Human Lung Epithelial Cell Line Research Articles

Anti-cancer activity of hierarchical ZSM-5 zeolites synthesized from rice-based waste materials

We report the organic template-free synthesis of hierarchical ZSM-5 zeolite using waste materials, by a simple hydrothermal method and their excellent anti-cancer activity against A549 human lung epithelial cell lines.

Cathepsin B plays a key role in optimal production of the influenza A virus.

Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as 'flu'. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. CTSB-deficient (CTSB-/-) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. CTSB-/- macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection.

The Cytotoxic Constituents ofBetula platyphyllaand their Effects on Human Lung A549 Cancer Cells

During the screening for cytotoxic compounds from plants grown in Korea, Betula platyphylla (BP) showed potent activity against the adenocarcinomic human alveolar basal epithelial A549 cell line. To identify the cytotoxic components from BP, the CH2Cl2 fraction with the most significant cytotoxic effect was applied to the column chromatographies. Seven compounds were isolated: lupeol (1), betulinic acid (2), (-)-rhododendrol (3), platyphyllenone (4), platyphyllone (5), (-)-centrolobol (6), and oleanolic acid (7). Among them, three diarylheptanoids (4 - 6) exhibited cytotoxicity toward A549 cells. Especially, 50 μM of 4 reduced A549 cell viability to 18.93 ± 0.82% compared to control (100.00 ± 21.48%). Lactate dehydrogenase (LDH) leakage and intracellular reactive oxygen species (ROS) production were also induced by 50 μM 4. This is the first report on the cytotoxic effect of BP-derived diarylheptanoids 4-6 against A549 cells. The compound 4 may be useful for the development of early hit compounds for non-small cell lung carcinoma, but the consideration about selectivity of 4 is required since 4 also showed the cytotoxicity in the human normal lung epithelial BEAS-2B cell line.

Mannose-functionalized solid lipid nanoparticles are effective in targeting alveolar macrophages

Mannose receptor is highly expressed on alveolar macrophages, being a potential target to promote the specific local drug delivery of anti-tuberculosis agents through the use of functionalized nanocarriers. In this work, isoniazid (Isn)-loaded solid lipid nanoparticles (SLN), reinforced with stearylamine (SA) were produced by double emulsion technique and further surface-functionalized with mannose in a straightforward chemical approach. Upon pre-formulation assessment, SLN close to 500 nm average size, positively charged and with association efficiency of ISN close to 50% were obtained. Functionalization with mannose was performed after SLN production and confirmed by Fourier transform infrared spectroscopy (FTIR). Both functionalized and non-functionalized SLN demonstrated to devoid of toxicity when tested in human lung epithelial cell line (NCI-H441) and differentiated THP-1 (dTHP-1), reducing the intrinsic cytotoxicity of Isn when incorporated into SLN. Uptake studies were conducted on same macrophage-like cells and the results showed that fluorescent mannosylated SLN (M-SLN) were more efficient in be internalized comparatively to SLN. Moreover, the uptake of M-SLN was reduced when cells were pre-incubated with mannose, demonstrating the receptor-dependence internalization of functionalized SLN. These functionalized nanocarriers may represent a useful platform to target alveolar macrophages for delivering anti-infective drugs.

An in vitro cytotoxicity assessment of graphene nanosheets on alveolar cells

The collection of intrinsic properties possessed by graphene family nanomaterials (GFNs) results in their continuous exploitation for biomedical applications. The materials biomedical potential has motivated an upsurge in green preparation routes for the production of graphene like materials with limited toxicity. A number of bio-friendly reducing agents have been utilized for the preparation of chemically reduced graphene oxide (GO), and their resulting cytotoxic effects examined. However, the toxicology effects of one of the first biomolecules implemented for the reduction of GO, ascorbic acid (AA) has yet to be investigated. Herein, the toxicity of three distinct GFNs; GO, hydrazine reduced GO (H.rGO) and AA.rGO, prepared through diverse chemical routes are studied, to demonstrate the cytotoxic activity of a green reducer, in comparison to an established reduction method using hydrazine hydrate. The variation in atomic structure of GO, H.rGO and AA.rGO resulting from different synthesis techniques demonstrates the dependence of toxicity on particle shape and size. All GFNs induced high levels of alveolar cell toxicity. Interaction of AA.rGO with the A549 human lung epithelial carcinoma cell line resulted in increased leakage of lactate dehydrogenase, indicative of diminished cell membrane integrity. The uncharacteristic shape of the AA.rGO may be responsible for this proliferated release of the essential protein. The presented data therefore demonstrates that modification of synthetic processes significantly alter the biological activities of GFNs.

Stenotrophomonas maltophilia Serine Protease StmPr1 Induces Matrilysis, Anoikis, and Protease-Activated Receptor 2 Activation in Human Lung Epithelial Cells.

Stenotrophomonas maltophilia is an emerging, opportunistic nosocomial pathogen that can cause severe disease in immunocompromised individuals. We recently identified the StmPr1 and StmPr2 serine proteases to be the substrates of the Xps type II secretion system in S. maltophilia strain K279a. Here, we report that a third serine protease, StmPr3, is also secreted in an Xps-dependent manner. By constructing a panel of protease mutants in strain K279a, we were able to determine that StmPr3 contributes to the previously described Xps-mediated rounding and detachment of cells of the A549 human lung epithelial cell line as well as the Xps-mediated degradation of fibronectin, fibrinogen, and the cytokine interleukin-8 (IL-8). We also determined that StmPr1, StmPr2, and StmPr3 account for all Xps-mediated effects toward A549 cells and that StmPr1 contributes the most to Xps-mediated activities. Thus, we purified StmPr1 from the S. maltophilia strain K279a culture supernatant and evaluated the protease's activity toward A549 cells. Our analyses revealed that purified StmPr1 behaves more similarly to subtilisin than to trypsin. We also determined that purified StmPr1 likely induces cell rounding and detachment of A549 cells by targeting cell integrin-extracellular matrix connections (matrilysis) as well as adherence and tight junction proteins for degradation. In this study, we also identified anoikis as the mechanism by which StmPr1 induces the death of A549 cells and found that StmPr1 induces A549 IL-8 secretion via activation of protease-activated receptor 2. Altogether, these results suggest that the degradative and cytotoxic activities exhibited by StmPr1 may contribute to S. maltophilia pathogenesis in the lung by inducing tissue damage and inflammation.

Exposure to selected limonene oxidation products: 4-OPA, IPOH, 4-AMCH induces oxidative stress and inflammation in human lung epithelial cell lines

Limonene oxidation products (LOPs) have gained interest on their harmful health effects over time. Recently, studies have shown that the selected LOPs: 4-oxopentanal (4-OPA), 3-isopropenyl-6-oxo-heptanal (IPOH) and 4-acetyl-1-methylcyclohexene (4-AMCH) have sensory irritation effects in mice and inflammatory effects in human lung cells. This study was therefore undertaken to investigate the potential capacity of 4-OPA, IPOH and 4-AMCH to cause cell membrane damage, oxidative stress and inflammation in human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines.Overall results suggest that 4-OPA, IPOH have cytotoxic effects on human lung cells that might be mediated by ROS: the highest concentration applied of IPOH [500 μM] enhanced ROS generation by 100-fold ± 7.7 (A549) and 230-fold ± 19.9 (16HBE14o-) compared to the baseline. 4-OPA [500 μM] increased ROS levels by 1.4-fold ± 0.3 (A549) and by 127-fold ± 10.5 (16HBE14o-), while treatment with 4-AMCH [500 μM] led to 0.9-fold ± 0.2 (A549) and 49-fold ± 12.8 (16HBE14o-) increase. IPOH [500 μM] caused a decrease in the thiol-state balance (e.g. after 2 h, GSH:GSSG was reduced by 37% compared to the untreated 16HBE14o-cells). 4-OPA [500 μM] decreased the GSH:GSSG by 1.3-fold change in A549 cells and 1.4-fold change in 16HBE14o-cells. No statistically significant decrease in the GSH:GSSG in A549 and 16HBE14o-cell lines was observed for 4-AMCH [500 μM]. In addition, IPOH and 4-OPA [31.2 μM] increased the amount of the inflammatory markers: RANTES, VEGF and EGF. On the other hand, 4-AMCH [31.2 μM] did not show inflammatory effects in A549 or 16HBE14o-cells.The 4-OPA, IPOH and 4-AMCH treatment concentration and time-dependently induce oxidative stress and/or alteration of inflammatory markers on human bronchial and alveolar cell lines.

Syk inhibitor R406 downregulates inflammation in an in vitro model of Pseudomonas aeruginosa infection.

As Pseudomonas aeruginosa infections are characterized by strong inflammation of infected tissues, anti-inflammatory therapies in combination with antibiotics have been considered for the treatment of associated diseases. Syk tyrosine kinase is an important regulator of inflammatory responses, and its specific inhibition was explored as a therapeutic option in several inflammatory conditions; however, this has not been studied in bacterial infections. We used a model of in vitro infection of human monocytic cell line THP-1 and lung epithelial cell line H292 with both wild-type and flagella-deficient mutant of P. aeruginosa strain K, as well as with clinical isolates from cystic fibrosis patients, to study the effect of a small molecule Syk inhibitor R406 on inflammatory responses induced by this pathogen. One-hour pretreatment of THP-1 cells with 10 μmol/L R406 resulted in a significant downregulation of the expression of the adhesion molecule ICAM-1, pro-inflammatory cytokines TNF-α and IL-1β, and phosphorylated signaling proteins ERK2, JNK, p-38, and IκBα, as well as significantly decreased TNF-α release by infected H292 cells. The results suggest that Syk is involved in the regulation of inflammatory responses to P. aeruginosa, and R406 may potentially be useful in dampening the damage caused by severe inflammation associated with this infection.

Chronic polycyclic aromatic hydrocarbon exposure causes DNA damage and genomic instability in lung epithelial cells.

Cell exposure to atmospheric polycyclic aromatic hydrocarbons (PAHs) is closely associated with DNA damage and genomic instability. We assessed the mechanisms of chronic and acute PAH exposure-induced genotoxicity in two human lung epithelial cell lines, A549 and NC-H1975. We sampled atmospheric PAHs at the Xixi Campus, Zhejiang University in Hangzhou, China during August (non-haze episode) and November (haze episode) 2015. We identified vehicle emissions as a dominant anthropogenic PAH source in our study. PAHs were extracted according to the United States Environmental Protection Agency Method TO-13A. We found that chronic PAH exposure saturated lung cell xenobiotic metabolic pathways, promoting intercellular reactive oxygen species production and accumulation. Chronic alteration of the cellular redox status resulted in DNA damage and genomic instability. Chronic PAH exposure also perturbed the cellular DNA damage response, inducing S phase arrest and inhibiting apoptosis. Dysregulation of PAH metabolism and the DNA damage response altered cellular homeostasis and increased cell susceptibility to subsequent PAH exposures, thereby enhancing the likelihood of genomic mutation and instability.

Early Assessment and Correlations of Nanoclay's Toxicity to Their Physical and Chemical Properties.

Nanoclays' functionalization with organic modifiers increases their individual barrier properties, thermal stability, and mechanical properties and allows for ease of implementation in food packaging materials or medical devices. Previous reports have shown that, while organic modifiers integration between the layered mineral silicates leads to nanoclays with different degrees of hydrophobicity that become easily miscible in polymers, they could also pose possible effects at inhalation or ingestion routes of exposure. Through a systematic analysis of three organically modified and one pristine nanoclay, we aimed to relate for the first time the physical and chemical characteristics, determined via microscopical and spectroscopical techniques, with the potential of these nanoclays to induce deleterious effects in in vitro cellular systems, i.e. in immortalized and primary human lung epithelial cell lines. To derive information on how functionalization could lead to toxicological profiles throughout nanoclays' life cycle, both as-received and thermally degraded nanoclays were evaluated. Our analysis showed that the organic modifiers chemical composition influenced both the physical and chemical characteristics of the nanoclays as well as their toxicity. Overall, when cells were exposed to nanoclays with organic modifiers containing bioreactive groups, they displayed lower cellular numbers as well more elongated cellular morphologies relative to the pristine nanoclay and the nanoclay containing a modifier with long carbon chains. Additionally, thermal degradation caused loss of the organic modifiers as well as changes in size and shape of the nanoclays, which led to changes in toxicity upon exposure to our model cellular systems. Our study provides insight into the synergistic effects of chemical composition, size, and shape of the nanoclays and their toxicological profiles in conditions that mimic exposure in manufacturing and disposal environments, respectively, and can help aid in safe-by-design manufacturing of nanoclays with user-controlled functionalization and lower toxicity levels when food packaging applications are considered.

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses.

Influenza A viruses pose a constant potential threat to human health. In view of the innate antiviral activity of interferons (IFNs) and their potential use as anti-influenza agents, it is important to know whether viral resistance to these antiviral proteins can arise. To examine the likelihood of emergence of IFN-λ1-resistant H1N1 variants, we serially passaged the A/California/04/09 (H1N1) strain in a human lung epithelial cell line (Calu-3) in the presence of increasing concentrations of recombinant IFN-λ1 protein. To monitor changes associated with adaptation of this virus to growth in Calu-3 cells, we also passaged the wild-type virus in the absence of IFN-λ1. Under IFN-λ1 selective pressure, the parental virus developed two neuraminidase (NA) mutations, S79L and K331N, which significantly reduced NA enzyme activity (↓1.4-fold) and sensitivity to IFN-λ1 (↓˃20-fold), respectively. These changes were not associated with a reduction in viral replication levels. Mutants carrying either K331N alone or S79L and K331N together induced weaker phosphorylation of IFN regulatory factor 3 (IRF3), and, as a consequence, much lower expression of the IFN genes (IFNB1, IFNL1 and IFNL2/3) and proteins (IFN-λ1 and IFN-λ2/3). The lower levels of IFN expression correlated with weaker induction of tyrosine-phosphorylated STAT1 and reduced RIG-I protein levels. Our findings demonstrate that influenza viruses can develop increased resistance to the antiviral activity of type III interferons.

Contribution of pilus type 2b to invasive disease caused by a Streptococcus agalactiae ST-17 strain

BackgroundGroup B Streptococcus (GBS) is a major cause of invasive disease especially in neonates. In GBS three structurally distinct pilus polymers have been identified as important virulence factors and promising vaccine candidates. The vast majority of Group B Streptococci belonging to the hypervirulent serotype III ST-17 lineage bear pilus types 1 and 2b. The purpose of this study was to investigate the relative contribution of these two pilus types to the pathogenesis of a ST-17 strain.ResultsWe performed in vivo and in vitro analysis of isogenic knockout mutants derived from the GBS COH1 ST-17 strain deprived of either pilus type 1 or 2b. We compared the two pilus mutants with the wild type strain in a mouse model of invasive disease, in vitro survival in macrophages, and adherence/invasion assays using human brain endothelial and lung epithelial cell lines. Significantly less of the pilus 2b mutant was recovered from the blood, lungs and brain tissue of infected mice compared to the wild-type and pilus 1 mutant strains. Further, while the pilus 2b mutant survived similarly in murine macrophages, it exhibited a lower capacity to adhere and invade human brain epithelial and lung endothelial cell lines.ConclusionsThe data suggest an important role of pilus 2b in mediating GBS infection and host cell interaction of strains belonging to the hypervirulent GBS ST-17 lineage.

Multiple endpoints to evaluate pristine and remediated titanium dioxide nanoparticles genotoxicity in lung epithelial A549 cells

Titanium dioxide nanoparticles (TiO2 NP) are broadly used in a wide range of applications. Several studies have reported that TiO2 NP possess cytotoxic and genotoxic properties that could induce adverse health effects in humans. The FP7 Sanowork project was aimed to minimize occupational hazard and exposure to engineered nanomaterials (ENM), including TiO2 NP, through the surface modification in order to avoid possible adverse toxic effects for humans. In this study we investigated cytotoxicity, genotoxicity and epigenetic properties of TiO2 NP uncoated and coated with silica or citrate, as well as of the benchmark material P25. We used a panel of in vitro assays in the human lung epithelial cell line A549, in order to better understand if the remediation strategy adopted was able to counteract possible toxic effects of uncoated TiO2 NP. Our results showed that the uncoated TiO2 NP were both cytotoxic and genotoxic, and the remediation strategy adopted did not reduce the adverse effects of uncoated TiO2 NP. In particular, the presence of citrate was able to increase their cytotoxicity and genotoxicity, exerting also epigenotoxic effects, as evaluated by the marked reduction of LINE-1 methylation levels.

Enhancing Tumor Cell Response to Multidrug Resistance with pH-Sensitive Quercetin and Doxorubicin Conjugated Multifunctional Nanoparticles

Classical chemotherapy uses chemotherapeutic agents as a mainstay of anticancer treatment. However, the development of multidrug resistance to chemotherapy limits the effectiveness of current cancer treatment. Nanosized bioconjugates combining a chemotherapeutic agent with a pharmacological approach may improve the curative effect of chemotherapeutic agents. Herein I addressed this issue by describing the synthesis, and testing of, pH-responsive Fe3O4@SiO2(FITC)-BTN/QUR/DOX multifunctional nanoparticles. The particles were designed to modulate resistance-mediating factors and to potentiate the efficacy of DOX against chemoresistance. The physicochemical properties of the nanoparticles were characterized based on the combination of several techniques: dynamic light scattering (DLS), zeta-potential measurement, Fourier transform infrared spectroscopy (FTIR), electron microscopy techniques (SEM and STEM with EDX) and an in vitro pH-dependent release study. Cellular uptake and cytotoxicity experiments demonstrated enhanced intracellular delivery and retention of nanoparticles in the cytoplasm and efficient reduction of cancer cell viability in drug-resistant lung carcinoma A549/DOX cell lines. This did not affect internalization and viability of an immortalized human lung epithelial cell line BEAS-2B. Moreover, proapoptotic and antiproliferative studies showed that Fe3O4@SiO2(FITC)-BTN/QUR/DOX nanoparticles can promote apoptosis, inhibit tumor cell proliferation, and enhance the chemotherapeutic effects of DOX against multidrug resistance. These results confirm that this multifunctional platform possesses significant synergy between QUR and DOX and is promising for development as an antitumor treatment in cancer therapy.

Effect of biodiesel fuel on "real-world", nonroad heavy duty diesel engine particulate matter emissions, composition and cytotoxicity.

Biodiesel is regarded by many as a "greener" alternative fuel to petroleum diesel with potentially lower health risk. However, recent studies examining biodiesel particulate matter (PM) characteristics and health effects are contradictive, and typically utilize PM generated by passenger car engines in laboratory settings. There is a critical need to analyze diesel and biodiesel PM generated in a "real-world" setting where heavy duty-diesel (HDD) engines and commercially purchased fuel are utilized. This study compares the mass concentrations, chemical composition and cytotoxicity of real-world PM from combustion of both petroleum diesel and a waste grease 20% biodiesel blend (B20) at a community recycling center operating HDD nonroad equipment. PM was analyzed for metals, elemental/organic carbon (EC/OC), polycyclic aromatic hydrocarbons (PAHs), and nitro-polycyclic aromatic hydrocarbons (N-PAHs). Cytotoxicity in a human lung epithelial cell line (BEAS-2B) following 24h exposure to the real-world particles was also evaluated. On average, higher concentrations for both EC and OC were measured in diesel PM. B20 PM contained significantly higher levels of Cu and Mo whereas diesel PM contained significantly higher concentrations of Pb. Principal component analysis determined Mo, Cu, and Ni were the metals with the greatest loading factor, suggesting a unique pattern related to the B20 fuel source. Total PAH concentration during diesel fuel use was 1.9 times higher than during B20 operations; however, total N-PAH concentration was 3.3 times higher during B20 use. Diesel PM cytotoxicity was 8.5 times higher than B20 PM (p<0.05) in a BEAS-2B cell line. This study contributes novel data on real-world, nonroad engine sources of metals, PAH and N-PAH species, comparing tailpipe PM vs. PM collected inside the equipment cabin. Results suggest PM generated from burning petroleum diesel in nonroad engines may be more harmful to human health, but the links between exposure, composition and toxicity are not straightforward.

GRP78 silencing enhances hyperoxia-induced alveolar epithelial cell apoptosis via CHOP pathway.

Hyperoxia is one of the primary causes of bronchopulmonary dysplasia, which may occur in premature infants following supplemental oxygen therapy. Glucose regulated protein 78 (GRP78), which is a molecular chaperone located in the lumen of the endoplasmic reticulum (ER), has been reported to regulate hyperoxia‑associated ER stress. The role of GRP78 in lung epithelial cells during hyperoxia remains to be elucidated. In the present study, the A549 cultured human lung epithelial cell line was exposed to hyperoxic conditions, and then transfected with short interfering (si)RNA targeted to GRP78. siRNA or pEGFP‑N1 plasmid were used to knockdown or overexpress specific genes, reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect RNA and protein levels of gene expression, and flow cytometry was used to detect apoptosis. The expression levels of ER stress‑associated genes were determined, and a significant increase in C/EBP homologous protein (CHOP) expression and apoptosis of A549 cells was observed, following GRP78 knockdown. The overexpression of CHOP downregulated B‑cell lymphoma (Bcl)‑2 expression levels, upregulated BCL2 associated X (Bax), and increased apoptosis of A549 cells under conditions of hyperoxia. CHOP knockdown demonstrated the opposite effect on Bcl‑2 and Bax expression levels. These results suggested that GRP78 silencing promoted lung epithelial cell apoptosis during hyperoxia, via regulation of the CHOP pathway.

Overexpression of MicroRNA-27b Inhibits Proliferation, Migration, and Invasion via Suppression of MET Expression.

MicroRNA-27b (miR-27b) was recently found to be significantly downregulated in different human cancers. However, evidence of the function of miR-27b in non-small cell lung cancer (NSCLC) remains limited. In this study, we aimed to investigate novel miR-27b-mediated targets or signaling pathways associated with the tumorigenesis and metastasis of NSCLC. Real-time (RT) PCR was performed to examine miR-27b expression in NSCLC specimens. MTT assay, wound-healing assay, and Transwell assay were used to determine cell proliferation, migration, and invasion. Our data indicated that the miR-27b levels were significantly decreased in NSCLC specimens and cell lines (SK-MES-1, H358, H460, A549, and H1229) when compared to matched normal adjacent tissues and normal human lung epithelial cell lines, respectively. Restoration of miR-27b significantly inhibited the proliferation, migration, and invasion of A549 cells. We then conducted in silico analysis and luciferase reporter gene assay and identified MET, a receptor tyrosine kinase, as a direct target of miR-27b in NSCLC cells. Moreover, overexpression of MET rescued the suppressive effect of miR-27b on the proliferation, migration, and invasion of A549 cells, suggesting that MET acts as a downstream effecter of miR-27b in NSCLC cells. In summary, our study identified a novel miR-27b/MET signaling pathway involved in the cell proliferation, migration, and invasion of NSCLC, and identification of miR-27b-mediated novel signaling pathways may help reveal the molecular mechanism underlying the development and malignant progression of this disease.

Activity of Pericampylus glaucus and periglaucine A in vitro against nasopharangeal carcinoma and anti-inflammatory activity.

Ethnopharmacological relevancePericampylus glaucus is a climbing plant found across Asia and used in traditional medicine to treat a number of conditions including splenomegaly, fever, cough, laryngitis, pulmonary disease, asthma, headache, hair loss, snake bite, boar bite, factures, boils, tumours, tetanus, rheumatic pain, itches and eclampsia. Aim of the studyTo test extracts of P. glaucus in a number of bioassays and determine the legitimacy of its traditional use. Materials and methodsThe stems, leaves, roots and fruits of P. glaucus were collected and extracted sequentially with hexane, chloroform and ethanol, respectively. The anti-inflammatory activity was assessed by testing the ability of the extracts to inhibit heat induced protein denaturation, stabilise human red blood cells under hypotonic stress and by testing the inhibitory activity of the extracts against cyclooxygenases 1 and 2. Cytotoxicity was tested using the human lung epithelial cell line MRC-5 and nasopharangeal carcinoma cell line HK1 in the MTT assay. ResultsMany of the samples showed an ability to prevent heat induced protein denaturation, as well as prevent lysis of red blood cells. Most of the extracts demonstrated inhibitory activity towards both of the COX enzymes. The ethanol extracts tended to demonstrate greater toxicity than other extracts, with some of the other extracts significantly enhancing growth and metabolism of the cells. ConclusionThe benefit of P. glaucus for the treatment of diseases related to inflammation and cancer was supported by the in vitro assays adopted in this study.

IL-35 Suppresses Lipopolysaccharide-Induced Airway Eosinophilia in EBI3-Deficient Mice

EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27-deficient mice or WSX-1/IL-27Rα subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-α and IL-1β. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.

Hydroxysafflor Yellow A Alleviates Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Mice.

Hydroxysafflor yellow A (HSYA) is an effective ingredient of the Chinese herb Carthamus tinctorius L. The present study investigated the protective effect of HSYA on lipopolysaccharide (LPS)-induced acute respiratory distress syndrome in mice, and the underlying mechanisms involved. HSYA (14, 28, 56 mg/kg) was intraperitoneally injected to mice once daily from day 1 to 10 after LPS administration. HSYA attenuated the body weight loss, the augmented left index and the increase of pathologic changes in pulmonary inflammation caused by LPS. Treatment with HSYA also alleviated increased expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, transforming growth factor (TGF)-β1, collagen (Col) I, Col III, α-smooth muscle actin (α-SMA), myeloid differentiation (MD)-2, Toll-like receptor 4 (TLR4) and cluster differentiation (CD)14 at the mRNA (RT-PCR) and protein levels (Western blot and enzyme-linked immuno sorbent assay). Moreover, HSYA inhibited the elevated levels of nuclear factor (NF)-κB and α-SMA in lung tissue (immunohistochemistry), and alleviated the slight collagen deposition in pulmonary tissues (Masson's trichrome staining). HSYA inhibited the specific binding of fluorescein isothiocyanate (FITC)-LPS on human lung epithelial cell line (A549) or human umbilical vein cell line (Eahy926) cells (flow cytometry). These findings suggested that HSYA has a protective effect on acute respiratory distress syndrome (ARDS) induced by LPS through blocking the TLR4/NF-κB pathway, and that the TLR4 receptor might be a target of HSYA on the cell membrane.