Myrtenol Inhibits Biofilm Formation and Virulence in the Drug-Resistant Acinetobacter baumannii: Insights into the Molecular Mechanisms.

Background: blaNDM-1-producing Acinetobacter baumannii (BP-AB) remains a critical problem in nosocomial infections, because of its resistance mediated by the biofilm formation and virulence factors. No studies have confirmed myrtenol's efficacy in inhibiting the biofilm formation and virulence associated with biofilm of BP-AB. Methods: The tested concentrations of myrtenol were wild type (A), 50 µg/mL (B), 100 µg/mL (C), 200 µg/mL (D), 250 µg/mL (E), and 300 µg/mL (F). Results: The BP-AB biofilm inhibition was significantly higher in the D, E, and F groups than in the A, B, and C groups. Myrtenol significantly reduced the air-liquid interface ring formation in glass tubes. It also effectively inhibited the attachment of BP-AB strains on polystyrene surfaces as shown by crystal violet staining. Microscopy showed a significant reduction in biofilm formation with dispersed BP-AB strains. The confocal laser scanning microscopy analysis showed a significant reduction in the biofilm's biomass, covered surface area, and thickness. The scanning electron microscopy analysis revealed significantly fewer BP-AB aggregates on the coverslip surface. In the CompStat analysis, the biofilm's biomass, maximum thickness, and surface-to-volume ratio were significantly reduced. The qPCR analysis revealed a significant down-regulation of bfmR, bap, csuA/B, and ompA expression, which positively correlated with the biofilm's biomass, maximum thickness, and surface-to-volume ratio in BP-AB strains. Myrtenol significantly improved the susceptibility of BP-AB to the antibiotics amikacin, piperacillin/tazobactam, cefoperazone/sulbactam, and ceftazidime. Conclusion: Myrtenol attenuates the BP-AB biofilm formation and virulence by suppressing the expression of bfmR, bap, csuA/B, and ompA.

A cohort autopsy study defines COVID-19 systemic pathogenesis.

Severe COVID-19 disease caused by SARS-CoV-2 is frequently accompanied by dysfunction of the lungs and extrapulmonary organs. However, the organotropism of SARS-CoV-2 and the port of virus entry for systemic dissemination remain largely unknown. We profiled 26 COVID-19 autopsy cases from four cohorts in Wuhan, China, and determined the systemic distribution of SARS-CoV-2. SARS-CoV-2 was detected in the lungs and multiple extrapulmonary organs of critically ill COVID-19 patients up to 67 days after symptom onset. Based on organotropism and pathological features of the patients, COVID-19 was divided into viral intrapulmonary and systemic subtypes. In patients with systemic viral distribution, SARS-CoV-2 was detected in monocytes, macrophages, and vascular endothelia at blood-air barrier, blood-testis barrier, and filtration barrier. Critically ill patients with long disease duration showed decreased pulmonary cell proliferation, reduced viral RNA, and marked fibrosis in the lungs. Permanent SARS-CoV-2 presence and tissue injuries in the lungs and extrapulmonary organs suggest direct viral invasion as a mechanism of pathogenicity in critically ill patients. SARS-CoV-2 may hijack monocytes, macrophages, and vascular endothelia at physiological barriers as the ports of entry for systemic dissemination. Our study thus delineates systemic pathological features of SARS-CoV-2 infection, which sheds light on the development of novel COVID-19 treatment.

The Degradation of Airway Epithelial Tight Junctions in Asthma Under High Airway Pressure Is Probably Mediated by Piezo-1.

Full functioning of the airway physical barrier depends on cellular integrity, which is coordinated by a series of tight junction (TJ) proteins. Due to airway spasm, edema, and mucus obstruction, positive end-expiratory alveolar pressure (also termed auto-PEEP) is a common pathophysiological phenomenon, especially in acute asthma attack. However, the influence of auto-PEEP on small airway epithelial TJs is currently unclear. We performed studies to investigate the effect of extra pressure on small airway epithelial TJs and its mechanism. The results first confirmed that a novel mechanosensitive receptor, piezo-1, was highly expressed in the airway epithelium of asthmatic mice. Extra pressure induced the degradation of occludin, ZO-1 and claudin-18 in primary human small airway epithelial cells (HSAECs), resulting in a decrease in transepithelial electrical resistance (TER) and an increase in cell layer permeability. Through in vitro investigations, we observed that exogenous pressure stimulation could elevate the intracellular calcium concentration ([Ca2+] i ) in HSAECs. Downregulation of piezo-1 with siRNA and pretreatment with BAPTA-AM or ALLN reduced the degradation of TJs and attenuated the impairment of TJ function induced by exogenous pressure. These findings indicate the critical role of piezo-1/[Ca2+] i /calpain signaling in the regulation of small airway TJs under extra pressure stimulation.

Single inhaler triple therapy (FF/UMEC/VI) versus FF/VI and UMEC/VI in patients with COPD: subgroup analysis of the China cohort in the IMPACT trial.

OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is becoming a leading cause of morbidity and mortality in China. In the IMPACT trial, fluticasone furoate[FF]/umeclidinium[UMEC]/vilanterol[VI] single-inhaler triple therapy demonstrated lower rates of moderate/severe exacerbations than dual therapy with FF/VI or UMEC/VI in patients with symptomatic COPD and a history of exacerbations. This analysis investigates the China cohort and its consistency with the overall ITT population. METHODS: 10,355 patients were randomized 2:2:1 to once-daily FF/UMEC/VI 100/62.5/25 µg, FF/VI 100/25 µg, or UMEC/VI 62.5/25 µg for 52 weeks. Endpoints included: annual rates of exacerbations, time-to-first on-treatment moderate/severe exacerbation and change from baseline in trough forced expiratory volume in 1 s (FEV1) at Week-52. Clinical trial registration is NCT02164513 (CTT116855). RESULTS: 535 patients (5.2%) were from China. Annual on-treatment moderate/severe exacerbation rate was 0.81 with FF/UMEC/VI versus 0.96 with FF/VI (rate ratio: 0.84; 95% confidence interval [CI]: 0.64, 1.11; p = .227) and 0.80 with UMEC/VI (rate ratio: 1.02; 95% CI: 0.72, 1.44; p = .929). Hazard ratio for time-to-first moderate/severe exacerbation was 0.84 (95% CI: 0.63, 1.11; p = .218) for FF/UMEC/VI versus FF/VI and 0.89 (95% CI: 0.62, 1.27; p = .516) versus UMEC/VI. Significant improvements in mean change from baseline in trough FEV1 were observed for FF/UMEC/VI versus FF/VI (treatment difference 137 mL; 95% CI: 86, 188; p < .001) and UMEC/VI (63 mL; 0, 125; p = .050). Health status was improved with FF/UMEC/VI versus both dual therapies. Results were similar to the overall ITT population. No new safety signals were identified. CONCLUSIONS: Single-inhaler triple therapy with FF/UMEC/VI versus FF/VI or UMEC/VI reduced the rate and risk of exacerbations, and improved lung function and health status in the China cohort similar to the overall ITT population. No new safety signals were identified.

The role of the XBP-1/AGR2 signaling pathway in the regulation of airway Mucin5ac hypersecretion under hypoxia.

Oversecretion of Mucin5ac (MUC5AC), which is primarily synthesized by goblet cells and is the major gel-forming mucin, is a hallmark of various pulmonary inflammatory diseases. Hypoxia is considered a common pathophysiologic feature in various pulmonary inflammatory diseases. It has been suggested that hypoxia-inducible factor 1α (HIF-1α) acts as a key factor in hypoxia-induced MUC5AC hypersecretion; however, the exact mechanisms that maintain the stability of HIF-1α and support oversecretion by airway epithelial cells under hypoxia are still unclear. With immunohistochemistry, we found overexpression of anterior gradient 2 (AGR2) in the bronchial epithelial cells of hypoxia-treated mice. With specific shRNA transduction, AGR2 was demonstrated to be a key factor in MUC5AC hypersecretion in vitro. Additionally, co-immunoprecipitation, cell immunochemistry and confocal microscopy experiments were performed to explore the interaction between HIF-1α and AGR2 during hypoxia-induced MUC5AC hypersecretion in vitro. The results indicated increased binding and intracytoplasmic colocation of HIF-1α and AGR2. Our findings suggest that AGR2 acts as a key regulator in hypoxia-induced airway MUC5AC hypersecretion by increasing the stability of HIF-1α. Additionally, the elevated expression of AGR2 induced by hypoxia in bronchial epithelial cells likely depends on an XBP-1-associated pathway.

Munc13­4 mediates human neutrophil elastase­induced airway mucin5AC hypersecretion by interacting with syntaxin2.

The overexpression and hypersecretion of mucus is a hallmark of chronic pulmonary inflammatory disease. Mucin5AC (MUC5AC) is a major component of airway gel­forming mucin. Members of the Unc13 (Munc13) protein family act as important activators of granule exocytosis from various types of mammalian cells. The present study aimed to determine the role of Munc13 family proteins in MUC5AC secretion via an in vitro study with BEAS­2B and Calu­3 cell lines. Reverse transcription­quantitative polymerase chain reaction and western blotting indicated that stimulation of the cells with 100 nM human neutrophil elastase (hNE) for 1 h did not affect the expression of either unc13 homolog B (Munc13­2) or unc13 homolog D (Munc13­4), but immunofluorescence analysis demonstrated that hNE treatment was associated with the recruitment of Munc13­4 to the plasma membrane. Co­immunoprecipitation analysis indicated increased binding between Munc13­4 and syntaxin2 followingh NE stimulation; however, Munc13­2 formed a stable interaction with syntaxin2 with or without hNE stimulation. Subsequently, Munc13­2 and Munc13­4 expression levels were downregulated in BEAS­2B and Calu­3 cells using small interfering RNA (siRNA). ELISAs and immunofluorescence analysis were performed to assess MUC5AC secretion and intracellular retention, respectively. Munc13­2 siRNA transfection did not alter the expression levels of intracellular or secreted MUC5AC following hNE stimulation in either cell line; however, it increased the baseline intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC. Conversely, Munc13­4 siRNA transfection increased the intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC following hNE stimulation, but did not affect their baseline quantities. The results of the present study indicate that Munc13­2 may be an essential regulator of basal MUC5AC exocytosis, while Munc13­4 appears to be a Munc13 protein subtype that may to be sensitive to hNE stimulation during airway MUC5AC hypersecretion.

Bisdemethoxycurcumin sensitizes cisplatin-resistant lung cancer cells to chemotherapy by inhibition of CA916798 and PI3K/AKT signaling.

Curcumin, a dietary supplement or herbal medicine from Curcuma longa, has shown antitumor activity in different cancer cell lines and clinical trials. CA916798, a novel protein, is overexpressed in multidrug-resistant tumor cells. This study aimed to assess the effects of curcumin on regulating chemosensitivity in cisplatin-resistant non-small cell lung cancer (NSCLC) cells in vitro and to explore the underlying molecular mechanisms. Human cisplatin-sensitive A549 and cisplatin-resistant A549/CDDP lung adenocarcinoma cells were treated with curcumin to assess cell viability and gene modulations using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. CA916798 shRNA and point mutations were used to assess the CA916798 functions and phosphorylation sites. Bisdemethoxycurcumin sensitized cisplatin-resistant lung cancer cells to various chemotherapeutic agents, including cisplatin. Bisdemethoxycurcumin reduced the levels of CA916798 mRNA and protein in A549 and A549/CDDP cells, while it also suppressed phosphatidylinositol-3-kinase (PI3K)/AKT signaling. CA916798, as a downstream gene, interacted with AKT after bisdemethoxycurcumin treatment in A549 and A549/CDDP cells. Moreover, A549/CDDP cells expressing the point-mutated CA916798-S20D protein were more resistant to cisplatin and bisdemethoxycurcumin, whereas tumor cells expressing CA916798-S20A, CA916798-S31A, CA916798-S60A, CA916798-S93A, or CA916798-T97A (different sites of amino acid phosphorylation) showed similar sensitivity or resistance to cisplatin and bisdemethoxycurcumin, compared with the control cells. Bisdemethoxycurcumin is able to sensitize cisplatin-resistant NSCLC cells to chemotherapeutic agents by inhibition of CA916798 and PI3K/AKT activities. Moreover, phosphorylation of CA916798 at the S20 residue plays a critical role in mediating bisdemethoxycurcumin antitumor activity.

Bombesin receptor-activated protein regulates neutrophil elastase-induced mucin5AC hypersecretion in human bronchial epithelial cells.

Bombesin receptor-activated protein (BRAP) is highly expressed in human bronchial epithelial cells. Recent studies have shown that BRAP reduces oxidative stress, inhibits airway inflammation and suppresses nuclear factor kappaB (NF-κB) activity. Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. Neutrophil elastase (NE) is a potent inducer of mucin5AC (MUC5AC), which is considered the predominant mucin secreted by human airway epithelial cells. Here, we hypothesize that BRAP may regulate NE-induced MUC5AC hypersecretion in a bronchial epithelial cell line (HBE16). We also investigated the underlying mechanism involved in the process. In this study, we found that BRAP was present in HBE16 human bronchial epithelial cells and was significantly increased by NE. Next, we found that the up-regulation of BRAP by pEGFP-N1-BRAP caused a significant decrease in the increased levels of MUC5AC expression, NF-κB activity, and the phosphorylation of extracellular signal-regulated kinases (ERK) and epidermal growth factor receptor (EGFR) induced by NE. Meanwhile, there was a significant decrease in ROS, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) levels when BRAP was up-regulated by pEGFP-N1-BRAP. Moreover, when cells were transfected with pEGFP-N1-BRAP and pretreated with NF-κB, ERK or EGFR inhibitors before the NE stimulation, there were further decreased in MUC5AC expression, NF-κB activity, and the phosphorylation of ERK and EGFR. These results suggest that BRAP plays an important role in airway inflammation and its overexpression may regulate NE-induced MUC5AC hypersecretion in HBE16 cells via the EGFR/ERK/NF-κB signaling pathway.

Saxifragifolin D attenuates phagosome maturation arrest in Mycobacterium tuberculosis-infected macrophages via an AMPK and VPS34-dependent pathway.

Saxifragifolin D (SD), a traditional Chinese medicine, is a pentacyclic triterpenoid compound first isolated from Androsace umbellata. Various plant triterpenoids have been reported to exhibit antitubercular activity. In this study, THP-1-derived macrophages were infected with an attenuated M. tuberculosis (M.tb) strain, H37Ra. Intracellular replication of M.tb was evaluated by counting the colonies after 4 weeks of incubation. The results indicated that SD treatment reduced the intracellular replication of M.tb in THP-1-derived macrophages but not in A549 cells. We performed a phagosome maturation test using confocal microscopy and found that SD treatment partially attenuated the phagosome arrest induced by M.tb infection. These effects were dependent on a VPS34-associated pathway. Immunoprecipitation assays showed that SD increased intracellular UVRAG-linked VPS34, the active VPS34 complex II. However, SD had no effect on the total VPS34 pool. Moreover, the results indicated that the SD-mediated increase in VPS34 complex II activity was mediated by an AMPK-dependent pathway. Collectively, these data indicate that SD may be a promising candidate for treatment of M.tb.

Inhibition of autophagy enhances heat-induced apoptosis in human non-small cell lung cancer cells through ER stress pathways.

The occurrence and mechanisms of autophagy induced by heat stress are not well known in lung cancer cells. Here, we have demonstrated that heat stress induces autophagy in A549 and NCI-H460 cells through morphological and biochemical analyses. The inhibition of autophagy by chloroquine, 3-methyladenine and Beclin 1 siRNA enhanced heat-induced apoptosis. Moreover, the combination of chloroquine and heat stress inhibited tumor growth and enhanced apoptosis in vivo experiments. In addition, heat-induced autophagy involved the ER stress pathway (PERK- or IRE1-dependent). Further, heat treatment led to the increased phosphorylation of AMPK and the decreased phosphorylation of mTOR in vitro and in vivo. Knockdown of GRP78 inhibited the AMPK-mTOR pathway, and the AMPK inhibitor compound C decreased heat-induced autophagy, suggesting that activation of ER stress was involved in autophagy induction and promotion of the AMPK-mTOR pathway. In conclusion, our data suggested that the heat treatment of lung cancer cells triggered protective autophagy, as mediated by ER stress. Thus, inhibition of autophagy can be a promising strategy to enhance hyperthermia in the treatment of lung cancer patients.

Acid-induced autophagy protects human lung cancer cells from apoptosis by activating ER stress.

An acidic tumor microenvironment exists widely in solid tumors. However, the detailed mechanism of cell survival under acidic stress remains unclear. The aim of this study is to clarify whether acid-induced autophagy exists and to determine the function and mechanism of autophagy in lung cancer cells. We have found that acute low pH stimulated autophagy by increasing LC3-positive punctate vesicles, increasing LC3 II expression levels and reducing p62 protein levels. Additionally, autophagy was inhibited by the addition of Baf or knockdown of Beclin 1, and cell apoptosis was increased markedly. In mouse tumors, the expression of cleaved caspase3 and p62 was enhanced by oral treatment with sodium bicarbonate, which can raise the intratumoral pH. Furthermore, the protein levels of ER stress markers, including p-PERK, p-eIF2α, CHOP, XBP-1s and GRP78, were also increased in response to acidic pH. The antioxidant NAC, which reduces ROS accumulation, alleviated acid-mediated ER stress and autophagy, and knocking down GRP78 reduced autophagy activation under acidic conditions, which suggests that autophagy was induced by acidic pH through ER stress. Taken together, these results indicate that the acidic microenvironment in non-small cell lung cancer cells promotes autophagy by increasing ROS-ER stress, which serves as a survival adaption in this setting.

Role of Wnt Inhibitory Factor-1 in Inhibition of Bisdemethoxycurcumin Mediated Epithelial-to-Mesenchymal Transition in Highly Metastatic Lung Cancer 95D Cells.

BACKGROUND: Bisdemethoxycurcumin (BDMC) is an active component of curcumin and a chemotherapeutic agent, which has been suggested to inhibit tumor growth, invasion and metastasis in multiple cancers. But its contribution and mechanism of action in invasion and metastasis of non-small cell lung cancer (NSCLC) are not very clear. Therefore, we tried to study the effects of BDMC on regulation of epithelial-to-mesenchymal transition (EMT), which is closely linked to tumor cell invasion and metastasis. METHODS: In this study, we first induced transforming growth factor-ß1 (TGF-ß1) mediated EMT in highly metastatic lung cancer 95D cells. Thereafter, we studied the effects of BDMC on invasion and migration of 95D cells. In addition, EMT markers expressions were also analyzed by western blot and immunofluorescence assays. The contribution of Wnt inhibitory factor-1 (WIF-1) in regulating BDMC effects on TGF-ß1 induced EMT were further analyzed by its overexpression and small interfering RNA knockdown studies. RESULTS: It was observed that BDMC inhibited the TGF-ß1 induced EMT in 95D cells. Furthermore, it also inhibited the Wnt signaling pathway by upregulating WIF-1 protein expression. In addition, WIF-1 manipulation studies further revealed that WIF-1 is a central molecule mediating BDMC response towards TGF-ß1 induced EMT by regulating cell invasion and migration. CONCLUSIONS: Our study concluded that BDMC effects on TGF-ß1 induced EMT in NSCLC are mediated through WIF-1 and elucidated a novel mechanism of EMT regulation by BDMC.

Autophagy Accompanied with Bisdemethoxycurcumin-induced Apoptosis in Non-small Cell Lung Cancer Cells.

OBJECTIVE: To investigate the effects of bisdemethoxycurcumin (BDMC) on non-small cell lung cancer (NSCLC) cell line, A549, and the highly metastatic lung cancer 95D cells. METHODS: CCK-8 assay was used to assess the effect of BDMC on cytotoxicity. Flow cytometry was used to evaluate apoptosis. Western blot analysis, electron microscopy, and quantification of GFP-LC3 punctuates were used to test the effect of BDMC on autophagy and apoptosis of lung cancer cells. RESULTS: BDMC inhibited the viability of NSCLC cells, but had no cytotoxic effects on lung small airway epithelial cells (SAECs). The apoptotic cell death induced by BDMC was accompanied with the induction of autophagy in NSCLC cells. Blockage of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) repressed the growth inhibitory effects and induction of apoptosis by BDMC. In addition, BDMC treatment significantly decreased smoothened (SMO) and the transcription factor glioma-associated oncogene 1 (Gli1) expression. Furthermore, depletion of Gli1 by siRNA and cyclopamine (a specific SMO inhibitor) induced autophagy. CONCLUSION: Aberrant activation of Hedgehog (Hh) signaling has been implicated in several human cancers, including lung cancers. The present findings provide direct evidence that BDMC-induced autophagy plays a pro-death role in NSCLC, in part, by inhibiting Hedgehog signaling.

Discriminating Lamiophlomis rotata According to Geographical Origin by (1)H-NMR Spectroscopy and Multivariate Analysis.

INTRODUCTION: Lamiophlomis rotata (Duyiwei) is a folk herbal medicine that traditionally has been used in China as a hemostatic agent. Raw plant materials used for medicinal products from different geographical regions are often inconsistent in chemical composition. Metabolic fingerprinting provides a new approach for distinguishing the geographical origins of L. rotata. OBJECTIVE: To identify metabolites that contribute to the different geographical regions of L. rotata samples. METHODS: Lamiophlomis rotata metabolomics were performed by (1)H-nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analyses. The L. rotata metabolic profile was prepared for NMR measurements using methanol-d4 solvent. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to analyse the L. rotata (1)H-NMR spectroscopy data. RESULTS: Nine iridoid glycosides, one flavonoid and three phenylpropanoid glycosides were detected in L. rotata by (1)H-NMR spectroscopy. (1)H-NMR measurements and multivariate analysis were used to successfully discriminate samples from three different locations. CONCLUSION: The NMR-based analysis of L. rotata is a more comprehensive approach than traditional chromatographic methods. Simple sample preparation, rapidity and reproducibility of are additional advantages of NMR analysis.

Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells.

BACKGROUND/AIM: Increased mucin secretion is a characteristic feature of many chronic airway diseases, particularly during periods of exacerbation; however, the exact mechanism of mucin secretion remains unclear. Ezrin, which is a specific marker of apical membranes, is predominantly concentrated in exocyst-rich cell surface structures, crosslinking the actin cytoskeleton with the plasma membrane. In the present study, we examined whether Ezrin is involved in mucin 5AC (MUC5AC) secretion after neutrophil elastase (NE) attack, and we investigated the role of the exocyst complex docking protein Sec3 in this process. METHODS: NE was used as a stimulator in a 16HBE14o- cell culture model. The expression and location of Ezrin and Sec3 were investigated, and the interaction between Ezrin and Sec3 in 16HBE14o-cells was assayed after treatment with NE, Ezrin siRNA, Sec3 siRNA, neomycin or PIP2-Ab. RESULTS: We found that Ezrin was highly expressed in the bronchi of humans with chronic airway diseases. NE induced robust MUC5AC protein secretion. The Ezrin siRNA, Sec3 siRNA, and neomycin treatments led to impaired MUC5AC secretion in cells. Both Ezrin and Sec3 were recruited primarily to the cytoplasmic membrane after NE stimulation, and the neomycin and PIP2-Ab treatments abrogated this effect. Immunoprecipitation analysis revealed that Ezrin and Sec3 combined to form complexes; however, these complexes could not be detected in Ezrin∆1-333 mutant-transfected cells, even when PIP2 was added. CONCLUSIONS: These results demonstrate that Ezrin/Sec3 complexes are essential for MUC5AC secretion in NE-stimulated airway epithelial cells and that PIP2 is of critical importance in the formation of these complexes.

Metabolic discrimination of Swertia mussotii and Swertia chirayita known as "Zangyinchen" in traditional Tibetan medicine by (1)H NMR-based metabolomics.

Swertia mussotii Franch. and Swertia chirayita Buch.-Ham. have been commonly used under the same name "Zangyinchen" for the treatment of liver and gallbladder diseases in traditional Tibetan medicine. Detailed characterization and comparison of the complete set of metabolites of these two species are critical for their objective identification and quality control. In this study, a rapid, simple and comprehensive (1)H NMR-based metabolomics method was first developed to differentiate the two species. A broad range of metabolites, including iridoid glycosides, xanthones, triterpenoids, flavonoids, carbohydrates, and amino acids, were identified. Statistical analysis showed evident differences between the two species, and the major markers responsible for the differences were screened. In addition, quantitative (1)H NMR method (qHNMR) was used for the target analysis of the discriminating metabolites. The results showed that S. mussotii had significantly higher contents of gentiopicrin, isoorientin, glucose, loganic acid, and choline, whereas S. chirayita exhibited higher levels of swertiamarin, oleanolic acid, valine, and fatty acids. These findings indicate that (1)H NMR-based metabolomics is a reliable and effective method for the metabolic profiling and discrimination of the two Swertia species, and can be used to verify the genuine origin of Zangyinchen.

Increased pro-angiogenic factors, infiltrating neutrophils and CD163(+) macrophages in bronchoalveolar lavage fluid from lung cancer patients.

Infiltration of inflammatory cells and production of pro-angiogenic factors are important in lung cancer immunity. The distributions of those cells and their contributions to the production of pro-angiogenic factors and the activation phenotype of macrophages in bronchoalveolar lavage fluid (BALF) from lung cancer patients remain unclear. We analyzed the presence of distinct inflammatory cells and the macrophage activation phenotype together with the levels of vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8) within BALF from 54 smoking lung cancer patients including 36 squamous cell carcinoma (SCC), 9 adenocarcinoma (AC), and 9 small cell lung cancer (SCLC) in comparison with those from 13 non-smoking and 7 smoking patients with nonspecific chronic inflammation and 8 non-smoking normal controls. We found a significantly lower percentage of total macrophages and a much higher percentage of neutrophils among all inflammatory cells in BALF from lung cancer and non-specific chronic inflammation patients. BALF from AC patients had a significantly higher percentage of lymphocytes. CD163(+)) macrophages predominantly existed in BALF from SCLC patients. BALF of lung cancer patients had markedly higher levels of IL-8 and VEGF. Interestingly, IL-8 level was positively correlated to the numbers of neutrophils and lymphocytes. VEGF level was inversely correlated to the number of lymphocytes but positively to cancer cells in SCC cases, whereas no correlation existed between CD163(+)) macrophages and the levels of IL-8 and VEGF. Our results suggest that the detection of infiltrating inflammatory cells and pro-angiogenic factors in BALF will be helpful for diagnosis of cancerous inflammation in lungs.

EMT and acquisition of stem cell-like properties are involved in spontaneous formation of tumorigenic hybrids between lung cancer and bone marrow-derived mesenchymal stem cells.

The most deadly phase in cancer progression is metastatic conversion. Epithelial-to-mesenchymal transition (EMT) is a key process by which cancer cells acquire invasive and metastatic phenotypes. In order to spawn macroscopic metastases, disseminated cancer cells would seem to require self-renewal capability. However, the underlying mechanism defining these processes is poorly understood. One possible mechanism underlying metastasis is fusion between myeloid cells and cancer cells. In this study, we found that spontaneously-formed tumorigenic hybrids between bone marrow-derived mesenchymal stem cells (MSCs) and three different non-small cell lung cancer (NSCLC) cell lines contributed to highly malignant subpopulations with both EMT and stem cell-like properties. Hybrids lost their epithelial morphology and assumed a fibroblast-like appearance. Up-regulation of vimentin, α-smooth muscle actin (α-SMA), and fibronectin, and down-regulation of E-cadherin and pancytokeratin were observed in tumorigenic hybrids. These cells also exhibited increased expression of the stem cell marker prominin-1 (CD133) and over-expression of transcription factors OCT4, Nanog, BMI1, Notch1, ALDH1 as well as Sox2, all genes responsible for regulating and maintaining the stem cell phenotype. In addition, in spontaneously-formed tumorigenic hybrids, increased pneumosphere-forming capacity and tumor-forming ability in NOD/SCID mice were detectable. Thus, cell fusion between lung cancer cells and MSCs provides a nonmutational mechanism that could contribute to aberrant gene expression patterns and give rise to highly malignant subpopulations both capable of EMT and with properties of cancer stem cells (CSCs).

The pathophysiological mechanisms underlying mucus hypersecretion induced by cold temperatures in cigarette smoke-exposed rats.

In a recent study, we demonstrated that transient receptor potential melastatin 8 (TRPM8), a calcium-permeable cation channel that is activated by cold temperatures, is localized in the bronchial epithelium and is upregulated in subjects with chronic obstructive pulmonary disease, which causes them to be more sensitive to cold air. In the present study, we found that exposure to cold temperatures induced ciliary ultrastructural anomalies and mucus accumulation on the epithelial surface. Male Sprague-Dawley rats were exposed to cold temperatures to determine the effects of cold air on ultrastructural changes in cilia and the airway epithelial surface. The rats were also exposed to cigarette smoke and/or cold temperatures to determine the effects of smoke and cold air on TRPM8 expression and the role of cold air in cigarette smoke-induced mucus hypersecretion. Following real-time RT-PCR and western blot analysis, we observed a high expression of TRPM8 mRNA and protein in the bronchial tissue following cigarette smoke inhalation. As shown by ELISA, concurrent cold air enhanced the levels of mucin 5AC (MUC5AC) protein, as well as those of inflammatory factors [tumor necrosis factor (TNF)-α and interleukin (IL)-8] that were induced by cigarette smoke inhalation to a greater extent than stimulation with separate stimuli (cold air and cigarette smoke separately). The results suggest that cold air stimuli are responsible for the ultrastructural abnormalities of bronchial cilia, which contribute to abnormal mucus clearance. In addition, cold air synergistically amplifies cigarette smoke-induced mucus hypersecretion and the production of inflammatory factors through the elevated expression of the TRPM8 channel that is initiated by cigarette smoke inhalation.

Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion.

The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA-AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT-PCR (reverse transcription-PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA-AM and RB-2, while EGTA did not have a suppressive effect. BAPTA-AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+-dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2.