Polystyrene microplastics induce pulmonary fibrosis by promoting alveolar epithelial cell ferroptosis through cGAS/STING signaling.

Polystyrene microplastics (PS-MPs) are new types of environmental pollutant that have garnered significant attention in recent years since they were found to cause damage to the human respiratory system when they are inhaled. The pulmonary fibrosis is one of the serious consequences of PS-MPs inhalation. However, the impact and underlying mechanisms of PS-MPs on pulmonary fibrosis are not clear. In this study, we studied the potential lung toxicity and PS-MPs-developed pulmonary fibrosis by long-term intranasal inhalation of PS-MPs. The results showed that after exposing to the PS-MPs, the lungs of model mouse had different levels of damage and fibrosis. Meanwhile, exposing to the PS-MPs resulted in a markedly decrease in glutathione (GSH), an increase in malondialdehyde (MDA), and iron overload in the lung tissue of mice and alveolar epithelial cells (AECs). These findings suggested the occurrence of PS-MP-induced ferroptosis. Inhibitor of ferroptosis (Fer-1) had alleviated the PS-MPs-induced ferroptosis. Mechanically, PS-MPs triggered cell ferroptosis and promoted the development of pulmonary fibrosis via activating the cGAS/STING signaling pathway. Inhibition of cGAS/STING with G150/H151 attenuated pulmonary fibrosis after PS-MPs exposure. Together, these data provided novel mechanistic insights of PS-MPs-induced pulmonary fibrosis and a potential therapeutic paradigm.

1G6-D7 Inhibits Homologous Recombination Repair by Targeting Extracellular HSP90α to Promote Apoptosis in Non-Small Cell Lung Cancer.

Despite recent advances in treatment, non-small cell lung cancer (NSCLC) continues to have a high mortality rate. Currently, NSCLC pathogenesis requires further investigation, and therapeutic drugs are still under development. Homologous recombination repair (HRR) repairs severe DNA double-strand breaks. Homologous recombination repair deficiency (HRD) occurs when HRR is impaired and causes irreparable double-strand DNA damage, leading to genomic instability and increasing the risk of cancer development. Poly(ADP-ribose) polymerase (PARP) inhibitors can effectively treat HRD-positive tumors. Extracellular heat shock protein 90α (eHSP90α) is highly expressed in hypoxic environments and inhibits apoptosis, thereby increasing cellular tolerance. Here, we investigated the relationship between eHSP90α and HRR in NSCLC. DNA damage models were established in NSCLC cell lines (A549 and H1299). The activation of DNA damage and HRR markers, apoptosis, proliferation, and migration were investigated. In vivo tumor models were established using BALB/c nude mice and A549 cells. We found that human recombinant HSP90α stimulation further activated HRR and reduced DNA damage extent; however, eHSP90α monoclonal antibody, 1G6-D7, effectively inhibited HRR. HRR inhibition and increased apoptosis were observed after LRP1 knockdown; this effect could not be reversed with hrHSP90α addition. The combined use of 1G6-D7 and olaparib caused significant apoptosis and HRR inhibition in vitro and demonstrated promising anti-tumor effects in vivo. Extracellular HSP90α may be involved in HRR in NSCLC through LRP1. The combined use of 1G6-D7 and PARP inhibitors may exert anti-tumor effects by inhibiting DNA repair and further inducing apoptosis of NSCLC cells.

IL-17A promotes tumorigenesis and upregulates PD-L1 expression in non-small cell lung cancer.

BACKGROUND: The tumor microenvironment plays a key role in non-small cell lung cancer (NSCLC) development and also influences the effective response to immunotherapy. The pro-inflammatory factor interleukin-17A mediates important immune responses in the tumor microenvironment. In this study, the potential role and mechanisms of IL-17A in NSCLC were investigated. METHODS: We detected IL-17A by immunohistochemistry (IHC) in 39 NSCLC patients. Its expression was correlated with the programmed cell death-ligand1 (PD-L1). IL-17A knockdown and overexpression in A549 and SPC-A-1 cell models were constructed. The function of IL-17A was examined in vitro by wound healing, migration, invasion, plate colony formation and T cell killing assay. Western blot analysis, immunofluorescence assay and IHC were performed to investigate the regulation effects of IL-17A on autophagy in A549 and SPC-A-1. The effect of IL-17A on ROS/Nrf2/p62 signaling pathway was detected. Subcutaneous tumor models were established to examine the tumor-promoting effect of IL-17A in vivo and its effect on immunotherapy. RESULTS: We found a prevalent expression of IL-17A in NSCLC tumor tissues and it was positively correlated with PD-L1 expression (r = 0.6121, p < 0.0001). In vitro, IL-17A promotes lung cancer cell migration, invasion and colony formation ability. Moreover, IL-17A upregulated N-cadherin, Twist, and Snail, and downregulated E-cadherin in NSCLC cells. IL-17A enhanced cell survival in the T cell killing assay. Mechanistically, IL-17A induced ROS production and increased Nrf2 and p62 expression, thereby inhibiting autophagy and reducing PD-L1 degradation. In vivo experiments, anti-IL-17A monoclonal antibody alone slowed the growth of subcutaneous tumors in mice. When combined with anti-PD-L1 monoclonal antibody, tumor tissue expression of PD-L1 was reduced and the therapeutic effect was diminished. CONCLUSION: We found that IL-17A promoted NSCLC progression and inhibited autophagy through the ROS/Nrf2/p62 pathway leading to increased PD-L1 expression in cancer cells. Modulation of IL-17A may affect the therapeutic efficacy of immunotherapy.

HDM induce airway epithelial cell ferroptosis and promote inflammation by activating ferritinophagy in asthma.

Asthma is a disease characterized by airway epithelial barrier destruction, chronic airway inflammation, and airway remodeling. Repeated damage to airway epithelial cells by allergens in the environment plays an important role in the pathophysiology of asthma. Ferroptosis is a novel form of regulated cell death mediated by lipid peroxidation in association with free iron-mediated Fenton reactions. In this study, we explored the contribution of ferroptosis to house dust mite (HDM)-induced asthma models. Our in vivo and in vitro models showed labile iron accumulation and enhanced lipid peroxidation with concomitant nonapoptotic cell death upon HDM exposure. Treatment with ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (Fer-1) illuminated the role of ferroptosis and related damage-associated molecular patterns in HDM-treated airway epithelial cells. Furthermore, DFO and Fer-1 reduced HDM-induced airway inflammation in model mice. Mechanistically, NCOA4-mediated ferritin-selective autophagy (ferritinophagy) was initiated during ferritin degradation in response to HDM exposure. Together, these data suggest that ferroptosis plays an important role in HDM-induced asthma and that ferroptosis may be a potential treatment target for HDM-induced asthma.

Extracellular HSP90α promotes cellular senescence by modulating TGF-ß signaling in pulmonary fibrosis.

Recent findings suggest that extracellular heat shock protein 90α (eHSP90α) promotes pulmonary fibrosis, but the underlying mechanisms are not well understood. Aging, especially cellular senescence, is a critical risk factor for idiopathic pulmonary fibrosis (IPF). Here, we aim to investigate the role of eHSP90α on cellular senescence in IPF. Our results found that eHSP90α was upregulated in bleomycin (BLM)-induced mice, which correlated with the expression of senescence markers. This increase in eHSP90α mediated fibroblast senescence and facilitated mitochondrial dysfunction. eHSP90α activated TGF-ß signaling through the phosphorylation of the SMAD complex. The SMAD complex binding to p53 and p21 promoters triggered their transcription. In vivo, the blockade of eHSP90α with 1G6-D7, a specific eHSP90α antibody, in old mice attenuated the BLM-induced lung fibrosis. Our findings elucidate a crucial mechanism underlying eHSP90α-induced cellular senescence, providing a framework for aging-related fibrosis interventions.

CBX4 Regulates Long-Form Thymic Stromal Lymphopoietin-mediated Airway Inflammation through SUMOylation in House Dust Mite-induced Asthma.

Thymic stromal lymphopoietin presents in two distinct isoforms: short-form (sfTSLP) and long-form (lfTSLP). lfTSLP promotes inflammation, whereas sfTSLP inhibits inflammation, in allergic asthma. However, little is known about the regulation of lfTSLP and sfTSLP during allergic attack in the asthma airway epithelium. Here, we report that small ubiquitin-like modifier (SUMOylation) was enhanced in house dust mite-induced allergic asthma airway epithelium. Inhibition of SUMOylation significantly alleviated airway T-helper cell type 2 inflammation and lfTSLP expression. Mechanistically, chromobox 4 (CBX4), a SUMOylation E3 ligase, enhanced lfTSLP mRNA translation, but not sfTSLP, through the RNA-binding protein muscle excess (MEX)-3B. MEX-3B promoted lfTSLP translation by binding the lfTSLP mRNA through its K homology domains. Furthermore, CBX4 regulated MEX-3B transcription in human bronchial epithelial cells through enhancing SUMOylation concentrations of the transcription factor TFII-I. In conclusion, we demonstrate an important mechanism whereby CBX4 promotes MEX-3B transcription through enhancing TFII-I SUMOylation and MEX-3B enhances the expression of lfTSLP through binding to the lfTSLP mRNA and promoting its translation. Our findings uncover a novel target of CBX4 for therapeutic agents for lfTSLP-mediated asthma.

Short isoform thymic stromal lymphopoietin reduces inflammation and aerobic glycolysis of asthmatic airway epithelium by antagonizing long isoform thymic stromal lymphopoietin.

BACKGROUND: Up-regulation of aerobic glycolysis has been reported as a characterization of asthma and facilitates airway inflammation. We has been previously reported that short isoform thymic stromal lymphopoietin (sTSLP) could reduce inflammation in asthmatic airway epithelial cells. Here we wanted to investigate whether the inhibition of sTSLP on asthma is related to aerobic glycolysis. METHODS: Asthmatic model was established in challenging Male BALB/c mice and 16-HBE (human bronchial epithelial) cell line with house dust mite (HDM). Indicators of glycolysis were assessed to measure whether involve in sTSLP regulating airway epithelial cells inflammation in asthmatic model in vivo and in vitro. RESULTS: sTSLP decreased inflammation of asthmatic airway and aerobic glycolysis in mice. HDM or long isoform thymic stromal lymphopoietin (lTSLP) promoted HIF-1α expression and aerobic glycolysis by miR-223 to target and inhibit VHL (von Hippel-Lindau) expression 16-HBE. Inhibition of aerobic glycolysis restrained HDM- and lTSLP-induced inflammatory cytokines production. sTSLP along had almost no potential to alter aerobic glycolysis of 16-HBE. But sTSLP decreased LDHA (lactate dehydrogenase A) and LD (Lactic acid) levels in BALF, and HIF-1α and LDHA protein levels in airway epithelial cells of asthma mice model. lTSLP and sTSLP both induced formation of TSLPR and IL-7R receptor complex, and lTSLP obviously facilitated phosphorylation of JAK1, JAK2 and STAT5, while sTSLP induced a little phosphorylation of JAK1 and STAT5. CONCLUSION: We identified a novel mechanism that lTSLP could promote inflammatory cytokines production by miR-223/VHL/HIF-1α pathway to upregulate aerobic glycolysis in airway epithelial cells in asthma. This pathway is suppressed by sTSLP through occupying binding site of lTSLP in TSLPR and IL-7R receptor complex.

RAGE mediates airway inflammation via the HDAC1 pathway in a toluene diisocyanate-induced murine asthma model.

BACKGROUND: Exposure to toluene diisocyanate (TDI) is a significant pathogenic factor for asthma. We previously reported that the receptor for advanced glycation end products (RAGE) plays a key role in TDI-induced asthma. Histone deacetylase (HDAC) has been reported to be important in asthmatic pathogenesis. However, its effect on TDI-induced asthma is not known. The aim of this study was to determine the role of RAGE and HDAC in regulating airway inflammation using a TDI-induced murine asthma model. METHODS: BALB/c mice were sensitized and challenged with TDI to establish an asthma model. FPS-ZM1 (RAGE inhibitor), JNJ-26482585 and romidepsin (HDAC inhibitors) were administered intraperitoneally before each challenge. In vitro, the human bronchial epithelial cell line 16HBE was stimulated with TDI-human serum albumin (TDI-HSA). RAGE knockdown cells were constructed and evaluated, and MK2006 (AKT inhibitor) was also used in the experiments. RESULTS: In TDI-induced asthmatic mice, the expression of RAGE, HDAC1, and p-AKT/t-AKT was upregulated, and these expressions were attenuated by FPS-ZM1. Airway reactivity, Th2 cytokine levels in lymph supernatant, IgE, airway inflammation, and goblet cell metaplasia were significantly increased in the TDI-induced asthmatic mice. These increases were suppressed by JNJ-26482585 and romidepsin. In addition, JNJ-26482585 and romidepsin ameliorated the redistribution of E-cadherin and ß-catenin in TDI-induced asthma. In TDI-HSA-stimulated 16HBE cells, knockdown of RAGE attenuated the upregulation of HDAC1 and phospho-AKT (p-AKT). Treatment with the AKT inhibitor MK2006 suppressed TDI-induced HDAC1 expression. CONCLUSIONS: These findings indicate that RAGE modulates HDAC1 expression via the PI3K/AKT pathway, and that inhibition of HDAC prevents TDI-induced airway inflammation.

Belief in a just world, health-related quality of life, and mental health among Chinese patients with chronic obstructive pulmonary disease.

PURPOSE: Personal belief in a just world (PBJW) has been demonstrated to protect mental health. However, whether general belief in a just world (GBJW) serves adaptive functions for mental health across different groups and cultures remains unclear. This study explored the effects of PBJW and GBJW on mental health and moderating effects of PBJW and GBJW on the relation between health-related quality of life and mental health among patients with chronic obstructive pulmonary disease (COPD) in China. METHODS: A total of 147 patients with COPD (90.5% male; mean age = 64.44 years) completed measures of health-related quality of life, depression, anxiety, PBJW, and GBJW and provided pulmonary function data. RESULTS: Younger age and female sex were related to higher depression; female sex, living with others, and high financial burden were associated with higher anxiety. Worse health-related quality of life and lower PBJW were associated with higher depression and anxiety. An interaction between health-related quality of life and BJW was revealed. For patients with low PBJW, lower health-related quality of life was correlated with higher depression. For patients with stronger endorsement of GBJW, worse health-related quality of life was associated with higher depression and anxiety, but the variance of anxiety caused by interaction was insignificant. CONCLUSIONS: The findings suggest that for patients with COPD experiencing health deterioration, holding strong PBJW but weak GBJW may be beneficial for mental health. Our study advances our understanding of the different functions of PBJW and GBJW in mental health across different groups and cultures.

The role of secreted Hsp90α in HDM-induced asthmatic airway epithelial barrier dysfunction.

BACKGROUND: The dysfunction of airway epithelial barrier is closely related to the pathogenesis of asthma. Secreted Hsp90α participates in inflammation and Hsp90 inhibitor protects endothelial dysfunction. In the current study, we aimed to explore the role of secreted Hsp90α in asthmatic airway epithelial barrier function. METHODS: Male BALB/c mice were sensitized and challenged with HDM to generate asthma model. The 16HBE and Hsp90α-knockdown cells were cultured and treated according to the experiment requirements. Transepithelial Electric Resistance (TEER) and permeability of epithelial layer in vitro, distribution and expression of junction proteins both in vivo and in vitro were used to evaluate the epithelial barrier function. Western Blot was used to evaluate the expression of junction proteins and phosphorylated AKT in cells and lung tissues while ELISA were used to evaluate the Hsp90α expression and cytokines release in the lung homogenate. RESULTS: HDM resulted in a dysfunction of airway epithelial barrier both in vivo and in vitro, paralleled with the increased expression and release of Hsp90α. All of which were rescued in Hsp90α-knockdown cells or co-administration of 1G6-D7. Furthermore, either 1G6-D7 or PI3K inhibitor LY294002 suppressed the significant phosphorylation of AKT, which caused by secreted and recombinant Hsp90α, resulting in the restoration of epithelial barrier function. CONCLUSIONS: Secreted Hsp90α medicates HDM-induced asthmatic airway epithelial barrier dysfunction via PI3K/AKT pathway, indicating that anti-secreted Hsp90α therapy might be a potential treatment to asthma in future.

Blockade of extracellular heat shock protein 90α by 1G6-D7 attenuates pulmonary fibrosis through inhibiting ERK signaling.

Pulmonary fibrosis is characterized by lung fibroblast activation and ECM deposition and has a poor prognosis. Heat shock protein 90 (Hsp90) participates in organ fibrosis, and extracellular Hsp90α (eHsp90α) promotes fibroblast activation and migration. This study aimed to investigate whether a selective anti-Hsp90α monoclonal antibody, 1G6-D7, could attenuate lung fibrosis and whether 1G6-D7 presents a protective effect by inactivating the profibrotic pathway. Our results showed that eHsp90α was increased in mice with BLM-induced pulmonary fibrosis and that 1G6-D7 attenuated inflammation and collagen deposition in the lung. TGF-ß1 induced eHsp90α secretion, concomitantly promoting HFL-1 activation and ECM synthesis. 1G6-D7-mediated inhibition of eHsp90α significantly blocked these effects, meanwhile inhibiting downstream profibrotic pathways such as ERK, Akt, and P38. Human recombinant (hr)Hsp90α mimicked the effects of TGF-ß1, by activating profibrotic pathways and by upregulating LRP-1. Moreover, ERK inhibition effectively blocked the effect of (hr)Hsp90α. In conclusion, 1G6-D7 significantly protects against BLM-induced pulmonary fibrosis by ameliorating fibroblast activation and ECM production, which may be through blocking ERK signaling. Our results suggest a safer molecular therapy, 1G6-D7, in pulmonary fibrosis.

Hsp90α and Hsp90ß together operate a hypoxia and nutrient paucity stress-response mechanism during wound healing.

When tissues are injured and blood vessels clot, the local environment becomes ischemic, meaning that there is a lack of adequate supply of oxygen and glucose delivered to the surrounding cells. The heat shock protein-90 (Hsp90) family proteins protect tissues from various environmental insults and participate in the repair of damaged tissue. Here, we report discovery of a new ischemia-responsive mechanism in which the two Hsp90 isoforms Hsp90α and Hsp90ß (also known as HSP90AA1 and HSP90AB1, respectively) work together to promote cell motility in wounded skin and accelerate wound closure. We demonstrate that Hsp90α and Hsp90ß have distinct and non-exchangeable functions during wound healing. Under hypoxia and when there is a lack of serum factors, Hsp90ß binds to the cytoplasmic tail of the LDL receptor-related protein-1 (LRP-1) and stabilizes the receptor at the cell surface. Hsp90α, however, is secreted by the cell into extracellular space where it binds and signals through the LRP-1 receptor to promote cell motility, leading to wound closure. In addition to skin injury, we suggest that this repair mechanism applies broadly to other non-cutaneous injured tissues.

1,25-Dihydroxyvitamin D3 targeting VEGF pathway alleviates house dust mite (HDM)-induced airway epithelial barrier dysfunction.

BACKGROUND: In our previous studies, we have indentified that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can alleviate toluene diisocyanate-induced airway epithelial barrier disruption and we also found that vascular endothelial growth factor (VEGF) derived from airway epithelials cells could disrupt epithelial barrier. OBJECTIVE: The study aimed to investigate whether 1,25(OH)2D3 can inhibit house dust mite (HDM) induced airway epithelial barrier dysfunction by regulating the VEGF pathway. METHOD: The 16HBE and BEAS-2B cells were cultured and treated according to the experiment requirement. Trans Epithelial Electric Resistance (TEER), permeability of epithelial layer, and distribution and expression of junction proteins were used to evaluate the cell layer barrier function, Western Blot was used to evaluate the expression of junction proteins and phosphorylated Akt in the cells, RT-PCR and ELISA were used to evaluate the VEGF gene expression and protein release in the cells. Recombinant VEGF165 was used to determine the role of the VEGF pathway in the epithelial barrier function. RESULTS: HDM resulted in a decline in TEER and increase of cell permeability, following abnormal distribution and expression of junction proteins (E-Cadherin and zona occludens (ZO)-1), accompanied by a significant upregulation of VEGF and phosphorylated Akt, which were all partly recovered by treatment with either 1,25(OH)2D3 or PI3K inhibitor LY294002. VEGF165-induced barrier dysfunction was accompanied by disruption of the epithelial E-cadherin and ß-catenin, pretreatment of 1,25(OH)2D3 and LY294002 markedly attenuated VEGF-induced airway barrier disruption in 16HBE cells. CONCLUSION: 1,25(OH)2D3 can alleviate HDM-induced airway epithelial barrier dysfunction by inhibiting PI3K pathway-dependent VEGF release.

TSLP signaling blocking alleviates E-cadherin dysfunction of airway epithelium in a HDM-induced asthma model.

Recent studies have indicated that Thymic stromal lymphopoietin (TSLP) plays an important role in the prevention and treatment of asthma. However the role of TSLP in dysfunction of airway epithelial adherens junctions E-cadherin in house dust mite (HDM)-induced asthma has not been addressed. We hypothesized that TSLP contributed to HDM-induced E-cadherin dysfunction in asthmatic BALB/c mice and 16HBE cells. In vivo, a HDM-induced asthma mouse model was set up for 8weeks. Mice inhaled an anti-TSLP monoclonal antibody (mAb) before HDM. The mice treated with the anti-TSLP mAb ameliorated airway inflammation, the decreasing and aberrant distribution of E-cadherin and ß-catenin as well as phosphorylation(p)-AKT induced by HDM. In vitro, HDM increased the expression of TSLP and E-cadherin dysfunction by PI3K/Akt signaling pathway. The exposure of 16HBE to TSLP resulted in redistribution of E-cadherin. These results indicate that TSLP may be an important contributor in E-cadherin dysfunction of HDM-induced asthma. TSLP signaling blocking shows a protective effect in mice and that the PI3K/Akt pathway may play a role in this process.

Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.

BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.

Bevacizumab reduced auto-phosphorylation of VEGFR2 to protect HDM-induced asthma mice.

Vascular endothelial growth factor (VEFG) is a major angiogenic factor involved in both normal physiological processes, such as embryonic development and wound healing, and in diseases, like cancer. Recent studies have revealed the functions of VEGF in inflammation and immunoregulation. Asthma is a chronic inflammation of the airways characterized by airway epithelial barrier dysfunction and imbalance in T-helper (Th) 1/Th2 during immunoregulation. We hypothesized that VEGF plays an important role in asthma. Utilizing a house dust mite extract (HDM)-induced murine model of asthma, we investigated whether bevacizumab, a humanized anti-VEGF monoclonal antibody, could protect the epithelial barrier in murine airways. We found that bevacizumab reduced airway hyper-responsiveness (AHR) and airway inflammation induced by HDM. In addition, HDM exposure promoted expression of VEGF, and caused AHR, disruptions of the epithelial barrier, and airway inflammation. Bevacizumab ameliorated AHR and the release of Th2 cytokines, thereby protecting the epithelial barrier. Our data suggest that bevacizumab may be a new therapeutic strategy for asthma.

1α,25-dihydroxyvitamin D3 counteracts the effects of cigarette smoke in airway epithelial cells.

Cigarette smoke extracts (CSE) alter calpain-1 expression via ERK signaling pathway in bronchial epithelial cells. 1α,25-dihydroxyvitamin D3 (1,25D3) inhibits cigarette smoke-induced epithelial barrier disruption. This study was aimed to explore whether the 1,25D3 counteracted the CSE effects in a human bronchial epithelial cell line (16HBE). In particular, transepithelial electrical resistance (TER) and permeability, expression and distribution of E-cadherin and ß-catenin, calpain-1 expression, and ERK phosphorylation were assessed in the CSE-stimulated 16HBE cells. The CSE induced the ERK phosphorylation, improved the calpain-1 expression, increased the distribution anomalies and the cleaving of E-cadherin and ß-catenin, and resulted in the TER reduction and the permeability increase. The 1,25D3 reduced these pathological changes. The 1,25D3 mediated effects were associated with a reduced ERK phosphorylation. In conclusion, the present study provides compelling evidences that the 1,25D3 may be considered a possible valid therapeutic option in controlling the cigarette smoke-induced epithelial barrier disruption.

[Polarization of neutrophils from patients with asthma, chronic obstructive pulmonary disease and asthma-chronic obstructive pulmonary disease overlap syndrome].

OBJECTIVE: To explore the polarization of migration dynamics of neutrophils isolated from patients with asthma, chronic obstructive pulmonary disease (COPD) and asthma-COPD overlap syndrome (ACOS) compared with healthy smoking and non-smoking controls. METHODS: Recruited volunteers were classified as healthy controls, healthy smokers, asthma, COPD and ACOS at Nanfang Hospital from April 2013 to June 2014 according to the Global Strategy for the Diagnosis, Management and Prevention of COPD 2011, Global Strategy for Asthma Management and Prevention 2011 and Consensus on Overlap Phenotype COPD-asthma in COPD 2012. Neutrophils were freshly isolated from whole blood with density gradient technique. The proportion of polarized cells with gradient concentration of formyl-Met-Leu-Phe (fMLP) in Zigmond chamber and vital component of Store Operated Calcium Entry (SOCE) (stromal interaction molecule (STIM) 1, 2 and Orai1) in neutrophils was detected by Western blot. RESULTS: Asthma, COPD and ACOS neutrophils demonstrated a higher spontaneous polarization rate versus healthy controls and healthy smokers ((25.05 ± 4.06)%, (16.20 ± 4.46)%, (29.43 ± 5.53)% vs (7.27 ± 0.99)%, (7.06 ± 3.12)%, all P < 0.01), asthma and ACOS neutrophils showed a higher directed polarization rate ((14.62 ± 2.26)%, (8.00 ± 1.75)%, all P < 0.05), but COPD had a relatively lower rate of directional polarization rate than healthy controls and healthy smokers ((2.45 ± 0.54)% vs (5.12 ± 1.28)%, (5.24 ± 1.34)%, all P < 0.01). The vital component of SOCE in neutrophils from asthma, COPD and ACOS were all up-regulated versus healthy controls and healthy smokers (STIM1: 1.63 ± 0.14, 0.88 ± 0.41, 1.29 ± 0.22 vs 0.26 ± 0.14, 0.38 ± 0.12; STIM2: 0.52 ± 0.19, 0.22 ± 0.13, 0.24 ± 0.10 vs 0.05 ± 0.03, 0.10 ± 0.06; Orai1: 0.56 ± 0.04, 0.39 ± 0.05, 0.48 ± 0.05 vs 0.13 ± 0.04, 0.13 ± 0.03) (all P < 0.01). CONCLUSIONS: Asthma, COPD and ACOS neutrophils are intrinsically different than counterparts from healthy control subjects and healthy smokers. And vital components of SOCE from patient neutrophils are intrinsically up-regulated.

Human epididymis protein 4, a novel potential biomarker for diagnostic and prognosis monitoring of lung cancer.

OBJECTIVE: This study aimed to explore the application value of human epididymis protein 4 (HE4) in diagnosing and monitoring the prognosis of lung cancer. METHODS: First, TCGA (The Cancer Genome Atlas) databases were used to analyze whey-acidic-protein 4-disulfide bond core domain 2 (WFDC2) gene expression levels in lung cancer tissues. Then, a total of 160 individuals were enrolled, categorized into three groups: the lung cancer group (n = 80), the benign lesions group (n = 40), and the healthy controls group (n = 40). Serum HE4 levels and other biomarkers were quantified using an electro-chemiluminescent immunoassay. Additionally, the expression of HE4 in tissues was analyzed through immunohistochemistry (IHC). In vitro cultures of human airway epithelial (human bronchial epithelial [HBE]) cells and various lung cancer cell lines (SPC/PC9/A594/H520) were utilized to detect HE4 levels via western blot (WB). RESULTS: Analysis of the TCGA and UALCAN (The University of Alabama at Birmingham Cancer Data Analysis Portal) databases showed that WFDC2 gene expression levels were upregulated in lung cancer tissues (p < 0.01). Compared with the control group and the benign group, HE4 was significantly higher in the serum of patients with lung cancer (p < 0.001). Receiver operating characteristic (ROC) analysis confirmed that HE4 had better diagnostic efficacy than classical markers in the differential diagnosis of lung cancer and benign lesions and had the highest diagnostic value in lung adenocarcinoma (area under the ROC curve [AUC] = 0.826). HE4 increased in early lung cancer and positively correlated with poor prognosis (p < 0.001). Moreover, the results of WB and IHC revealed that the expression of HE4 was increased in lung cancer cells (SPC/A549/H520) and lung cancer tissues but decreased in PC9 cells with a lack of exon EGFR19 (p < 0.05). CONCLUSION: Serum HE4 emerges as a promising novel biomarker for the diagnosis and prognosis assessment of lung cancer.

Tetrandrine alleviates pulmonary fibrosis by inhibiting alveolar epithelial cell senescence through PINK1/Parkin-mediated mitophagy.

Idiopathic pulmonary fibrosis (IPF) is a fatal and insidious interstitial lung disease. So far, there are no effective drugs for preventing the disease process. Cellular senescence plays a critical role in the development of IPF, with the senescence and insufficient mitophagy of alveolar epithelial cells being implicated in its pathogenesis. Tetrandrine is a natural alkaloid which is now produced synthetically. It was known that the tetrandrine has anti-fibrotic effects, but the efficacy and mechanisms are still not well evaluated. Here, we reveal the roles of tetrandrine on AECs senescence and the antifibrotic effects by using a bleomycin challenged mouse model of pulmonary fibrosis and a bleomycin-stimulated mouse alveolar epithelial cell line (MLE-12). We performed the ß-galactosidase staining, immunohistochemistry and fluorescence to assess senescence in MLE-12 cells. The mitophagy levels were detected by co-localization of LC3 and COVIX. Our findings indicate that tetrandrine suppressed bleomycin-induced fibroblast activation and ultimately blocked the increase of collagen deposition in mouse model lung tissue. It has significantly inhibited the bleomycin-induced senescence and senescence-associated secretory phenotype (SASP) in alveolar epithelial cells (AECs). Mechanistically, tetrandrine suppressed the decrease of mitochondrial autophagy-related protein expression to rescue the bleomycin-stimulated impaired mitophagy in MLE-12 cells. We revealed that knockdown the putative kinase 1 (PINK1) gene by a short interfering RNA (siRNA) could abolish the ability of tetrandrine and reverse the MLE-12 cells senescence, which indicated the mitophagy of MLE-12 cells is PINK1 dependent. Our data suggest the tetrandrine could be a novel and effective drug candidate for lung fibrosis and senescence-related fibrotic diseases.