Apoptosis In Lung Epithelial Cells Research Articles

Stenotrophomonas maltophilia Encodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, Including Pseudomonas aeruginosa.

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed that S. maltophilia also encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved among S. maltophilia strains and, looking beyond the Stenotrophomonas genus, is most similar to the T4SS of Xanthomonas To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains of Pseudomonas aeruginosa, the T4SS promoted the growth of S. maltophilia and reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS required S. maltophilia contact with its target. Thus, S. maltophilia VirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before.

Blockade of EGFR Activation Promotes TNF-Induced Lung Epithelial Cell Apoptosis and Pulmonary Injury.

Pneumonitis is the leading cause of death associated with the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) against non-small cell lung cancer (NSCLC). However, the risk factors and the mechanism underlying this toxicity have not been elucidated. Tumor necrosis factor (TNF) has been reported to transactivate EGFR in pulmonary epithelial cells. Hence, we aimed to test the hypothesis that EGFR tyrosine kinase activity regulates TNF-mediated bronchial epithelial cell survival, and that inhibition of EGFR activity increases TNF-induced lung epithelial cell apoptosis. We used surfactant protein C (SPC)-TNF transgenic (tg) mice which overexpress TNF in the lungs. In this model, gefitinib, an EGFR-TKI, enhanced lung epithelial cell apoptosis and lymphocytic inflammation, indicating that EGFR tyrosine kinase prevents TNF-induced lung injury. Furthermore, IL-17A was significantly upregulated by gefitinib in SPC-TNF tg mice and p38MAPK activation was observed, indicative of a pathway involved in lung epithelial cell apoptosis. Moreover, in lung epithelial cells, BEAS-2B, TNF stimulated EGFR transactivation via the TNF-α-converting enzyme in a manner that requires heparin binding (HB)-EGF and transforming growth factor (TGF)-α. These novel findings have significant implications in understanding the role of EGFR in maintaining human bronchial epithelial cell homeostasis and in NSCLC treatment.

The effects of Sestrin2 on apoptosis of heat-exposed lung epithelial cells and its mechanism

To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism. Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1+. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells. With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure. Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.

Chlorogenic acid prevents paraquat-induced apoptosis via Sirt1-mediated regulation of redox and mitochondrial function

Paraquat (PQ) is a widely used agro-chemical in agriculture and highly toxic to humans. Although the mechanism of PQ poisoning is not clear, it has been well documented that reactive oxygen species (ROS) generation and apoptosis play pivotal roles. Alternatively, chlorogenic acid (CA) is a biologically active dietary polyphenol, playing several therapeutic roles. However, it is not known whether CA has protective effect on PQ-induced apoptosis. Here, we investigated the effect of CA in preventing PQ-induced apoptosis and explored the underlying mechanisms. A549 cells were pretreated with 100 µM CA for 24 h and then exposed to 160 µM PQ for 24 h. We found that CA was effective in preventing PQ-induced apoptotic features, including the release of cytochrome c from the mitochondria to cytoplasm, the cleavages of caspase 3 and caspase 9, and the increases in levels of Bcl-2-associated X protein (Bax) and intracellular calcium ions. CA alleviated ROS production and prevented the reduction of antioxidant capacity in cells exposed to PQ by increasing NF-E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2) and glutathione levels. In addition, CA also attenuated PQ-induced alterations of mitochondrial structure and function (such as the decreases in membrane potential and adenosine triphosphate level), and the impaired autophagic flux was improved by CA. Down-regulation of sirtuin 1 (Sirt1) by short hairpin RNA reversed the protective effects of CA. Thus, CA may be viewed as a potential drug to treat PQ-induced lung epithelial cell apoptosis and other disorders with similar pathologic mechanisms.

Role of phospholipase D in bleomycin-induced mitochondrial reactive oxygen species generation, mitochondrial DNA damage, and pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a pernicious lung disease characterized by alveolar epithelial apoptosis, dysregulated repair of epithelial injury, scar formation, and respiratory failure. In this study, we identified phospholipase D (PLD)-generated phosphatidic acid (PA) signaling in the development of pulmonary fibrosis (PF). Of the PLD isoenzymes, the protein expression of PLD2, but not PLD1, was upregulated in lung tissues from IPF patients and bleomycin challenged mice. Both PLD1 (Pld1-/-)- and PLD2 (Pld2-/-)-deficient mice were protected against bleomycin-induced lung inflammation and fibrosis, thereby establishing the role of PLD in fibrogenesis. The role of PLD1 and PLD2 in bleomycin-induced lung epithelial injury was investigated by infecting bronchial airway epithelial cells (Beas2B) with catalytically inactive mutants of PLD (hPLD1-K898R or mPld2-K758R) or downregulation of expression of PLD1 or PLD2 with siRNA. Bleomycin stimulated mitochondrial (mt) superoxide production, mtDNA damage, and apoptosis in Beas2B cells, which was attenuated by the catalytically inactive mutants of PLD or PLD2 siRNA. These results show a role for PLD1 and PLD2 in bleomycin-induced generation of mt reactive oxygen species, mt DNA damage, and apoptosis of lung epithelial cells in mice. Thus, PLD may be a novel therapeutic target in ameliorating experimental PF in mice.

Effects of Rg_1 on LPS-induced apoptosis and autophagy of lung epithelial cells

This paper aimed to study the protective effect of ginsenoside Rg_1 on endotoxin(LPS)-induced apoptosis of lung epithelial cells and its mechanism of action. Mouse lung epithelial cells(MLE-12) were first treated with LPS. The autophagy changes and apoptosis and the relationship with concentration and time of LPS were observed. Then,the level of autophagy in MLE-12 was regulated at a specific concentration and action time of LPS,and the changes of apoptosis were observed. Secondly,ginsenoside Rg_1 and autophagy inhibitor 3-MA were added respectively at the same concentration and action time of LPS. The lung epithelial cells were grouped to observe the effect of ginsenoside Rg_1 on LPS-induced apoptosis of lung epithelial cells and its mechanism. In the animal experiment,the mice were grouped and tested by apoptosis protein,lung injury score and HE staining section to verify whether ginsenoside Rg_1 has a protective effect on LPS-induced lung injury. The results showed that apoptosis and autophagy increased as the rise of concentration after treatment with LPS for 12 h. The apoptosis increased gradually,and the autophagy increased first and then decreased over time at the LPS concentration of 25 g·L-1. The apoptosis of LPS group was higher than that of control group,and LPS+3-MA group increased further,while apoptosis decreased significantly in LPS+RAM(rapamycin,autophagy promoter) group. The autophagy increased in LPS group,decreased in LPS+3-MA group and increased in LPS+RAM group. The apoptosis of LPS group was higher than that of control group,and the apoptosis of LPS+Rg_1 group decreased. The apoptosis of LPS+Rg_1+3-MA group increased again. The autophagy of LPS group further increased after administration of ginsenoside Rg_1,but decreased after administration of 3-MA. In the in vivo experiments in mice,the apoptosis of LPS group increased significantly compared with the control group,while LPS + ginsenoside Rg_1 group decreased. Lung injury score and HE staining also conformed to the above trend. LPS can induce the apoptosis of lung epithelial cells in a time-dependent and concentration-dependent manner. The autophagy of lung epithelial cells increases with the rise of LPS concentration. At the specific concentration of LPS,autophagy increases first and then decreases after 12-16 hours. Proper increase of autophagy in lung epithelial cells within a certain period of time can reduce the apoptosis induced by LPS,while inhibition of autophagy can increase apoptosis. Ginsenoside Rg_1 has a protective effect on lung cancer epithelial cell apoptosis induced by autophagy.

Polyhexamethylene guanidine phosphate-induced ROS-mediated DNA damage caused cell cycle arrest and apoptosis in lung epithelial cells.

Polyhexamethylene guanidine phosphate (PHMG-p) is an active ingredient of humidifier disinfectants and causes severe lung injury resulting in pulmonary fibrosis. Current evidence indicates that pulmonary fibrosis is initiated as a result of epithelial damage, which can lead to an inflammatory response and fibrotic cell infiltration; however, the toxic mechanism of PHMG-p on the epithelium is still unknown. In this study, the toxic response of PHMG-p on human lung epithelial cells was evaluated, and its mechanisms associated with reactive oxygen species (ROS), DNA damage, and its relationship with p53 activation were investigated. The toxic responses of epithelial cells were assessed by flow cytometry analysis and western blot analysis. The results revealed that PHMG-p induced G1/S arrest and apoptosis in A549 cells. Interestingly, p53 was activated by PHMG-p treatment and p53 knockdown suppressed PHMG-p-induced apoptosis and cell cycle arrest. PHMG-p promoted ROS generation and consequently increased the expression of DNA damage markers such as ATM and H2AX phosphorylation. The antioxidant N-acetylcysteine reduced the expression of phosphorylated ATM and H2AX, and the ATM inhibitor, caffeine, inhibited p53 activation. Taken together, our results demonstrate that PHMG-p triggered G1/S arrest and apoptosis through the ROS/ATM/p53 pathway in lung epithelial cells.

CHAC1 Is Differentially Expressed in Normal and Cystic Fibrosis Bronchial Epithelial Cells and Regulates the Inflammatory Response Induced by Pseudomonas aeruginosa.

In cystic fibrosis (CF), Pseudomonas aeruginosa (Pa) colonizes the lungs, leading to chronic inflammation of the bronchial epithelium. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) mRNA is differentially expressed in primary human airway epithelial cells from bronchi (hAECBs) from patients with CF and healthy patients at baseline and upon infection with Pa. CHAC1 degrades glutathione and is associated with ER stress and apoptosis pathways. In this study, we examined the roles of CHAC1 in the inflammatory response and apoptosis in lung epithelial cells. First, we confirmed by reverse transcription quantitative polymerase chain reaction that CHAC1 mRNA was overexpressed in hAECBs from patients without CF compared with the expression in hAECBs from patients with CF upon Pa (PAK strain) infection. Moreover, the Pa virulence factors LPS and flagellin were shown to induce CHAC1 expression in cells from patients without CF. Using NCI-H292 lung epithelial cells, we found that LPS-induced CHAC1 mRNA expression was PERK-independent and involved ATF4. Additionally, using CHAC1 small interfering RNA, we showed that reduced CHAC1 expression in the context of LPS and flagellin stimulation was associated with modulation of inflammatory markers and alteration of NF-κB signaling. Finally, we showed that Pa was not able to induce apoptosis in NCI-H292 cells. Our results suggest that CHAC1 is involved in the regulation of inflammation in bronchial cells during Pa infection and may explain the excessive inflammation present in the respiratory tracts of patients with CF.

Protective effect of ginsenoside Rg1 on LPS-induced apoptosis of lung epithelial cells

Sepsis-induced acute lung injury (ALI) is a life-threatening medical condition with high mortality and morbidity in the critical care units. Though, it was commonly accepted that inflammation and apoptosis of lung epithelial cells played an essential role in the pathogenesis of ALI, the underlying mechanism remain unknown. In our study, we found that LPS-induced cell apoptosis could be counteracted by elevated cell autophagy. In LPS-treated MLE-12 cells, suppression of autophagy via 3-MA could aggravate LPS-induced apoptosis, while activation of autophagy via Rapamycin could effectively impair the apoptosis of MLE-12 cells induced by LPS. In order to further discover the molecular regulation mechanism between apoptosis and autophagy in LPS-treated MLE-12 cells, we demonstrated that autophagy could induced the expression of Nrf2, followed with the decrease of p-p65. Targeted inhibition of Nrf2 could induce enlarged cell apoptosis via increasing the level of p-p65. In addition, we demonstrated that ginsenoside Rg1 protected MLE-12 cells from LPS-induced apoptosis via augmenting autophagy and inducing the expression of Nrf2. Our data implicates that activation of autophagy and Nrf2 by ginsenoside Rg1 may provide a preventive and therapeutic strategy for ALI.

238 - Antioxidants ameliorate hyperoxia-induced lung injury by inhibition of HMGB1

Hyperoxia-induced acute lung injury (HALI) is mediated by the production of excessive reactive oxygen species (ROS), which overwhelms intracellular antioxidant systems and induces oxidative stress. This damages macromolecules critical for cell survival, inducing apoptosis in lung epithelial cells and compromising alveolar macrophage phagocytosis. In this study, we demonstrate that antioxidants protect against HALI and improve macrophage dysfunction by reducing ROS and inhibiting the release of the damage associated molecular pattern (DAMP), HMGB1. While hyperoxia exposure (≥98% O2, 72 h) alone induced alveolar thickening, leukocyte infiltration, and an increase in total protein in the broncheolar lavage fluid (BALF) of C57BL/6 mice, ascorbic acid (AA) supplementation reversed these hallmarks of HALI. Reduced lung injury was correlated to reduced oxidative redox potential and nitric oxide production in BALF and in RAW 264.7 macrophages, and decreased accumulation of airway HMGB1. Increased activation of the endogenous antioxidant regulator, Nrf-2, by sulforaphane (SUL) similarly showed protection against hyperoxia-compromised cell function in RAW 264.7 cells and in bone marrow derived macrophages (BMDM) by reducing both intracellular levels of ROS and the release of HMGB1. Our results suggest that supplementation of antioxidants during oxygen therapy can prevent lung damage and preserve macrophage function, providing a preventive measure for HALI.

WISP-3/CCN6 inhibits apoptosis by regulating caspase pathway after hyperoxia in lung epithelial cells

Cell death is a normal phenomenon in the course of biological development, moreover, which is also a prominent feature in lung exposed to hyperoxia. Severe hypoxia occurs in ALI/ARDS patients, who generally require high concentration oxygen therapy assisted by mechanical ventilation. Nevertheless, high oxygen can cause excessive reactive oxygen species (ROS), leading to apoptosis in lung epithelial cells, which has been reported in our previous study. Herein, the correlation between increments of ROS and CCN6 expression was negative in CCN6-mediated the mitochondria dependent, intrinsic apoptotic pathway. Our latest research explained that CCN6 can inhibit caspase-8 mediated extrinsic apoptotic pathway to protect cells from hyperoxia-induced apoptosis. As demonstrated by Western Blot Analysis, Caspase 8 cleavage and Caspase 3 cleavage in CCN6-depleted cells exceeded the control group treated with high oxygen (48 h). And deletion of CCN6 enhanced caspase-8 activation after hyperoxia shown by Flow Cytometry. Although, it is unclear how CCN6 participated in the regulation of apoptotic pathways, the future targeted therapy drugs inhibiting CCN6 may be useful in the treatment of ALI/ARDS.

Porphyromonasgingivalis induced inflammatory responses and promoted apoptosis in lung epithelial cells infected with H1N1 via the Bcl‑2/Bax/Caspase‑3 signaling pathway.

The aim of the present study was to investigate the effects of Porphyromonas gingivalis (P. gingivalis) on inflammatory cytokine and nitic oxide (NO) production in lung epithelial cells infected with H1N1, and the underlying mechanisms. Lung epithelial cells were co-infected with P. gingivalis and H1N1. The concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were detected via an ELISA, and the concentration of NO was detected by the nitrate reductive enzymatic method at 4, 8, 12 and 24 h following infection. The expression levels of inducible NO synthase (iNOS) was detected by western blotting. The apoptotic rate of lung epithelial cells was detected by flow cytometry. The relative protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 in lung epithelial cells were detected by western blotting. Compared with the control group, the concentration of the inflammatory cytokines TNF-α, IL-1β and IL-6 exhibited a significant increase (P<0.05) in the viral-infected, bacterial-infected and co-infected groups. The concentration of NO also increased significantly (P<0.05), along with the rise in the expression levels of iNOS (P<0.05) and the increase in the apoptosis rate of lung epithelial cells (P<0.05). The relative expression levels of caspase-3 and Bax proteins were increased significantly in the viral- and bacterial-infected groups when compared with the control. The relative expression levels of Bcl-2 protein exhibited a significant decrease in lung epithelial cells following the co-infection with P. gingivalis and H1N1 compared with the control (P<0.05). The results of the present study revealed that the combination of P. gingivalis and H1N1 infection in lung epithelial cells may promote the production of inflammatory cytokines and increase NO production, leading to increased levels of apoptosis in lung epithelial cells via the Bcl-2/Bax/caspase-3 signaling pathway.

MMP-12 activates protease-activated receptor-1, upregulates placenta growth factor, and leads to pulmonary emphysema.

Because of the expansion of aging and smoking populations, chronic obstructive pulmonary disease (COPD) is predicted to be the third leading cause of death worldwide in 2030. Therefore, it is pertinent to develop effective therapy to improve management for COPD. Cigarette smoke-mediated protease-antiprotease imbalance is a major pathogenic mechanism for COPD and results in massive pulmonary infiltration of neutrophils and macrophages, releasing excessive neutrophil elastase (NE) and matrix metalloproteinases (MMPs). Our previous studies indicated that placenta growth factor (PGF) and PGF-triggered downstream signaling molecules mediate NE-induced lung epithelial cell apoptosis, which is a major pathogenic mechanism for pulmonary emphysema. However, the relationship between MMP-directed COPD and PGF remains elusive. We hypothesize that MMPs may upregulate PGF expression and be involved in MMP-mediated pathogenesis of COPD. In this study, we demonstrate that only MMP-12 can increase the expression of PGF by increasing early-growth response protein 1 (Egr-1) level through the activation of protease-activated receptor 1 (PAR-1). The PGF-mediated downstream signaling molecules drive caspase-3 and caspase-9-dependent apoptosis in bronchial epithelial cells. Both the upregulation of PGF by MMP-12 and PGF downstream signaling molecules with pulmonary apoptosis and emphysema were also demonstrated in animals. Given these findings, we suggest that both human COPD-associated elastases, NE, and MMP-12, upregulate PGF expression and promote the progression of emphysema and COPD.

Long non-coding RNA CASC2 improved acute lung injury by regulating miR-144-3p/AQP1 axis to reduce lung epithelial cell apoptosis

Background and objectiveApoptosis of lung epithelial cell is implicated in the pathogenesis of acute lung injury (ALI). To study the protective effect and mechanism of cancer susceptibility candidate 2 (CASC2) on reducing lung epithelial cell apoptosis after LPS inducing acute lung injury in mice.Methods and resultsThe ALI mice model was performed by intratracheally instilling with lipopolysaccharide (LPS). The CASC2 expression detected by quantitative real-time polymerase chain reaction was significantly decreased in LPS-induced A549 cell and ALI mice model. LPS induced A549 cell apoptosis, while transfection with pcDNA-CASC2 reversed the increased cell apoptosis, suggesting overexpression of CASC2 inhibited LPS-induced A549 cell apoptosis. In addition, we found that miR-144-3p expression were opposite to CASC2, while Aquaporin-1 (AQP1) expression was opposite to miR-144-3p in LPS-induced A549 cell and ALI mice model. The RNA immunoprecipitation and RNA pull-down assay demonstrated that CASC2 could function as a miR-144-3p decoy. The luciferase reporter assay revealed that AQP1 was a target of miR-144-3p in A549 cell. And then, further in vitro studied showed that CASC2 controlled AQP1 expression by regulating miR-144-3p, and LPS induced A549 cell apoptosis by regulating CASC2/miR-144-3p/AQP1 axis. At last, after injection with lentivirus-expressing CASC2 or control lentivirus, the mice were intratracheally instilled with LPS. Comparing to the mice injected with pcDNA, the mice injected with pcDNA-CASC2 had a significantly reduced lung wet–dry weight ratio.ConclusionsLong non-coding RNA CASC2 improved acute lung injury by regulating miR-144-3p/AQP1 axis to reduce lung epithelial cell apoptosis.

Mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model

BackgroundMesenchymal stem (stromal) cells (MSCs) mediate their immunoregulatory and tissue repair functions by secreting paracrine factors, including extracellular vesicles (EVs). In several animal models of human diseases, MSC-EVs mimic the beneficial effects of MSCs. Influenza viruses cause annual outbreaks of acute respiratory illness resulting in significant mortality and morbidity. Influenza viruses constantly evolve, thus generating drug-resistant strains and rendering current vaccines less effective against the newly generated strains. Therefore, new therapies that can control virus replication and the inflammatory response of the host are needed. The objective of this study was to examine if MSC-EV treatment can attenuate influenza virus-induced acute lung injury in a preclinical model.MethodsWe isolated EVs from swine bone marrow-derived MSCs. Morphology of MSC-EVs was determined by electron microscopy and expression of mesenchymal markers was examined by flow cytometry. Next, we examined the anti-influenza activity of MSC-EVs in vitro in lung epithelial cells and anti-viral and immunomodulatory properties in vivo in a pig model of influenza virus.ResultsMSC-EVs were isolated from MSC-conditioned medium by ultracentrifugation. MSC-EVs were round-shaped and, similarly to MSCs, expressed mesenchymal markers and lacked the expression of swine leukocyte antigens I and II. Incubation of PKH-26-labeled EVs with lung epithelial cells revealed that MSC-EVs incorporated into the epithelial cells. Next, we examined the anti-influenza and anti-inflammatory properties of MSC-EVs. MSC-EVs inhibited the hemagglutination activity of avian, swine, and human influenza viruses at concentrations of 1.25–5 μg/ml. MSC-EVs inhibited influenza virus replication and virus-induced apoptosis in lung epithelial cells. The anti-influenza activity of MSC-EVs was due to transfer of RNAs from EVs to epithelial cells since pre-incubation of MSC-EVs with RNase enzyme abrogated the anti-influenza activity of MSC-EVs. In a pig model of influenza virus, intratracheal administration of MSC-EVs 12 h after influenza virus infection significantly reduced virus shedding in the nasal swabs, influenza virus replication in the lungs, and virus-induced production of proinflammatory cytokines in the lungs of influenza-infected pigs. The histopathological findings revealed that MSC-EVs alleviated influenza virus-induced lung lesions in pigs.ConclusionsOur data demonstrated in a relevant preclinical large animal model of influenza virus that MSC-EVs possessed anti-influenza and anti-inflammatory properties and that EVs may be used as cell-free therapy for influenza in humans.

Hesperidin induces apoptosis and G0/G1 arrest in human non-small cell lung cancer A549 cells.

Lung cancer has high incidence and mortality rates worldwide. In the present study, the mechanisms by which hesperidin decreases the viability and induces the apoptosis of human non-small cell lung cancer(NSCLC) A549 cells were investigated. Initially, MTT and flow cytometric assays were performed to evaluate the effects of hesperidin on the viability and apoptosis of A549 cells and human normal lung epithelial BEAS-2B cells. The results revealed that hesperidin has no negative effects on the human normal lung epithelial BEAS-2B cells and the viability of cells treated with various concentrations of hesperidin was inhibited in a time- and dose-dependent manner compared with the control groups. Subsequently, the expression levels of proteins involved in the mitochondria-associated apoptotic pathway were studied by western blot analysis. Hesperidin was identified to induce A549 cell apoptosis by downregulating the levels of B-cell lymphoma-2(Bcl-2) and Bcl extra large protein and simultaneously upregulating the levels of Bcl-2-associated Xprotein, BH3 interacting-domain death agonist(Bid), tBid, cleaved caspase-9, cleaved caspase-3 and cleaved poly(adenosine diphosphate ribose)polymerase. The effect of hesperidin on the cell cycle was assessed by flow cytometry. Hesperidin was observed to cause G0/G1 arrest of A549 cells by decreasing the expression of cyclinD1 and increasing the expression of p21 and p53. In summary, it was demonstrated that hesperidin induced apoptosis through the mitochondrial apoptotic pathway and induced G0/G1 arrest in human NSCLC A549 cells. Therefore, hesperidin may be developed as a potential therapeutic drug for the treatment of NSCLC.

SOCS-1 ameliorates smoke inhalation-induced acute lung injury through inhibition of ASK-1 activity and DISC formation

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of apoptosis and pro-inflammatory cytokine signaling, two major contributors to the pathogenesis of ALI. We have found that SOCS-1 protects lung epithelial cells from smoke-induced apoptosis through two mechanisms. One is that SOCS-1 enhances degradation of ASK-1 and diminishes cleavage of pro-caspase-3 to repress smoke-triggered apoptosis in lung epithelial cells. The other is that SOCS-1 represses smoke-triggered DISC formation through altering TRADD-caspase-8 interaction rather than TNFR-1-TRADD interaction or TNFR-1-TRAF-2 interaction. In conclusion, SOCS-1 relieves smoke inhalation-induced lung injury by repressing ASK-1 and DISC-mediated epithelium apoptosis.

Highly pathogenic avian influenza A virus H5N1 non-structural protein 1 is associated with apoptotic activation of the intrinsic mitochondrial pathway.

Outbreaks of avian influenza A (H5N1) virus infection have significant health and economic consequences. Non-structural protein 1 (NS1) is an essential virulence factor of the highly pathogenic H5N1 avian influenza virus and of the apoptosis associated with the pathogenesis of H5N1. Previous studies have revealed that the NS1 protein is able to induce apoptosis via an extrinsic pathway. However, it remains unclear whether the intrinsic pathway is also associated with this apoptosis. The present study used a clone of the NS1 gene from avian influenza A/Jiangsu/1/2007 and observed the localization of the NS1 protein and cytochrome c release from mitochondria and the change of mitochondrial membrane potential (MMP) in lung cancer cells. Cytotoxicity was detected using an MTT assay and the number of apoptotic cells was counted using a flow cytometer. Following the isolation of mitochondria, western blotting was performed to compare cytochrome c release from the mitochondria in cells before and after apoptosis. The change of MMP was detected using JC-1 staining. Furthermore, the results reveal that the majority of the NS1 protein was localized in the cell nucleus, and that it may induce apoptosis of human lung epithelial cells. The apoptosis occurred with marked cytochrome c release from mitochondria and a change of the MMP. This indicated that the NS1 protein may be associated with apoptosis induced by an intrinsic mitochondrial pathway.

Pseudomonas Quinolone Signal Induces Oxidative Stress and Inhibits Heme Oxygenase-1 Expression in Lung Epithelial Cells.

Pseudomonasaeruginosa causes lung infections in patients with cystic fibrosis (CF). The Pseudomonas quinolone signal (PQS) compound is a secreted P. aeruginosa virulence factor that contributes to the pathogenicity of P. aeruginosa We were able to detect PQS in sputum samples from CF patients infected with P. aeruginosa but not in samples from uninfected patients. We then tested the hypothesis that PQS induces oxidative stress in host cells by determining the ability of PQS to induce the production of reactive oxygen species (ROS) in lung epithelial cells (A549 and primary normal human bronchial epithelial [NHBE]) cells and macrophages (J774A.1 and THP-1). ROS production induced by PQS was detected with fluorescent probes (dichlorodihydrofluorescein diacetate, dihydroethidium, and MitoSOX Red) in conjunction with confocal microscopy and flow cytometry. PQS induced ROS production in lung epithelial (A549 and NHBE) cells and macrophages (J774A.1 and THP-1 cells). NHBE cells were sensitive to PQS concentrations as low as 500 ng/ml. PQS significantly induced early apoptosis (P < 0.05, n = 6) in lung epithelial cells, as measured by annexin/propidium iodide detection by flow cytometry. However, no change in apoptosis upon PQS treatment was seen in J774A.1 cells. Heme oxygenase-1 (HO-1) protein is an antioxidant enzyme usually induced by oxidative stress. Interestingly, incubation with PQS significantly reduced HO-1 and NrF2 expression in A549 and NHBE cells but increased HO-1 expression in J774A.1 cells (P < 0.05, n = 3), as determined by immunoblotting and densitometry. These PQS effects on host cells could play an important role in the pathogenicity of P. aeruginosa infections.

Cathelicidins display conserved direct antiviral activity towards rhinovirus

Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in immunocompromised individuals and patients with pre-existing pulmonary conditions. The therapeutic options available are extremely limited and therefore novel therapeutics for HRV infections are of significant interest. Cathelicidins have been shown to have potent antiviral activity against a range of pathogens and are known to be key immunomodulatory mediators during infection. We therefore assessed the antiviral potential of cathelicidins from humans and other mammalian species against HRV, together with the potential for the human cathelicidin to modulate apoptotic pathways and alter cell viability during HRV infection. We demonstrate that LL-37, the porcine cathelicidin Protegrin-1, and the ovine cathelicidin SMAP-29 display potent antiviral activity towards HRV and that this activity is visible when either the virus is exposed to the peptides prior to cell infection or after cells have been infected. We further demonstrate that, in contrast to established findings with bacterial infection models, LL-37 does not induce apoptosis or necrosis in HRV-infected lung epithelial cells at physiological or superphysiological concentrations, but does reduce the metabolic activity of infected cells compared to uninfected cells treated with similar peptide concentrations. Collectively, the findings from this study demonstrate that the mechanism of action of cathelicidins against rhinovirus is by directly affecting the virus and we propose that the delivery of exogenous cathelicidins, or novel synthetic analogues, represent an exciting and novel therapeutic strategy for rhinovirus infection.