Phenolic Characterization and Antioxidant Activity of Chrysanthemum indicum and Opisthopappus Shih during Different Growth Stages.

Recently, much attention has been devoted to natural phenolics because of their ideal structure and chemistry for free radical scavenging activities, which may play important roles in long-term health and a reduction in the risk of developing chronic degenerative diseases. Chrysanthemum indicum (C. indicum) has been widely used as a health food and as a popular herb in China for many centuries. Opisthopappus Shih (O. shih) often takes the place of its related genera, C. indicum, in functional tea or medicine prescriptions in place of origin. In this article, a comparative study on the phenolics and antioxidant activity of C. indicum and O. shih during different growth stages was investigated. The antioxidant properties of plant extracts were tested using DPPH and ABTS assays. The characterization of potential phytochemicals was carried out using Fourier transform infrared (FT-IR) spectroscopy. Total phenolics (TPC) and total flavonoid content (TFC) were measured using Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. An HPLC method was used to simultaneously quantify five phenolic compounds, including chlorogenic acid, luteolin, rutin, quercetin, and apigenin. Results indicated that the Trolox equivalent antioxidant activity (TEAC) values of C. indicum and O. shih had extremely large variations at different growth stages. The most abundant phenolics and potent antioxidant activity of two related plants appear at the early vegetative and then flowering stages. Antioxidant activities and phenolic content of O. shih were higher than those of corresponding organs of C. indicum at the same collection time. The whole plant of O. shih, especially its leaves and flowers, are good candidates for obtaining nutraceuticals and functional food ingredients.

NLRP3 Inflammasome Mediates Silica-induced Lung Epithelial Injury and Aberrant Regeneration in Lung Stem/Progenitor Cell-derived Organotypic Models.

Silica-induced lung epithelial injury and fibrosis are vital pathogeneses of silicosis. Although the NOD-like receptor protein 3 (NLRP3) inflammasome contributes to silica-induced chronic lung inflammation, its role in epithelial injury and regeneration remains unclear. Here, using mouse lung stem/progenitor cell-derived organotypic systems, including 2D air-liquid interface and 3D organoid cultures, we investigated the effects of the NLRP3 inflammasome on airway epithelial phenotype and function, cellular injury and regeneration, and the potential mechanisms. Our data showed that silica-induced NLRP3 inflammasome activation disrupted the epithelial architecture, impaired mucociliary clearance, induced cellular hyperplasia and the epithelial-mesenchymal transition in 2D culture, and inhibited organoid development in 3D system. Moreover, abnormal expression of the stem/progenitor cell markers SOX2 and SOX9 was observed in the 2D and 3D organotypic models after sustained silica stimulation. Notably, these silica-induced structural and functional abnormalities were ameliorated by MCC950, a selective NLRP3 inflammasome inhibitor. Further studies indicated that the NF-κB, Shh-Gli and Wnt/ß-catenin pathways were involved in NLRP3 inflammasome-mediated abnormal differentiation and dysfunction of the airway epithelium. Thus, prolonged NLRP3 inflammasome activation caused injury and aberrant lung epithelial regeneration, suggesting that the NLRP3 inflammasome is a pivotal target for regulating tissue repair in chronic inflammatory lung diseases.

Epithelium-derived cystatin SN inhibits house dust mite protease activity in allergic asthma.

BACKGROUND: Allergen source-derived proteases are a critical factor in the formation and development of asthma. The cysteine protease activity of house dust mite (HDM) disrupts the epithelial barrier function. The expression of cystatin SN (CST1) is elevated in asthma epithelium. CST1 inhibits the cysteine protease activity. We aimed to elucidate the role of epithelium-derived CST1 in the development of asthma caused by HDM. METHODS: CST1 protein levels in sputum supernatants and serum of patients with asthma and healthy volunteers were measured by ELISA. The ability of CST1 protein to suppress HDM-induced bronchial epithelial barrier function was examined in vitro. The effects of exogenous CST1 protein on abrogating HDM-induced epithelial barrier function and inflammation were examined in mice in vivo. RESULTS: CST1 protein levels were higher in sputum supernatants (142.4 ± 8.95 vs 38.87 ± 6.85 ng/mL, P < 0.0001) and serum (1129 ± 73.82 vs 703.1 ± 57.02 pg/mL, P = 0.0035) in patients with asthma than in healthy subjects. The levels were significantly higher in patients with not well- and very poorly controlled asthma than those with well-controlled asthma. Sputum and serum CST1 protein levels were negatively correlated with lung function in asthma. CST1 protein levels were significantly lower in the serum of HDM-specific IgE (sIgE)-positive asthmatics than in sIgE-negative asthmatics. The HDM-induced epithelial barrier function disruption was suppressed by recombinant human CST1 protein (rhCST1) in vitro and in vivo. CONCLUSION: Our data indicated that human CST1 protein suppresses asthma symptoms by protecting the asthmatic bronchial epithelial barrier through inhibiting allergenic protease activity. CST1 protein may serve as a potential biomarker for asthma control.

Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma.

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.

Wogonin attenuates neutrophilic inflammation and airway smooth muscle proliferation through inducing caspase-dependent apoptosis and inhibiting MAPK/Akt signaling in allergic airways.

BACKGROUND: Severe neutrophilic asthma is often characterized by persistent airway inflammation and irreversible airway remodeling, which are overstimulated by the high-mobility group box protein 1 (HMGB1). Although wogonin, an O-methylated flavone, has been widely used to treat inflammatory and allergic diseases, its therapeutic effects and potential mechanisms on severe neutrophilic asthma remain elusive. OBJECTIVE: To evaluate whether wogonin alleviates airway neutrophilia through inducing neutrophil apoptosis and attenuates airway smooth muscle cells (ASMCs) proliferation and migration. METHODS: The effect of wogonin on reducing neutrophilic airway inflammation, including neutrophil infiltration and inflammatory mediators, was examined in a mouse model of severe neutrophilic asthma sensitized with ovalbumin and lipopolysaccharide. Also, the effect of wogonin on inducing human neutrophil apoptosis was manifested using cellular morphology, flow cytometry, and caspase inhibition assays. Furthermore, the effect of wogonin on inhibiting HMGB1-mediated ASMCs proliferation and migration was determined. RESULTS: Wogonin reduced the frequency of neutrophils and inhibited the production of multiple inflammatory mediators, including ovalbumin-specific IgE, tumor necrosis factor-α, interleukin-6, and HMGB1, in bronchoalveolar lavage fluid and lung tissues of the neutrophilic asthmatic mouse model. These data strongly support a significantly suppressed neutrophilic airway inflammation, functionally consistent to the relieved airway hyperresponsiveness by wogonin in vivo. Wogonin induced human neutrophil apoptosis in a dose-dependent manner by activating caspase-8 and caspase-3 in vitro. Wogonin pretreatment abolished HMGB1-induced ASMCs proliferation and migration, which can be explained by the inhibition of phosphorylation in the mitogen-activated protein kinase (MAPK) /Akt singling pathways. CONCLUSION: Our findings demonstrate that wogonin augments caspase-dependent apoptosis in neutrophils to alleviate neutrophilic inflammatory responses and regulates intracellular signaling to inhibit HMGB1-mediated ASMCs activation, providing a promising therapeutic agent for severe neutrophilic asthma.

Langerin-expressing dendritic cells in pulmonary immune-related diseases.

Dendritic cells (DCs) are "frontline" immune cells dedicated to antigen presentation. They serve as an important bridge connecting innate and adaptive immunity, and express various receptors for antigen capture. DCs are divided into various subclasses according to their differential expression of cell surface receptors and different subclasses of DCs exhibit specific immunological characteristics. Exploring the common features of each sub-category has became the focus of many studies. There are certain amounts of DCs expressing langerin in airways and peripheral lungs while the precise mechanism by which langerin+ DCs drive pulmonary disease is unclear. Langerin-expressing DCs can be further subdivided into numerous subtypes based on the co-expressed receptors, but here, we identify commonalities across these subtypes that point to the major role of langerin. Better understanding is required to clarify key disease pathways and determine potential new therapeutic approaches.

Chloroquine Attenuates Asthma Development by Restoring Airway Smooth Muscle Cell Phenotype Via the ROS-AKT Pathway.

Switching of airway smooth muscle (ASM) cell phenotype from differentiated-contractile to dedifferentiated-proliferative/synthetic state often occurs in asthmatic subjects with airway dysfunction. Evidence has been provided that chloroquine (an agonist of bitter taste receptors) presented benefits to ASM cell function implicated in asthma. However, the underlying mechanism is unclear. House dust mite (HDM)-sensitized mice were administered with chloroquine or dexamethasone before challenge. BALF and lung tissue were obtained for cell counting, histological analysis or ELISA. Primary cultured ASM cells were stimulated with transforming growth factor (TGF)-ß1 or H2O2. Cells and supernatant were collected for the detection of ASM phenotype, ROS level, and proinflammatory cytokine production. In HDM-sensitized mice, chloroquine attenuated airway hyperresponsiveness (AHR), inflammation and remodeling with an inhibition of immunoglobulin E, IL-4/-13, and TGF-ß1 in BALF. ASM cell proliferation (PCNA), hypertrophy (α-SMA), and parasecretion (MMP-9 and MMP-13) were strongly suppressed by chloroquine, hinting the rebalance of the heterogeneous ASM populations in asthmatic airway. Our data in vitro indicated that chloroquine markedly restrained maladaptive alteration in ASM phenotype in concert with a remission of ROS. Using H2O2 and PI3K inhibitor (LY294002), we found that the inhibition of oxidative stress level and ROS-AKT signal by chloroquine may serve as a potential mechanism that dedicates to the restoration of the phenotypic imbalance in ASM cells. Overall, the present findings suggested that chloroquine improves asthmatic airway function by controlling ASM cell phenotype shift, sketching a novel profile of chloroquine as a new therapeutic candidate for airway remodeling.

Characteristics of alveolar macrophages in bronchioalveolar lavage fluids from active tuberculosis patients identified by single-cell RNA sequencing.

Tuberculosis (TB), is an infectious disease caused by Mycobacterium tuberculosis ( M. tuberculosis), and presents with high morbidity and mortality. Alveolar macrophages play an important role in TB pathogenesis although there is heterogeneity and functional plasticity. This study aimed to show the characteristics of alveolar macrophages from bronchioalveolar lavage fluid (BALF) in active TB patients. Single-cell RNA sequencing (scRNA-seq) was performed on BALF cells from three patients with active TB and additional scRNA-seq data from three healthy adults were established as controls. Transcriptional profiles were analyzed and compared by differential geneexpression and functional enrichment analysis. We applied pseudo-temporal trajectory analysis to investigate correlations and heterogeneity within alveolar macrophage subclusters. Alveolar macrophages from active TB patients at the single-cell resolution are described. We found that TB patients have higher cellular percentages in five macrophage subclusters. Alveolar macrophage subclusters with increased percentages were involved in inflammatory signaling pathways as well as the basic macrophage functions. The TB-increased alveolar macrophage subclusters might be derived from M1-like polarization state, before switching to an M2-like polarization state with the development of M. tuberculosis infection. Cell-cell communications of alveolar macrophages also increased and enhanced in active TB patients. Overall, our study demonstrated the characteristics of alveolar macrophages from BALF in active TB patients by using scRNA-seq.

The structure of blood-tumor barrier and distribution of chemotherapeutic drugs in non-small cell lung cancer brain metastases.

BACKGROUND: Brain metastasis is an important cause of increased mortality in patients with non-small cell lung cancer (NSCLC). In brain metastasis, the blood-brain barrier (BBB) is frequently impaired, forming blood-tumor barrier (BTB). The efficacy of chemotherapy is usually very poor. However, the characteristics of BTB and the impacts of BTB on chemotherapeutic drug delivery remain unclear. The present study investigated the structure of BTB, as well as the distribution of routine clinical chemotherapeutic drugs in both brain and peripheral tumors. METHODS: Bioluminescent image was used to monitor the tumor load after intracranial injection of lung cancer Lewis cells in mice. The permeability of BBB and BTB was measured by fluorescent tracers of evans blue and fluorescein sodium. Transmission electron microscopy (TEM), immunohistochemistry and immunofluorescence were performed to analyze structural differences between BBB and BTB. The concentrations of chemotherapeutic drugs (gemcitabine, paclitaxel and pemetrexed) in tissues were assayed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). RESULTS: Brain metastases exhibited increased BTB permeability compared with normal BBB detected by fluorescence tracers. TEM showed abnormal blood vessels, damaged endothelial cells, thick basement membranes, impaired intercellular endothelial tight junctions, as well as increased fenestrae and pinocytotic vesicles in metastatic lesions. Immunohistochemistry and immunofluorescence revealed that astrocytes were distributed surrounded the blood vessels both in normal brain and the tumor border, but no astrocytes were found in the inner metastatic lesions. By LC-MS/MS analysis, gemcitabine showed higher permeability in brain metastases. CONCLUSIONS: Brain metastases of lung cancer disrupted the structure of BBB, and this disruption was heterogeneous. Chemotherapeutic drugs can cross the BTB of brain metastases of lung cancer but have difficulty crossing the normal BBB. Among the three commonly used chemotherapy drugs, gemcitabine has the highest distribution in brain metastases. The permeability of chemotherapeutic agents is related to their molecular weight and liposolubility.

Melatonin modulates airway smooth muscle cell phenotype by targeting the STAT3/Akt/GSK-3ß pathway in experimental asthma.

Among the troika of clinicopathologic features of asthma, airway remodelling has gained sufficient attention for its contribution to progressive airway narrowing. Much effort has been directed at the management of airway smooth muscle cells (ASMCs), but few attempts have proven to prevent the progression of remodelling. Recently, accumulating data have shown the anti-inflammatory/anti-proliferative potency of melatonin (a crucial neurohormone involved in many physiological and pathological processes) in diverse cells. However, no evidence has confirmed its effect on ASMCs. The present study investigates the benefits of melatonin in asthma, with an emphasis on airway remodelling. The results indicated that melatonin significantly attenuated airway hyperresponsiveness (AHR), inflammation and remodelling in a house dust mite (HDM) model. Melatonin markedly alleviated goblet cell hyperplasia/metaplasia, collagen deposition and airway smooth muscle hyperplasia/hypertrophy, implying the achievement of remodelling remission. The data obtained in vitro further revealed that melatonin notably inhibited ASMCs proliferation, VEGF synthesis and cell migration induced by PDGF, which might depend on STAT3 signalling. Moreover, melatonin remarkably relieved ASMCs contraction and reversed ASMCs phenotype switching induced by TGF-ß, probably via the Akt/GSK-3ß pathway. Altogether, our findings illustrated for the first time that melatonin improves asthmatic airway remodelling by balancing the phenotypic proportions of ASMCs, thus highlighting a novel purpose for melatonin as a potent option for the management of asthma.

Exposure to Ambient Particles Alters the Evolution of Macrophage Phenotype and Amplifies the Inducible Release of Eotaxin-1 in Allergen-Sensitized Mice.

So far, epidemiological data have revealed that the elevation of fine ambient particulate matter (aerodynamic diameter ≤2.5 µm; PM2.5) is closely associated with an exacerbation of asthma while the underlying mechanism is poorly understood. Using a murine model, we investigated the impact of PM2.5 on the development of asthma. Before OVA challenge, mice were administrated with PM2.5, phosphate-buffer saline (PBS) or control filter extracts (CFE). Results showed that compared to PBS or CSF, PM2.5 co-exposure with OVA led to a higher airway hyperresponsiveness (AHR) and a severer eosinophils infiltration. Both alveolar and interstitial macrophages are alternatively activated in OVA-challenged mice with a propensity to M2, marked by arginase-1, CD206, and YM-1. PM2.5 co-exposure dramatically elicited a YM-1 upregulation, implying an aggravated M2 polarization and macrophages recruitment. Eotaxin-1 was predominantly detected in YM-1-positive macrophages, and the level as well as those of IgE, IL-4 or IL-13 was notably increased by PM2.5 co-exposure. With IL-4/IL-13-induced transformation of bone marrow-derived macrophages (BMDM), these M2-polarized macrophages were adoptively transferred into allergic mice. An increase of CD11b+ Siglec+eosinophils was observed in these mice while in vitro, no significant polarization of BMDM was found when treated with PM2.5. Together, our findings suggested that PM2.5 could exacerbate asthma by aggravating M2-polarization, highlighting for the first time that Eotaxin-1 released from M2 macrophages plays a crucial role in asthma pathogenesis.

Glucagon-like peptide-1 receptor activation alleviates lipopolysaccharide-induced acute lung injury in mice via maintenance of endothelial barrier function.

Glucagon-like peptide-1 (GLP-1), which is well known for regulating glucose homeostasis, exhibits multiple actions in cardiovascular disorders and renal injury. However, little is known about the effect of GLP-1 receptor (GLP-1R) activation on acute lung injury (ALI). In this study, we investigated the effect of GLP-1R on ALI and the potential underlying mechanisms with the selective agonist liraglutide. Our results show that GLP-1 levels decreased in serum, though they increased in bronchoalveolar lavage fluid (BALF) and lung tissue in a mouse model of lipopolysaccharide (LPS)-induced ALI. Liraglutide prevented LPS-induced polymorphonuclear neutrophil (PMN) extravasation, lung injury, and alveolar-capillary barrier dysfunction. In cultured human pulmonary microvascular endothelial cells (HPMECs), liraglutide protected against LPS-induced endothelial barrier injury by restoring intercellular tight junctions and adherens junctions. Moreover, liraglutide prevented PMN-endothelial adhesion by inhibiting the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and thereafter suppressed PMN transendothelial migration. Furthermore, liraglutide suppressed LPS-induced activation of Rho/NF-κB signaling in HPMECs. In conclusion, our results show that GLP-1R activation protects mice from LPS-induced ALI by maintaining functional endothelial barrier and inhibiting PMN extravasation. These results also suggest that GLP-1R may be a potential therapeutic target for the treatment of ALI.

The Superior Antitumor Effect of Self-Assembled Paclitaxel Nanofilaments for Lung Cancer Cells.

OBJECTIVES: Paclitaxel (Ptx) has been regarded as one of the most effective chemotherapeutic drugs for lung cancers. Increasing studies focused on the nano-delivery system of Ptx due to its poor solubility and hypersensitivity. The aim of the recent study was to investigate the antitumor effects of self-assembled Ptx nano-filaments for lung cancer cells. METHODS: In the present study, we designed and synthesized novel Ptx-loaded nano-filaments through conjugation of Ptx and succinic acid (SA) (Ptx-SA, P-NFs). Non-small cell lung cancer (NSCLC) A549 and H460 cells were used for detecting the antitumor effects of P-NFs, including cytotoxicity, apoptosis, and migration. Western blotting was performed for analyzing mechanism. RESULTS: P-NFs nano-filaments exerted superior antitumor effects against NSCLC cells compared with free Ptx using cytotoxicity tests. Furthermore, P-NFs nano-filaments were much more effective in inducing NSCLC cells apoptosis and inhibiting A549 cells migration than free Ptx. To elucidate the underlying mechanisms, the expression of apoptotic and endoplasmic reticulum (ER) stress proteins was detected. The results indicated that P-NFs nano-filaments enhanced the expression of bax/bcl-2, protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), phospho- c-Jun N-terminal kinase (p-JNK), and C/EPB homologous protein (CHOP), which suggested that the strong antitumor effect of P-NFs nano-filaments may be partially attributed to the activation ER stress. CONCLUSION: The current work demonstrated that P-NFs nano-filaments showed superior cytotoxicity of lung cancer cells, highlighting a novel profile of nano-filaments delivery systems as potential strategies for facilitating the therapeutic efficacy of Ptx in lung cancer treatment.

Inhibition of H3K27me3 demethylases attenuates asthma by reversing the shift in airway smooth muscle phenotype.

BACKGROUND: The shift in airway smooth muscle cells (ASMCs) phenotype between proliferation and contraction during asthma has been reported recently, highlighting a role of ASMCs plasticity in the pathophysiology of asthma. As an event involved in epigenetic post-translational modification, histone H3 lysine27 (H3K27) demethylation has attracted significant attention with respect to the epigenetic changes in diverse cells; however, little is known about its contribution to the switching of ASMCs phenotype in asthma. OBJECTIVE: To investigate the role of trimethylated H3K27 (H3k27me3) demethylation in ASM remodelling as well as the underling mechanism. METHODS: Mice were exposed five times a week to house dust mite (HDM) extract for 5 weeks. Lung function was measured following the final HDM challenge. Airway inflammation and remodelling were then assessed in lungs of individual mice. Human ASMCs were purchased from Sciencell Research Laboratories. Proliferation, synthesis, migration and contraction of ASMCs were analysed, respectively. RESULTS: We observed demethylation at H3k27me3 sites in lungs harvested from mice exposed to HDM extract. Administration of a selective inhibitor of H3K27 demethylase (GSK-J4) could ameliorate the classical hallmarks of asthma, such as airway hyperresponsiveness, airway inflammation and remodelling. We established a proliferative as well as a contractive model of human ASMCs to explore the impacts of H3K27 demethylase inhibition on ASMCs phenotype. Our results indicated that GSK-J4 decreased ASMCs proliferation and migration elicited by PDGF through the Akt/JNK signalling; GSK-J4 also prevented the upregulation of contractile proteins in ASMCs induced by TGF-ß through the Smad3 pathway. CONCLUSIONS: Inhibition of H3K27me3 demethylation alleviated the development of asthmatic airway disease in vivo and modulated ASMCs phenotype in vitro. Collectively, our findings highlight a role of H3K27me3 demethylation in experimental asthma and ASMCs phenotype switch.

New dynamic viewing of mast cells in pulmonary arterial hypertension (PAH): contributors or outsiders to cardiovascular remodeling.

BACKGROUND: In patients with pulmonary arterial hypertension (PAH), mast cells (MCs) are extensively observed around pulmonary vessels. However, their temporal and spatial variation during PAH development remains obscure. This study investigated the dynamic evolution of MCs in lungs and right ventricles (RV) to illuminate their role in pulmonary vascular and RV remodeling. METHODS: The PAH model was established by a single intra-peritoneal injection of monocrotaline (MCT, 60 mg/kg) in rats. On day 0, 3, 7, 14, and 28 after MCT injection, lung and RV tissues were harvested for staining with hematoxylin and eosin (HE), Gomori aldehyde fuchsin (GAF), toluidine blue (TB) and picrosirius red (PSR). Immunohistochemistry was performed to evaluate the levels of α-SMA, CD68 and tryptase. A simple RV remolding model was produced as well by pulmonary artery banding (PAB). RV tissues were collected to determine the degree of MCs infiltration. RESULTS: After MCT challenge, elevated mean pulmonary arterial pressure (mPAP), increased RV systolic pressure (RVSP), pulmonary arterial media hypertrophy as well as distal vascular muscularization gradually occurred with time. MCs recruitment along with CD68+ macrophages accumulation was observed around distal pulmonary vessels and in alveolar septa. Excessive infiltration and degranulation of MCs were detected in MCT-treated group in lung tissues but not in RV. In addition, no exacerbation of MCs infiltration and degranulation in RV was noted in PAB-treated rats, suggesting few contributions of MCs to RV remodeling. CONCLUSIONS: Our findings implied a crucial role of MCs in the remodeling of pulmonary vessels, not RV, which probably through releasing cytokines such as tryptase. The present study enriches the knowledge about PAH, providing a potential profile of MCs as a switch for the treatment of PAH.

AQP4­knockout alleviates the lipopolysaccharide­induced inflammatory response in astrocytes via SPHK1/MAPK/AKT signaling.

To date, aquaporin­4 (AQP4) has been considered as a critical contributor to neuroinflammation, but little is known about the underlying mechanism. Previous studies have shown that a critical enzyme involved in the sphingomyelin cycle, sphingosine kinase 1 (SPHK1), is implicated in inflammatory processes and contributes to chronic neuroinflammation. The present study investigated the role of AQP4 in proinflammatory cytokine release from astrocytes, with an emphasis on the SPHK1/mitogen­activated protein kinase (MAPK)/protein kinase B (AKT) pathway. Using primary cultures isolated from AQP4+/+ and AQP4­/­ embryos, the production of tumor necrosis factor­α (TNF­α)/interleukin­6 (IL­6) from astrocytes challenged by lipopolysaccharide (LPS) was compared. The results showed increased secretion of TNF­α/IL­6 in the two groups following LPS treatment, but a significantly lower level was observed in the AQP4­/­ group compared with that in the AQP4+/+ group. Although upregulation of SPHK1 was detected in the two genotypes, only a mild increase in SPHK1 was found in the AQP4­/­ genotype. The phosphorylation of MAPK/AKT was also confirmed to be attenuated in the AQP4­/­ group, suggesting decreased MAPK/AKT signaling over time in AQP4­/­ astrocytes. Overall, the study findings demonstrated that AQP4 deficiency alleviates proinflammatory cytokine release from astrocytes, in association with the SPHK1/MAPK/AKT pathway. This data improves our understanding of AQP4 in neuroinflammatory events, highlighting a novel profile of SPHK1 as a potential target for the treatment of CNS inflammation.

Hypoxia induces the dysfunction of human endothelial colony-forming cells via HIF-1α signaling.

Endothelial injury is considered as a trigger of pulmonary vascular lesions in the pathogenesis of hypoxic pulmonary hypertension (HPH). Although endothelial colony-forming cells (ECFCs) have vascular regeneration potential to maintain endothelial integrity, hypoxia-induced precise alteration in ECFCs function remains controversial. This study investigated the impact of hypoxia on human ECFCs function in vitro and the underlying mechanism. We found that hypoxia inhibited ECFCs proliferation, migration and angiogenesis. Compared with no treatment, the expression of hypoxia inducible factor-1α (HIF-1α) in hypoxia-treated ECFCs was increased, with an up-regulation of p27 and a down-regulation of cyclin D1. The over-secreted vascular endothelial growth factor (VEGF) was detected, with the imbalanced expression of fetal liver kinase 1 (flk-1) and fms related tyrosine kinase 1 (flt-1). Hypoxia-induced changes in ECFCs could be reversed by HIF-1α inhibitor KC7F2. These data suggest that HIF-1α holds the key in regulating ECFCs function which may open a new perspective of ECFCs in HPH management.

Oligomeric proanthocyanidins attenuate airway inflammation in asthma by inhibiting dendritic cells maturation.

To date, although a promising anti-inflammatory activity of oligomeric proanthocyanidins (OPCs) has been observed in asthma, the mechanism responsible for these immunomodulatory properties remains obscure. Dendritic cells (DCs) that reside in the airway have been widely perceived as an important contributor to asthma. Our study was to demonstrate OPCs' effects on maturation and immunoregulation of pulmonary CD11c+ dendritic cells (DCs). BALB/c mice were exposed to ovalbumin (OVA) to induce murine model of asthma. In addition, pulmonary DCs and bone marrow-derived DCs (BMDCs) cultures were used to evaluate impacts of OPCs on DCs function. The results obtained here indicated that OPCs treatment dramatically reduced airway inflammation, such as the infiltration of inflammatory cells and the levels of allergen-specific serum IgE and Th2 cytokines. The expression of co-stimulatory molecules especially CD86 distributed on pulmonary DCs and bone marrow-derived DCs (BMDCs) also markedly declined. The phosphorylation of cAMP responsive element-binding protein (CREB) was significantly inhibited while no changes were observed in the expression of cAMP responsive element modulator (CREM). By transferring BMDCs into the airways of naïve mice, we found that OPCs-treated DCs (DC+OVA+OPC) were much less potent in promoting CD4+ T cells proliferation than OVA-pulsed DCs (DC+OVA), followed by the ameliorated eosinophilic inflammation in airway. Our findings tailor a novel profile of OPCs in the regulation of DCs function, shedding new light on the therapeutic potential of OPCs in asthma management.

Foretinib Enhances the Radiosensitivity in Esophageal Squamous Cell Carcinoma by Inhibiting Phosphorylation of c-Met.

As a crucial event involved in the metastasis and relapse of esophageal cancer, c-Met overexpression has been considered as one of the culprits responsible for the failure in patients who received radiochemotherapy. Since c-Met has been confirmed to be pivotal for cell survival, proliferation and migration, little is known about its impact on the regulation of radiosensitivity in esophageal cancer. The present study investigated the radiosensitization effects of c-Met inhibitor foretinib in ECA-109 and TE-13 cell lines. Foretinib inhibited c-Met signaling in a dose-dependent manner resulting in decreases in the cell viability of ECA-109 and TE-13. Pretreatment with foretinib synergistically prompted cell apoptosis and G2/M arrest induced by irradiation. Moreover, decreases ability of DNA damage repair was also observed. In vivo studies confirmed that the combinatorial use of foretinib with irradiation significantly diminishes tumor burden compared to either treatment alone. The present findings implied a crucial role of c-Met in the modulation of radiosensitization in esophageal cancer, and foretinib increased the radiosensitivity in ECA-109 and TE-13 cells mainly via c-Met signaling, highlighting a novel profile of foretinib as a potential radiosensitizer for the treatment of esophageal cancer.

The effect of tetrandrine combined with cisplatin on proliferation and apoptosis of A549/DDP cells and A549 cells.

BACKGROUND: Non-small cell lung cancer comprises the majority of lung cancer cases and is insensitive to chemotherapy. Most patients develop drug resistance. Recently, tetrandrine (TET), a bis-benzylisoquinoline alkaloid, was identified as a novel anti-cancer agent. However, the effect of tetrandrine combined with cisplatin on lung cancer has not yet been studied. We aimed to identify a possible synergistic effect between tetrandrine and cisplatin, besides, to investigate the effects of TET in combination with DDP on proliferation and apoptosis in cisplatin-resistant and cisplatin-sensitive A549 cell lines, and to study the underlying mechanism. METHODS: Cell viability was confirmed with CCK8 assays, and the IC50 values for each treatment group were calculated. The synergistic interaction of these drugs was evaluated using an isobolographic analysis. Proliferation was assessed by EDU staining. Hoechst staining and flow cytometry were used to assess apoptosis. Apoptosis- and autophagy-associated proteins were analyzed by western blot. Transmission electron microscopy was used to detect autophagy, RFP-GFP-LC3 lentivirus was used to perform autophagic flux assay. RESULTS: Tetrandrine and cisplatin exerted synergistic cytotoxic effects on both cisplatin-resistant and cisplatin-sensitive A549 cell lines. The combination of tetrandrine and cisplatin induced apoptosis and inhibited proliferation in a synergistic manner. The formation of autophagosomes was evident by transmission electron microscopy. The autophagic flux of combination treatment was increased. CONCLUSIONS: Tetrandrine synergized with cisplatin to reduce the viability of cisplatin-resistant and cisplatin-sensitive A549 cells, tetrandrine could reverse the resistance of A549 cells to cisplatin. Tetrandrine combined with cisplatin could induce autophagy. Therefore, tetrandrine is a potent autophagy agonist and may be a promising drug for the treatment of non-small cell lung cancer.