (OPA1, Mfn1, Mfn2, Fis1 and Drp1) in rat lung; (3) PM2.5 inhibited Mn superoxide dismutase (MnSOD), Na+K+-ATPase and Ca2+-ATPase activities and elevated malondialdehyde (MDA) contents in rat lung mitochondria; (4) PM2.5 induced rat AMs to produce ROS, which was inhibited about 84.1 % by diphenyleneiodonium chloride (DPI), an important ROS generation inhibitor. It is suggested that the rat lung pathological injury exposed to PM2.5 is associated with the mitochondrial fusion-fission dysfunction, ROS generation, mitochondrial lipid peroxidation and cellular homeostasis imbalance. The lung mitochondrial damage may be one of the important mechanisms by which PM2.5 induced lung injury attributing to the respiratory diseases.
Background:
Urothelial cancer (UC) is a common cancer among males. Once metastatic or chemoresistant diseases develop, there is little effective treatment available. A fungal immunomodulatory protein, ganoderma tsugae (FIP-gts) possesses antitumor activity against solid tumors and inhibits telomerase activity. FIP-gts induces autophagy in cancer cells and may provide an alternative pathway against chemo-resistance.
Materials and methods:
Two UC cell lines were used to investigate the cytotoxicity effects and the autophagy regulation of FIP-gts using flow cytometry, acidic vesicular organelles (AVO) staining and western blotting.
Results:
MTT assay showed that FIP-gts and bafilomycin-A1 (Baf-A1) and or chloroquine (CQ) could enhance a significantly synergistic cytotoxicity. The treatment of UC cell lines with FIP-gts activated LC-3 II formation and AVO positive staining on western blot and flow cytometry. Interestingly, FIP-gts and Baf-A1 combined treatment was found to lead to enhancement of apoptosis along with inhibition of autophagy in parental and resistant UC cells.
Conclusion:
FIP-gts may have the potential to be utilized as a therapeutic adjuvant for the treatment of resistant UC cancer down-regulating Beclin-1 to activate autophagic cell death.
Diesel exhaust has been classified as a potential carcinogen and associated with various health effects. A previous study showed that the doses for manifesting the mutagenetic effects of diesel exhaust could be reduced when co-exposed with ultraviolet-A (UVA) in a cellular system. However, the mechanisms underlying synergistic effects remain to be clarified, especially in an in vivo system. In the present study, using Caenorhabditis elegans (C. elegans) as an in vivo system we studied the synergistic effects of diesel particulate extract (DPE) plus UVA, and the underlying mechanisms were dissected genetically using related mutants. Our results demonstrated that though co-exposure of wild type worms at young adult stage to low doses of DPE (20 μg/ml) plus UVA (0.2, 0.5 and 1.0 J/cm2) did not affect the worm development (mitotic germ cells and brood size), it resulted in a significant induction of germ cell death. Using the strain of hus-1::gfp, distinct foci of HUS-1::GFP was observed in proliferating germ cells, indicating the DNA damage after worms were treated with DPE plus UVA. Moreover, the induction of germ cell death by DPE plus UVA was alleviated in single-gene loss-of-function mutations of core apoptotic, checkpoint HUS-1, CEP-1/p53 and MAPK dependent signaling pathways. Using a ROS probe, it was found that the production of ROS in worms co-exposed to DPE plus UVA increased as a time-dependent manner. In addition, employing a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron spin resonance analysis, the increased 1O2 production was demonstrated in worms co-exposed to DPE plus UVA. These results indicated that UVA could enhance the apoptotic induction of DPE at low doses through a DNA damage-trigged pathway, and the production of ROS, especially 1O2, played a pivotal role in initiating the synergistic process.
Considerable evidence shows a key role for protein modification in the adverse effects of chemicals; however, the interaction of diesel exhaust particles (DEP) with proteins and the resulting biological activity remains unclear. DEP and carbon black (CB) suspensions with and without bovine serum albumin (BSA) were used to elucidate the biological effects of air pollutants. The DEP and CB samples were then divided into suspensions and supernatants. Two important goals of the interaction of DEP with BSA were as follows: (1) understanding BSA modification by particles and (2) investigating the effects of particles bound with BSA and the corresponding supernatants on cellular oxidative stress and inflammation. We observed significant free amino groups production was caused by DEP. Using liquid chromatography-mass spectrometry (LC-MS), we observed that BSA was significantly oxidised by DEP in the supernatants and that the peptides ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK, derived BSA-DEP conjugates, were also oxidised. In A549 cells, DEP-BSA suspensions and the corresponding supernatants reduced 8-hydroxy-2'-deoxyguanosine (8-OHdG) production and increased interleukin-6 (IL-6) levels when compared to DEP solutions without BSA. Our findings suggest that oxidatively modified forms of BSA caused by DEP could lead to oxidative stress and the activation of inflammation.
Ganoderma mushrooms (Lingzhi in Chinese) have well-documented health benefits. Ganoderma tsugae (G. tsugae), one of the ganoderma species, has been commercially cultivated as a dietary supplement. Because G. tsugae has high antioxidant activity and because oxidative stress is often associated with cardiac injury, we hypothesized that G. tsugae protects against cardiac injury by alleviating oxidative stress. We tested the hypothesis using a work-overload-induced myocardial injury model created by challenging mice with isoproterenol (ISO). Remarkably, oral G. tsugae protected the mice from ISO-induced myocardial injury. Moreover, the triterpenoid fraction of G. tsugae, composed of a mixture of nine structurally related ganoderic acids (GAs), provided cardioprotection by inhibiting the ISO-induced expression of Fas/Fas ligand, oxidative stress, and apoptosis. The antioxidant activity of GAs was tested in cultured cardio-myoblast H9c2 cells against the insult of H2 O2 . GAs dissipated the cellular reactive oxygen species imposed by H2 O2 and prevented cell death. Our findings uncovered the cardioprotective activity of G. tsugae and identified GAs as the bioactive components against cardiac insults.
Particulate pollution is a major public health concern because epidemiological studies have demonstrated that exposure to particles is associated with respiratory diseases and lung cancer. Diesel exhaust particles (DEP), which is classified as a human carcinogen (IARC, 2012), are considered a major contributor to traffic-related particulate matter (PM) in urban areas. DEP consists of various compounds, including PAHs and metals which are the principal components that contribute to the toxicity of PM. The present study aimed to investigate effects of PM on induction of oxidative DNA damage and inflammation by using lymphocytes in vitro and in human exposed to PM in the environment. Human lymphoblasts (RPMI 1788) were treated with DEP (SRM 2975) at various concentrations (25-100μg/ml) to compare the extent of responses with alveolar epithelial cells (A549). ROS generation was determined in each cell cycle phase of DEP-treated cells in order to investigate the influence of the cell cycle stage on induction of oxidative stress. The oxidative DNA damage was determined by measurement of 8-hydroxy-deoxyguanosine (8-OHdG) whereas the inflammatory responses were determined by mRNA expression of interleukin-6 and -8 (IL-6 and IL-8), Clara cell protein (CC16), and lung surfactant protein-A (SP-A). The results showed that RPMI 1788 and A549 cells had a similar pattern of dose-dependent responses to DEP in terms of particle uptake, ROS generation with highest level found in G2/M phase, 8-OHdG formation, and induction of IL-6 and IL-8 expression. The human study was conducted in 51 healthy subjects residing in traffic-congested areas. The effects of exposure to PM2.5 and particle-bound PAHs and toxic metals on the levels of 8-OHdG in lymphocyte DNA, IL-8 expression in lymphocytes, and serum CC16 were evaluated. 8-OHdG levels correlated with the exposure levels of PM2.5 (P<0.01) and PAHs (P<0.05), but this was not the case with IL-8. Serum CC16 showed significantly negative correlations with B[a]P equivalent (P<0.05) levels, but positive correlation with Pb (P<0.05). In conclusion, a similar pattern of the dose-dependent responses to DEP in the lymphoblasts and lung cells suggests that circulating lymphocytes could be used as a surrogate for assessing PM-induced oxidative DNA damage and inflammatory responses in the lung. Human exposure to PM leads to oxidative DNA damage whereas PM-induced inflammation was not conclusive and should be further investigated.
To evaluate the effect of Chinese herbs decoction on endocrine therapy- associated hot flashes symptom in breast cancer patients.
Sixty-six patients with breast cancer receiving adjuvant endocrine therapy were categorized to two groups, the control group received endocrine therapy alone, the other group is administered with Chinese herbs decoction besides the endocrine therapy: decoction was administered above 6 months per year for more than 2 years. Frequency of hot flashes per day was recorded, and the effect of decoction on hot flashes symptom being assessed with Kupperman Scoring Index.
Sixty cases were analyzed, 32 cases in endocrine therapy combining Chinese herbs decoction group, 28 cases in mere endocrine therapy group. For hot flashes symptom, in Chinese herbs decoction administration group, 7 cases (21.9%) reported symptom disappeared, 22 cases (68.7%) reported symptom alleviated, 3 cases (9.4%) reported symptom not changed; in endocrine therapy alone group, 5 cases (17.9%) reported symptom disappeared, 13 cases (46.4%) reported symptom alleviated, 10 cases (10/28, 35.7%) reported symptom not changed. The difference between two groups was statistically significant (=0.013). For sleeping disorder, in Chinese herbs decoction administration group, 27 cases (84.4%) reported symptom improved, 5 cases (15.6%) reported no change; in endocrine therapy alone group, 16 cases (57.1%) symptom improved, 12 cases (42.9%) reported no change in sleeping disorder (=0.019), significance statistically.
Long-term Chinese herbs decoction administration remarkably improved hot flashes symptom and sleeping disorder associated with endocrine therapy, meanwhile without definite toxicity and influence on the risk of recurrence of tumor.
Abstract This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. ApoE(-/-) mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas intratracheal instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta.
Inhalation of diesel exhaust particles (DEPs) is associated with pulmonary and cardiovascular disease. One contributor to pathogenesis is inhaled particles reaching and injuring the lung capillary endothelial cells, and possibly gaining access to the blood stream. Using in vitro capillary tubes as a simplified vascular model system for this process, it was previously shown that DEPs induce the redistribution of vascular endothelial cell-cadherin (VE-Cad) away from the plasma membrane to intracellular locations. This allowed DEPs into the cell cytoplasm and tube lumen, suggesting the tubes may have become permeable (Chao et al., 2011). Here some of the mechanisms responsible for endothelial tube changes after DEP exposure were examined. The results demonstrate that endothelial tube cells mounted an oxidative stress response to DEP exposure. Hydrogen peroxide and oxidized proteins were detected after 24h of exposure to DEPs. Particles induced relocalization of Nrf2 from the cytoplasm to the nucleus, upregulating the expression of the enzyme heme oxygenase-1 (HO-1). Surprisingly, vascular endothelial cell growth factor-A (VEGF-A), initially termed "vascular permeability factor" (VPF), was found to be up-regulated in response to the HO-1 expression induced by DEPs. Similar to DEPs, applied VEGF-A induced relocalization of VE-Cadherin from the cell membrane surface to an intracellular location, and relocalization of VE-cadherin was associated with permeability. These data suggest that the DEPs may induce or contribute to the permeability of capillary-like endothelial tube cells via induction of HO-1 and VEGF-A.
Particulate matter (PM) emissions involve a complex mixture of solid and liquid particles suspended in a gas, where it is noted that PM emissions from diesel engines are a major contributor to the ambient air pollution problem. While epidemiological studies have shown a link between increased ambient PM emissions and respiratory morbidity and mortality, studies of this design are not able to identify the PM constituents responsible for driving adverse respiratory health effects. This review explores in detail the physico-chemical properties of diesel PM (DPM) and identifies the constituents of this pollution source that are responsible for the development of respiratory disease. In particular, this review shows that the DPM surface area and adsorbed organic compounds play a significant role in manifesting chemical and cellular processes that if sustained can lead to the development of adverse respiratory health effects. The mechanisms of injury involved included inflammation, innate and acquired immunity, and oxidative stress. Understanding the mechanisms of lung injury from DPM will enhance efforts to protect at-risk individuals from the harmful respiratory effects of air pollutants.
Extracts of Ganoderma tsugae, also known as the Hemlock varnish shelf mushroom, and related Reishi mushrooms are well documented in traditional Chinese medicine. Several Ganoderma sp. are currently cultivated for use in coffee, teas, and dietary supplements. We now report on the isolation and characterization of an unprecedented benzofuran, ganodone (1), from the fruiting bodies of mature growth G. tsugae. This discovery provides a key next step in evaluating the active components in their associated herbal supplements.
15% of patients with severe endometriosis suffer from pain symptoms in spite of pharmacological and surgical treatments. In this retrospective study we aimed to assess the effectiveness of a combined therapy of Traditional Chinese medicine and hypnotherapy (systemic autoregulation therapy, SART) as a novel treatment for endometriosis-associated symptoms.
47 patients with severe endometriosis, which were treated with SART, were followed-up by standardized telephone interviews. Follow-up data were compared to baseline assessments.
median follow-up time was 5 years. The median intensity of endometriosis-associated pain had decreased from 8 to 3 points on a 0-10 point visual analogue scale (p < 0.001). 18 patients (38%) were free of pain, and the number of patients using pain medication had decreased from 38 to 19 (from 81% to 40%). 17 out of the 31 women trying for a pregnancy (55%) showed a total of 21 births at follow-up. Exploratory analyses revealed that larger decreases in pain scores correlated significantly with longer treatment durations, suggesting a dose-response relationship.
treatment of endometriosis with a holistic approach of Chinese medicine and hypnotherapy may result in a substantial reduction of pain as well as increased birth rates in patients with therapy-refractory endometriosis. Randomized controlled studies to further investigate the effectiveness of SART are highly warranted.
Whether diesel exhaust particles (DEPs) potentially have a direct effect on capillary endothelia was examined by following the adherens junction component, vascular endothelial cell cadherin (VE-cadherin). This molecule is incorporated into endothelial adherens junctions at the cell surface, where it forms homodimeric associations with adjacent cells and contributes to the barrier function of the vasculature (Dejana et al., 2008; Venkiteswaran et al., 2002; Villasante et al., 2007). Human umbilical vein endothelial cells (HUVECs) that were pre-formed into capillary-like tube networks in vitro were exposed to DEPs for 24h. After exposure, the integrity of VE-cadherin in adherens junctions was assessed by immunofluorescence analysis, and demonstrated that increasing concentrations of DEPs caused increasing redistribution of VE-cadherin away from the cell-cell junctions toward intracellular locations. Since HUVEC tube networks are three-dimensional structures, whether particles entered the endothelial cells or tubular lumens was also examined. The data indicate that translocation of the particles does occur. The results, obtained in a setting that removes the confounding effects of inflammatory cells or blood components, suggest that if DEPs encounter alveolar capillaries in vivo, they may be able to directly affect the endothelial cell-cell junctions.
Endothelial cells form cell-cell adhesive structures, called adherens and tight junctions, which maintain tissue integrity, but must be dynamic for leukocyte transmigration during the inflammatory response and cellular remodeling during angiogenesis. This review will focus on Vascular Endothelial (VE)-cadherin, an endothelial-specific cell-cell adhesion protein of the adherens junction complex. VE-cadherin plays a key role in endothelial barrier function and angiogenesis, and consequently VE-cadherin availability and function are tightly regulated. VE-cadherin also participates directly and indirectly in intracellular signaling pathways that control cell dynamics and cell cycle progression. Here we highlight recent work that has advanced our understanding of multiple regulatory and signaling mechanisms that converge on VE-cadherin and have consequences for endothelial barrier function and angiogenic remodeling.
Ganoderma tsugae is a medicinal fungus with several biological activities. It has long been used as a folk remedy for the promotion of health and longevity in China and other oriental countries. Here, a bioactive fraction of G. tsugae was progressively purified to be enriched in the activity of cytoprotective enzymes. The highest bioactivity was detected in the 20% EtOH-precipitated fraction, which was prepared from submerged fermentation filtrate of G. tsugae. Following further purification by gel filtration chromatography and acetone extraction, the most bioactive fraction, F5-2, was identified as a peptidoglycan-like compound. Extracts of G. tsugae (F5-2) induced heme oxygenase-1 (HO-1) and thioredoxin reductase-1 (TrxR1) expression in endothelial cells by increasing NF-E2-related factor-2 (Nrf2) nuclear translocation. Pretreatment with F5-2 increased intracellular glutathione (GSH) and protected against H(2)O(2), suggesting that induction of these antioxidant enzymes is important in protection against oxidative stress. Hence the bioactive peptidoglycan-like compound from G. tsugae might protect endothelial cells.
Heme oxygenase-1 apart from converting heme to carbon monoxide, iron and biliverdin has been shown to exert anti-inflammatory, antiapoptotic and antioxidant actions. The present review summarizes the most recent studies about heme oxygenase-1 involvement in atherosclerosis, neovascularization and endothelial progenitor cells biology.
Heme oxygenase-1 has been shown to be protective against atherosclerosis via decreasing ROS generation and proinflammatory cytokine production resulting in diminished lipid uptake and foam cell formation. Moreover, heme oxygenase-1 role in neovascularization and its involvement in response of endothelial progenitor cells to stromal cell derived factor-1 as well as endothelial cells to vascular endothelial growth factor has been stressed, recently. The detailed mechanisms of heme oxygenase-1 action in the processes of vasculogenesis and angiogenesis as well as the involvement of Nrf2 and KLF2 transcription factors in heme oxygenase-1-dependent vascular protection are among the subjects most intensively studied, currently.
Recent studies underscore the critical role of heme oxygenase-1 in neovascularization, implicating heme oxygenase-1 as an attractive therapeutic target for treatment of cardiovascular disease.
This chapter discusses medicinal benefits of the Mushroom Ganoderma. Recently, the fruit body and liquid-cultured mycelium of Ganoderma have been reported to contain polysaccharides which inhibit the growth of tumors. Identified as glucans, one of the major constituents in the cell wall of fungi, they appear to increase bodily resistance against the growth of tumors, induce the production of interferon, enhance the immunity function, and kill tumor cells within the body. The role of medicinal mushrooms in metabolic regulation is gaining greater attention. Their use in the development and application of beneficial biological activities offers an advantage in that the active principle is safe and can be tolerated by humans. Cultivation of such mushrooms provides an adequate supply but is unnecessary if mycelial cultures grown in large-scale fermentations can produce the same active principle. Finally, the search for new uses for the traditional medicinal fungi continues and the public should be educated about their potential value.
A systematic method of extraction, fractionation, and purification of polysaccharides from Songshan Lingzhi (Ganoderma tsugae) with antitumor activity was established. Seven glycans with strong antitumor activities were obtained from 14 water-soluble, and 15 water-insoluble fractions: FIo-a, FA-1, FII-1, FIII-2, and FIII-2-a, -b, and -c. FIo-a and FA-1 were protein-containing glucogalactans associated with mannose and fucose. FII-1 was a (1-->3)-beta-D-glucan having a lower protein content. The water-insoluble fractions FIII-2-a, -b, and -c were extracted with alkali, and were found to be protein-containing (1-->3)-beta-D-glucans showing the strongest activity. Chemical properties and structure of each antitumor polysaccharide were compared with three fungi of the Ganoderma family, Kofukitake (G. applanatum), Mannentake (G. lucidum), and Songshan Lingzhi (G. tsugae).
Residents of the dense urban core neighborhoods of New York City (NYC) have expressed increasing concern about the potential human health impacts of diesel vehicle emissions. We measured concentrations of particulate matter [less than/equal to] 2.5 micro in aerodynamic diameter (PM(2.5)) and diesel exhaust particles (DEP) on sidewalks in Harlem, NYC, and tested whether spatial variations in concentrations were related to local diesel traffic density. Eight-hour (1000-1800 hr) air samples for PM(2.5 )and elemental carbon (EC) were collected for 5 days in July 1996 on sidewalks adjacent to four geographically distinct Harlem intersections. Samples were taken using portable monitors worn by study staff. Simultaneous traffic counts for diesel trucks, buses, cars, and pedestrians were carried out at each intersection on [Greater/equal to] 2 of the 5 sampling days. Eight-hour diesel vehicle counts ranged from 61 to 2,467 across the four sites. Mean concentrations of PM(2.5) exhibited only modest site-to-site variation (37-47 microg/m(3)), reflecting the importance of broader regional sources of PM(2.5). In contrast, EC concentrations varied 4-fold across sites (from 1.5 to 6 microg/m(3)), and were associated with bus and truck counts on adjacent streets and, at one site, with the presence of a bus depot. A high correlation (r = 0.95) was observed between EC concentrations measured analytically and a blackness measurement based on PM(2.5) filter reflectance, suggesting the utility of the latter as a surrogate measure of DEP in future community-based studies. These results show that local diesel sources in Harlem create spatial variations in sidewalk concentrations of DEP. The study also demonstrates the feasibility of a new paradigm for community-based research involving full and active partnership between academic scientists and community-based organizations.
48 patients with liver fibrosis due to hepatitis B were treated for 2 years with the drugs for tonifying the kidney, supplementing qi, cooling and invigorating the blood and detoxification. The symptoms were markedly improved, and serum ALT and bilirubin were recovered and kept normal in most of the cases. The mean levels of serum hyaluronic acid, procollagen peptide III and circulating immune complex were decreased and returned to normal after the treatment. B-ultrasonography showed that the portal vein kept in normal size in 82% of the patients, the enlarged portal vein diminished in diameter, and the enlarged spleen reduced.
Episodes of increased air pollution are associated with increases in hospital admissions for cardiovascular disease. Even modest acute phase responses are associated with increased risk of coronary heart disease. The study investigates whether induction of an acute phase response by exposure to air pollution may contribute to cardiovascular pathology.
A prospective cohort study based on a survey in 1984/85 with a 3-year follow-up was conducted in 631 randomly selected men aged 45 to 64 years free of cardiovascular disease at entry 1984/85. Serum C-reactive protein concentrations were determined by a high sensitivity immunoradiometric assay. C-reactive protein concentration was increased in association with the 1985 air pollution episode. In multivariate analyses, elevated concentrations were independently associated with concentrations of total suspended particles and the sulphur dioxide episode. At ambient concentrations of pollution, as noted during the 1985 air pollution episode, the odds of observing C-reactive protein concentrations above 5.7 mg. l(-1)(>90th percentile) tripled, and increases of 26 microg. m(-3)total suspended particles (mean of 5 days) raised the odds of C-reactive protein levels 50% above the 90th percentile.
Exposure to current levels of particulate matter in the atmosphere elicits an acute phase response in randomly selected healthy middle-aged men, which may contribute to the increased cardiovascular risk caused by air pollution.
The endothelial cell (EC) lining of the pulmonary vasculature forms a semipermeable barrier between the blood and the interstitium of the lung. Disruption of this barrier occurs during inflammatory disease states such as acute lung injury and acute respiratory distress syndrome and results in the movement of fluid and macromolecules into the interstitium and pulmonary air spaces. These processes significantly contribute to the high morbidity and mortality of patients afflicted with acute lung injury. The critical importance of pulmonary vascular barrier function is shown by the balance between competing EC contractile forces, which generate centripetal tension, and adhesive cell-cell and cell-matrix tethering forces, which regulate cell shape. Both competing forces in this model are intimately linked through the endothelial cytoskeleton, a complex network of actin microfilaments, microtubules, and intermediate filaments, which combine to regulate shape change and transduce signals within and between EC. A key EC contractile event in several models of agonist-induced barrier dysfunction is the phosphorylation of regulatory myosin light chains catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase and/or through the activity of the Rho/Rho kinase pathway. Intercellular contacts along the endothelial monolayer consist primarily of two types of complexes (adherens junctions and tight junctions), which link to the actin cytoskeleton to provide both mechanical stability and transduction of extracellular signals into the cell. Focal adhesions provide additional adhesive forces in barrier regulation by forming a critical bridge for bidirectional signal transduction between the actin cytoskeleton and the cell-matrix interface. Increasingly, the effects of mechanical forces such as shear stress and ventilator-induced stretch on EC barrier function are being recognized. The critical role of the endothelial cytoskeleton in integrating these multiple aspects of pulmonary vascular permeability provides a fertile area for the development of clinically important barrier-modulating therapies.
Two nationwide surveys were carried out using an electronic poll of 2,000 GPs and postal questionnaires were sent to 30,000 patients with osteoarthritis (OA). Both surveys found a high level of gastro-intestinal (GI) side-effects in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs). Almost every GP (97%) reported experience of patients suffering GI symptoms while on an NSAID, 38% reported patients who had been hospitalised and 4% reported patients who had died owing to NSAID-induced side-effects. Most GPs (92%) said they were concerned about GI safety when prescribing an NSAID and almost a third (32%) said they were concerned about litigation from patients who had experienced a bleed. Use of NSAIDs in OA remained high, with 44% of GPs prescribing conventional NSAIDs to at least three quarters of their patients, 57% of GPs using simple analgesia and just 12% using a cyclo-oxygenase-2 (COX-2) selective inhibitor in over 74% of patients. Some 45% of patients reported receiving NSAIDs compared with 43% on simple analgesia and 4% on COX-2 selective inhibitors. Most GPs (69%) stated that their main therapeutic objective in using an NSAID to treat OA was to control pain without GI side-effects. Almost a quarter (24%) said they used low-dose NSAIDs in the hope that this would control pain without GI side-effects. Dissatisfaction with treatment was the most common reason reported by GPs for patients on an NSAID to re-present, with 73% citing either breakthrough pain or incomplete pain relief as the most common reason for patient dissatisfaction. This mirrored the patients' perception, with 63% citing inadequate pain relief as their main reason for dissatisfaction with current painkillers compared to 17% who cited stomach upsets or irritation. Patient and GP appear to be united in their concern at the GI risks of NSAID treatment. In the light of this and recent data on the efficacy, safety profile and cost-effectiveness of COX-2 selective inhibitors, GPs should re-examine their medical management of OA.
Particulate air pollution is associated with cardiorespiratory effects and ultrafine particles (UFPs, diameter < 100 nm) are believed to play an important role. We studied the acute (1 h) effect of intratracheally instilled unmodified (60 nm), negatively charged carboxylate-modified (60 nm), or positively charged amine-modified (60 or 400 nm) polystyrene particles on bronchoalveolar lavage (BAL) indices and on peripheral thrombosis in hamster. The latter was assessed by measuring the extent of photochemically induced thrombosis in a femoral vein via transillumination. Unmodified and negative UFPs did not modify thrombosis and BAL indices. Positive UFPs increased thrombosis at 500 microg per animal (+ 341 +/- 96%) and at 50 microg per animal (+ 533 +/- 122%), but not at 5 microg per animal. Neutrophils, lactate dehydrogenase, and histamine were increased in BAL at all these doses but protein concentration was increased only at 500 microg per animal. Positive 400-nm particles (500 microg per animal) did not affect thrombosis, although they led to a neutrophil influx and an increase in BAL proteins and histamine. Using the Platelet Function Analyser (PFA-100), the platelets of hamsters were activated by the in vitro addition of positive UFPs and 400-nm particles to blood. We conclude that intratracheally administered positive ultrafine and 400-nm particles induce pulmonary inflammation within 1 h. Positive UFPs, but not the 400-nm particles enhance thrombosis. Hence, particle-induced lung inflammation and thrombogenesis can be partially uncoupled.