Apoptosis Of BEAS-2B Cells Research Articles

Ebeiedinone and peimisine inhibit cigarette smoke extract-induced oxidative stress injury and apoptosis in BEAS-2B cells.

Ebeiedinone and peimisine are the major active ingredients of Fritillariae Cirrhosae Bulbus. In this study, we looked at how these two forms of isosteroidal alkaloids protect human bronchial epithelial BEAS-2B cells from oxidative stress and apoptosis caused by cigarette smoke extract (CSE). First, the cytotoxicity was determined using the CCK8 assay, and an oxidative stress model was established. Then the antioxidative stress activity and mechanism were investigated by ELISA, flow cytometry, and Western blotting. By the CCK-8 assay, exposure to CSE (20%, 40%, and 100%) reduced the viability of BEAB-2S cells. The flow cytometry findings indicated that CSE-induced production of ROS (0.5% to maximum) and treatments with 10μM ebeiedinone and 20μM peimisine attenuated the production of ROS. The western blot assay results indicate that ebeiedinone and peimisine reduce CSE-induced oxidative stress, DNA damage, apoptosis, and autophagy dysregulation by inhibiting ROS, upregulating SOD and GSH/GSSG, and downregulating MDA, 4-HNE, and 8-OHdG through the NRF2/KEAP1 and JNK/MAPK-dependent pathways, thereby delaying the pathological progression of COPD caused by CS.Our data suggest that CSE causes oxidative stress, DNA damage, and apoptosis in BEAS-2B cells, as well as the progression of COPD. Ebeiedinone and peimisine fight CS-induced COPD by suppressing autophagy deregulation and apoptosis.

TROP2 promotes PINK1-mediated mitophagy and apoptosis to accelerate the progression of senile chronic obstructive pulmonary disease by up-regulating DRP1 expression

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterised by irreversible airflow limitation. The elderly are a vulnerable population for developing COPD. With the growth of age, physiological degenerative changes occur in the thorax, bronchus, lung and vascular wall, which can lead to age-related physiological attenuation of lung function in the elderly, so the prevalence of COPD increases with age. Its pathogenesis has not yet been truly clarified. Mitophagy plays an important role in maintaining the stability of mitochondrial function and intracellular environment by scavenging damaged mitochondria. Currently, studies have shown that trophoblast antigen 2 (TROP2) expression is up-regulated in airway basal cells of patients with COPD, suggesting that TROP2 is involved in the progression of COPD. However, whether it is involved in disease progression by regulating mitochondrial function remains unclear. In this study, compared with non-smoking non-COPD patients, the expression of TROP2 in lung tissues of smoking non-COPD patients and patients with COPD increased, and TROP2 expression in patients with COPD was higher than that in smoking non-COPD patients. To further explore the role of TROP2, we stimulated BEAS-2B with cigarette smoke to construct an in vitro model. We found that TROP2 expression increased, whereas TROP2 silencing reversed the cigarette smoke extract-induced decrease in mitochondrial membrane potential, increased reactive oxygen species content, decreased adenosine triphosphate (ATP) production, increased inflammatory factor secretion and increased apoptosis. In addition, we searched online bioinformatics and screened the gene dynamin-related protein 1 (DRP1) related to mitophagy as the research object. Co-IP assay verified the binding relationship between DRP1 and TROP2. Further study found that TROP2 promoted mitophagy and apoptosis of BEAS-2B cells by up-regulating the expression of DRP1. In addition, PTEN-induced putative kinase 1 (PINK1) is a potential binding protein of DRP1, and DRP1 accelerated mitophagy and apoptosis of BEAS-2B cells by promoting the expression of PINK1. We established a COPD SD rat model by cigarette smoke exposure and LPS instillation and treated it by intraperitoneal injection of si-TROP2. The results showed that TROP2 silencing restored lung function and reduced the secretion of inflammatory factors in bronchoalveolar lavage fluid. In conclusion, TROP2 can be used as a new reference for COPD treatment.

Circ_0070934 promotes MGAT3 expression and inhibits epithelial-mesenchymal transition in bronchial epithelial cells by sponging miR-199a-5p

BackgroundCircular RNA (circRNA) has the potential to serve as a crucial regulator in the progression of bronchial asthma. The objective of this investigation was to elucidate the functional dynamics of the circ_0070934/miR-199a-5p/Mannoside acetylglucosaminyltransferase 3 (MGAT3) axis in the development of asthma.MethodsCirc_0070934, miR-199a-5p and MGAT3 in peripheral venous blood of 38 asthmatic patients and 43 healthy controls were detected by qRT-PCR, and the expression of MGAT3 protein was examined by ELISA. The GSE148000 dataset was analyzed for differences in MGAT3. The BEAS-2B cells were transfected with circ_0070934 plasmid and small interfering RNA, miR-199a-5p mimics and inhibitors. The apoptosis level was detected by flow cytometry and MGAT3 was detected by qRT-PCR and Western blot. The expression of E-cadherin, N-cadherin, Vimentin was examined by Western blot. Interleukin-4 (IL-4) and IL-13 were used to co-stimulate BEAS-2B cells as an asthmatic airway epithelial cell model. BEAS-2B cells exposed to type 2 cytokines (IL-4 and IL-13) were treated with circ_0070934 plasmid, and the expression of E-cadherin, N-cadherin, and Vimentin was detected by Western blot. The binding relationships were verified using dual-luciferase reporter assay and miRNA pull-down assay.ResultsThe expression of circ_0070934 and MGAT3 in peripheral venous blood of asthmatic patients was down-regulated, and the expression of miR-199a-5p was up-regulated. And the expression of MGAT3 was reduced in sputum of asthma patients. Down-regulating the expression of circ_0070934 could promote apoptosis of BEAS-2B cells and increase epithelial-mesenchymal transition (EMT), and this effect can be partially reversed by down-regulating miR-199a-5p. Circ_0070934 could inhibit the process of epithelial mesenchymal transition induced by IL-4 and IL-13 in BEAS-2B cells. In addition, miR-199a-5p could respectively bind to circ_0070934 and MGAT3.ConclusionThe findings of this study indicate that circ_0070934 may function as a competitive endogenous RNA (ceRNA) of miR-199a-5p, thereby modulating the expression of MGAT3 and impacting the process of EMT in bronchial epithelial cells. These results contribute to the establishment of a theoretical framework for advancing the prevention and treatment strategies for asthma.

Forced expression of microRNA-221-3p exerts protective effects against manganese-induced cytotoxicity in human lung epithelial cells

Manganese (Mn)-induced pulmonary toxicity and the underlying molecular mechanisms remain largely enigmatic. Further, in recent years, microRNAs (miRNAs) have emerged as regulators of several pollutants-mediated toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in manganese (II) chloride (MnCl2) (Mn2+)-induced cytotoxicity in lung epithelial cells. Growth inhibition of Mn2+ towards normal human bronchial epithelial (BEAS-2B) and adenocarcinomic human alveolar basal epithelial (A549) cells was analyzed by MTT assay following 24 or 48 h treatment. Reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), cell cycle arrest, and apoptosis were evaluated by flow cytometry. RT-qPCR and Western blot were performed to analyze the expression of cyclins, anti-oxidant genes, and miRNAs. We used small RNA sequencing to investigate Mn2+-induced changes in miRNA expression patterns. In both cell lines, Mn2+ treatment inhibited growth in a dose-dependent manner. Further, compared with vehicle-treated cells, Mn2+ (250 μM) treatment induced ROS generation, cell cycle arrest, apoptosis, and decreased ΔΨm as well as altered the expression of cyclins and anti-oxidant genes. Sequencing data revealed that totally 296 miRNAs were differentially expressed in Mn2+-treated cells. Among them, miR-221-3p was one of the topmost down-regulated miRNAs in Mn2+-treated cells. We further confirmed this association in A549 cells. In addition, transient transfection was performed to study gain-of-function experiments. Forced expression of miR-221-3p significantly improved cell viability and reduced Mn2+-induced cell cycle arrest and apoptosis in BEAS-2B cells. In conclusion, miR-221-3p may be the most likely target that accounts for the cytotoxicity of Mn2+−exposed lung epithelial cells.

Lonicera japonica Thunb extract ameliorates lipopolysaccharide-induced acute lung injury associated with luteolin-mediated suppression of NF-κB signaling pathway

ObjectiveLonicera japonica Thunb (LJT) is a commonly used herbal soup to treat inflammation-related diseases. However, the effect of LJT on ALI is unknown. The present study was aimed at investigating the protective effects of LJT extract (LTE) and its active ingredient luteolin (Lut) on lipopolysaccharide (LPS)-stimulated ALI and investigate its potential mechanism.Materials and methodsThe effects of LTE and Lut were explored in an ALI mouse model induced by intraperitoneal injection of lipopolysaccharide (LPS). Besides, the LPS-induced inflammation model in BEAS-2B cells was used to clarify the underlying mechanisms. The ALI pathological changes in lung tissues were tested through Haematoxylin and eosin (HE) staining. The apoptosis of cells in lung tissue and the cell model in vitro was evaluated by TUNEL assays, respectively. Meanwhile, the viability of cells in vitro was evaluated by Cell Counting Kit-8 (CCK-8) assay. The levels/concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and IL-10 in BALF were detected by enzyme-linked immunosorbent assay (ELISA). Besides, through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, the expression of the above-mentioned inflammatory factors and key factors in the NF-κB signaling pathway was examined. The distribution of inflammatory factors in tissue was observed through immunohistochemistry (IHC) assays .ResultsIn relative to LPS-stimulated group, the in vivo study showed that LTE and different concentrations of Lut dramatically alleviated LPS-evoked lung pathological injury and lung edema based on the changes in total protein levels and lung wet/dry (W/D) ratio in the bronchoalveolar lavage fluid (BALF) from ALI mice. LTE and different concentrations of Lut also suppressed the inflammatory response, as reflected by the variations of neutrophil accumulation and the production of proinflammatory and anti-inflammatory cytokines in the lung tissues and BALF of ALI mice. The in vitro research also demonstrated that LTE and Lut visibly facilitated cell viability and restrained the apoptosis of BEAS-2B cells stimulated by LPS. Lut hindered LPS-inducible activation of NF-κB pathway in BEAS-2B cells.ConclusionThe present study proved that LTE might suppress LPS-induced acute injury and inflammation in mice and BEAS-2B cells through the Lut-caused suppression of NF-κB signal path (Figure 1).

Tensile Overload Injures Human Alveolar Epithelial Cells through YAP/F-Actin/MAPK Signaling.

Explosion shockwaves can generate overloaded mechanical forces and induce lung injuries. However, the mechanism of lung injuries caused by tensile overload is still unclear. Flow cytometry was used to detect the apoptosis of human alveolar epithelial cells (BEAS-2B) induced by tensile overload, and cell proliferation was detected using 5-ethynyl-2'-deoxyuridine (EdU). Immunofluorescence and Western blot analysis were used to identify the tensile overload on the actin cytoskeleton, proteins related to the mitogen-activated protein kinase (MAPK) signal pathway, and the Yes-associated protein (YAP). Tensile overload reduced BEAS-2B cell proliferation and increased apoptosis. In terms of the mechanism, we found that tensile overload led to the depolymerization of the actin cytoskeleton, the activation of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase 1/2 (ERK1/2), and the upregulation of YAP expression. Jasplakinolide (Jasp) treatment promoted the polymerization of the actin cytoskeleton and reduced the phosphorylation of tension-overload-activated JNK and ERK1/2 and the apoptosis of BEAS-2B cells. Moreover, the inhibition of the JNK and ERK1/2 signaling pathways, as well as the expression of YAP, also reduced apoptosis caused by tensile overload. Our study establishes the role of the YAP/F-actin/MAPK axis in tensile-induced BEAS-2B cell injury and proposes new strategies for the treatment and repair of future lung injuries.

Geranylgeranylacetone, an inducer of heat shock protein 70, attenuates pulmonary fibrosis via inhibiting NF-κB/NOX4/ROS signalling pathway in vitro and in vivo

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive pulmonary disease which is characterized by epithelial cell damage and extracellular collagen deposition. To date, the therapeutic options for IPF are still very limited, so the relevant mechanisms need to be explored. Heat shock protein 70 (HSP70), which has protective versus antitumor effects on cells under stress, is a member of the heat shock protein family. In the current study, qRT–PCR, western blotting, immunofluorescence staining, and migration assays were used to explore the Epithelial-mesenchymal transition (EMT) process in BEAS-2B cells. Moreover, the role of GGA in the process of pulmonary fibrosis was detected by HE, Masson staining, pulmonary function test and immunohistochemistry in C57BL/6 mice. Our results indicated that GGA, as an inducer of HSP70, enhanced the transformation of BEAS-2B cells from epithelial to mesenchymal cells through the NF-κB/NOX4/ROS (reactive oxygen species) signalling pathway and could significantly reduce apoptosis of BEAS-2B cells induced by TGF-β1(Transforming growth factor β1) in vitro. In vivo studies demonstrated that HSP70-inducing drugs, such as GGA, attenuated pulmonary fibrosis progression induced by bleomycin (BLM). Collectively, these results suggested that overexpression of HSP70 attenuated pulmonary fibrosis induced by BLM in C57BL/6 mice and EMT process induced by TGF-β1 through NF-κB/NOX4/ROS pathway in vitro. Thus, HSP70 might be a potential therapeutic strategy for human lung fibrosis.

Safflor Yellow A Protects Beas-2B Cells Against LPS-Induced Injury via Activating Nrf2.

Acute lung injury and its severe form acute respiratory distress syndrome are lethal lung diseases. So far, effective therapy for the diseases is deficient and the prognosis is poor. Recently, it was found activating nuclear factor erythroid 2-related factor 2 could attenuate the injury including inflammation, oxidative stress, and apoptosis in those diseases. To discover novel therapy, we have evaluated safflor yellow A and explored the underlying mechanisms using Beas-2B cells injured by lipopolysaccharide. As a result, safflor yellow A could improve the viability of Beas-2B cells treated with lipopolysaccharide. Further investigations have revealed safflor yellow A suppressed oxidative stress induced by lipopolysaccharide via reducing reactive oxygen species and malondialdehyde, and elevating superoxide dismutase, catalase, and glutathione peroxidase. Meanwhile, the inflammation resulting from lipopolysaccharide was ameliorated through decreasing the pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6. It was also found nuclear factor κB was inactivated by safflor yellow A. In addition, safflor yellow A downregulated cysteinyl aspartate specific proteinase-3 and Bcl-2-associated X protein and upregulated B-cell lymphoma-2 to inhibited apoptosis of Beas-2B cells induced by lipopolysaccharide. The activation of nuclear factor erythroid 2-related factor 2 was observed in Beas-2B cells, which was associated with the protective effects of safflor yellow A. And molecular docking elucidated safflor yellow A interacted with Kelch-like ECH-associated protein 1 to activate nuclear factor erythroid 2-related factor 2. These results can provide evidences for the discovery of novel therapy for further evaluation of safflor yellow A in the treatment of acute lung injury and acute respiratory distress syndrome.

Black phosphorus quantum dots induce autophagy and apoptosis of human bronchial epithelial cells via endoplasmic reticulum stress

PurposeThe board application of black phosphorus quantum dots (BP-QDs) increases the risk of inhalation exposure in the manufacturing process. The aim of this study is to explore the toxic effect of BP-QDs on human bronchial epithelial cells (Beas-2B) and lung tissue of Balb/c mice. Methods: The BP-QDs were characterized using transmission electron microscopy (TEM) and a Malvern laser particle size analyzer. Cell Counting Kit-8 (CCK-8) and TEM were used to detect cytotoxicity and organelle injury. Damage to the endoplasmic reticulum (ER) was detected by using the ER-Tracker molecular probe. Rates of apoptosis were detected by AnnexinV/PI staining. Phagocytic acid vesicles were detected using AO staining. Western blotting and immunohistochemistry were used to examine the molecular mechanisms. Results: After treatment with different concentrations of BP-QDs for 24 h, the cell viability decreased, as well as activation of the ER stress and autophagy. Furthermore, the rate of apoptosis was increased. Inhibition of ER stress caused by 4-phenyl butyric acid (4-PBA) was shown to significantly inhibit both apoptosis and autophagy, suggesting that ER stress could be an upstream mediator of both autophagy and apoptosis. BP-QD-induced autophagy can also inhibit the occurrence of apoptosis using molecules related to autophagy including rapamycin (Rapa), 3-methyladenine (3-MA), and bafilomycin A1 (Bafi A1). In general, BP-QDs activate ER stress in Beas-2B cells, which further induces autophagy and apoptosis, and autophagy may be activated as a factor that protects against apoptosis. We also observed strong staining of related proteins of ER stress, autophagy, and apoptosis proteins in mouse lung tissue following intracheal instillation over the course of a week. Conclusion: BP-QD-induced ER stress facilitates autophagy and apoptosis in Beas-2B cells and autophagy may be activated as a protective factor against apoptosis. Under conditions of ER stress induced by BP-QDs, The interplay between autophagy and apoptosis determines cell fate.

LincRNA00612 inhibits apoptosis and inflammation in LPS-induced BEAS-2B cells via enhancing interaction between p-STAT3 and A2M promoter.

Long non-coding RNAs (lncRNAs) have been reported as key regulators of chronic obstructive pulmonary disease (COPD). This study aimed to figure out the regulatory mechanism as well as the effects of lncRNA00612 (LINC00612) in lipopolysaccharide (LPS)-induced inflammation and apoptosis in BEAS-2B cells. LINC00612 and its co-expressed gene alpha-2-macroglobulin (A2M) were strikingly downregulated in the peripheral venous blood of COPD patients. Overexpressed LINC00612 enhances BEAS-2B cells against apoptosis and inflammatory reactions mediated by LPS, however, an A2M knockdown can attenuate the degree of the enhancement. Bioinformatics analysis revealed putative binding sites between LINC00612, signal transducer and activator of transcription 3 (STAT3) and the A2M promoter, while RNA antisense purification and Chromatin immunoprecipitation were performed to confirm the prediction. Knockdown of LINC00612 impaired the binding of p-STAT3 to the promoter of A2M, which meant that LINC00612 was critical for the binding of STAT3 with the A2M promoter. Therefore, it can be concluded that LINC00612 ameliorates LPS-induced cell apoptosis and inflammation via recruiting STAT3 to bind to A2M. This conclusion will serve as a theoretical foundation for the treatment of COPD.

Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD.

Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, up-regulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis.

Arsenic trioxide promotes ERK1/2-mediated phosphorylation and degradation of BIMEL to attenuate apoptosis in BEAS-2B cells

Inorganic arsenic is highly toxic, widely distributed in the human environment and may result in multisystem diseases and several types of cancers. The BCL-2-interacting mediator of cell death protein (BIM) is a key modulator of the intrinsic apoptosis pathway. Interestingly, in the present study, we found that arsenic trioxide (As2O3) decreased BIMEL levels in human bronchial epithelial cell line BEAS-2B and increased BIMEL levels in human lung carcinoma cell line A549 and mouse Sertoli cell line TM4. Mechanismly, the 26S proteasome inhibitors MG132 and bortezomib could effectively inhibit BIMEL degradation induced by As2O3 in BEAS-2B cells. As2O3 activated extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, but only the ERK1/2 MAPK inhibitor PD98059 blocked BIMEL degradation induced by As2O3. Furthermore, As2O3 induced-phosphorylation of BIMEL at multiple sites was inhibited by ERK1/2 MAPK inhibitor PD98059. Inhibition of As2O3-induced ERK1/2 MAPK phosphorylation increased the levels of BIMEL and cleaved-caspase-3 proteins and decreased BEAS-2B cell viability. As2O3 also markedly mitigated tunicamycin-induced apoptosis of BEAS-2B cells by increasing ERK1/2 phosphorylation and BIMEL degradation. Our results suggest that As2O3-induced activation of the ERK1/2 MAPK pathway increases phosphorylation of BIMEL and promotes BIMEL degradation, thereby alleviating the role of apoptosis in As2O3-induced cell death. This study provides new insights into how to maintain the survival of BEAS-2B cells before malignant transformation induced by high doses of As2O3.

Diesel exhaust particle exposure accelerates oxidative DNA damage and cytotoxicity in normal human bronchial epithelial cells through PD-L1

Diesel exhaust particles (DEPs) are a major cause of cancer progression as well as a variety of acute and chronic diseases. It is well-known that programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that can induce immune escape in tumor cells. However, the function of PD-L1 in bronchial epithelial cells or how PD-L1 relates to cellular oxidation under DEPs-mediated oxidative stress is not well known. In this study, we investigated how PD-L1 affected DEPs-induced oxidative stress and cytotoxicity in human bronchial epithelial (HBE) cells, Beas-2B. DEPs not only induced intracellular reactive oxygen species (ROS) production, but also increased PD-L1 expression in HBE cells. Beas-2B cells overexpressing PD-L1 showed higher levels of ROS production, DNA damage, and apoptosis after DEPs treatment compared to control cells. In particular, the expression of an antioxidant enzyme heme-oxygenase-1 (HO-1) and nuclear translocation and transcriptional activity of Nrf2, a major regulator of HO-1, were lower in Beas-2B overexpressing PD-L1 cells than in control cells. DEPs-induced ROS generation, DNA damage and apoptosis in Beas-2B cells overexpressing PD-L1 were significantly restored by overexpressing HO-1. Collectively, our results suggest that DEPs can increase the expression of PD-L1 in HBE cells and that overexpressing PD-L1 might eventually promote DEPs-induced oxidative DNA damage and apoptosis.

Resveratrol Protects Against Nicotine-Induced Apoptosis by Enhancing Autophagy in BEAS-2B Lung Epithelial Cells

Background: Nicotine (Nic), the major component of tobacco products, can induce apoptosis in lung epithelial cells, and the resulting damage contributes to chronic obstructive pulmonary disease. Apoptosis is closely related to autophagy. Resveratrol (Res) can induce autophagy and inhibit apoptosis. Therefore, the present study investigated whether Nic induces apoptosis of lung epithelial cells by regulating autophagy and the effect of Res on apoptosis of Nic-exposed lung epithelial cells. Methods: The BEAS-2B lung epithelial cell line was used to study the harmful effects of Nic and the potential benefits of Res as well as the underlying mechanisms. Viability and apoptosis were examined using the Cell Counting Kit-8 and annexin V-propidium iodide staining, respectively. The expression of levels of apoptosis-related proteins, autophagy-related proteins, and members of the PI3K/Akt/mTOR pathway was measured by western blotting. Autophagic flux was detected via mRFP-GFP-LC3 double-labeled adenovirus transfection and transmission electron microscopy. Results: Nic significantly reduce the viability and increased the apoptosis of BEAS-2B cells in a concentration-dependent manner. Nic treatment also increased the numbers of autophagosomes in BEAS-2B cells and upregulated LC3II and p62 expression. Moreover, Res at concentration of 2, 10, and 50 μM protected BEAS-2B cells from Nic apoptosis, and the expression of LC3II increased further and p62 decreased in Res pretreatment group. Apart from this, Res reduced Akt and mTOR phosphorylation. Subsequently, upon inhibiting PI3K phosphorylation by PI3K inhibitors, BEAS-2B cell autophagy induced by Res was obviously abolished. Conclusions: Nic-induced BEAS-2B cell apoptosis by inhibiting the late-stage autophagic flux, but Res could protect BEAS-2B cells from the detrimental effects of nicotine by enhancing autophagy via the PI3K/Akt/mTOR pathway. These results will provide an experimental basis for the prevention and treatment of COPD.

SRY-related high-mobility group box 9 (SOX9) alleviates cigarette smoke extract (CSE)-induced inflammatory injury in human bronchial epithelial cells by suppressing stromal interaction molecule 1 (STIM1) expression.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease with airflow limitation and abnormal inflammatory response. It has been verified that SOX9 plays a key role in lung function of various lung diseases and SOX9 is closely associated with COPD. Additionally, literature has reported that STIM1 is involved in lung injury and is highly expressed in neutrophils from COPD patients. This study aimed to characterize the biological roles of SOX9 and STIM1 in the pathogenesis of COPD and to elucidate the regulatory mechanism. Human bronchial epithelial cells (BEAS-2B) were treated with CSE to construct in vitro COPD model. The levels of SOX9 and STIM1 in CSE-treated BEAS-2B cells were detected by western blot and RT-qPCR assay. Then, JASPAR datasets were utilized to analyze SOX9 binding sites in the promoter region of STIM1. Besides, luciferase reporter assay and ChIP assay were employed to validate the binding sites in STIM1 promoter region to SOX9. In addition, viability and apoptosis of BEAS-2B cells were assessed by utilizing MTT assay and TUNEL staining. ELISA kits and corresponding commercial kits were applied to measure the levels of TNF-α, IL-6, IL-1β, SOD, GSH-Px and MDA. CSE treatment dose- and time-dependently reduced SOX9 expression in BEAS-2B cells. SOX9 overexpression enhanced the viability and suppressed the apoptosis of CSE-treated BEAS-2B cells as well as attenuated CSE-induced inflammation and oxidative stress. Then, it was validated that SOX9 bound to the promoter region of STIM1. Moreover, SOX9 overexpression-mediated impacts on cell viability, cell apoptosis, inflammation and oxidative stress in CSE-treated BEAS-2B cells were partially abolished by upregulation of STIM1. To sum up, results here suggested that overexpression of SOX9 could mitigate inflammatory injury in CSE-treated bronchial epithelial cells by suppressing STIM1.

MiR-513a-3p promotes radiation-induced apoptosis of human lung cells by inhibiting glutathione S-transferase P1.

Radiotherapy is a common treatment for lung cancer. However, radiation pneumonitis caused by radiotherapy can affect the quality of life and prognosis of lung cancer patients. miR-513a-3p has been found to sensitize human lung adenocarcinoma cells to chemotherapy by targeting glutathione S-transferase P1 (GSTP1). Here, we found that x-ray induced the apoptosis of BEAS-2B and miR-513a-3p expression in a dose- and time-dependent manner, and miR-513a-3p-mimic significantly increased x-ray induced apoptosis, while miR-513a-3p-inhibitor significantly decreased x-ray induced apoptosis. Dual luciferase gene reporter system showed that miR-513a-3p targeted to inhibit the expression of GSTP1 in BEAS-2B cells. Moreover, knockdown of GSTP1 significantly increased, while overexpression of GSTP1 decreased the apoptosis of BEAS-2B induced by x-ray. Importantly, overexpression of GSTP1 significantly reduced miR-513a-3p-mimic elevated x-ray -induced apoptosis in BEAS-2B cells. In conclusion, x-ray caused increased expression of miR-513a-3p, and miR-513a-3p promoted x-ray-induced apoptosis of human lung cells by inhibiting GSTP1.

Rutaecarpine ameliorates lipopolysaccharide-induced BEAS-2B cell injury through inhibition of endoplasmic reticulum stress via activation of the AMPK/SIRT1 signaling pathway.

Rutaecarpine (RUT) is an alkaloid isolated from Tetradium ruticarpum, which has been reported to protect against several inflammatory diseases. However, to the best of our knowledge, the role of RUT in acute lung injury (ALI) and the specific molecular mechanism remain unknown. In the present study, an in vitro model of ALI was established in BEAS-2B cells by lipopolysaccharide (LPS) administration. Cell viability following RUT treatment with or without LPS stimulation was evaluated using a Cell Counting Kit-8 assay. The inflammatory response and oxidative stress were detected using ELISA kits and commercially available kits, respectively. TUNEL assay and western blotting were performed to assess cell apoptosis. The expression levels of endoplasmic reticulum (ER) stress-related proteins and AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signaling pathway-related proteins were measured by western blotting. The results revealed that RUT markedly improved cell viability after LPS treatment in a dose-dependent manner. In addition, RUT inhibited the LPS-induced inflammatory response and oxidative stress in BEAS-2B cells, and suppressed the LPS-induced apoptosis of BEAS-2B cells. Mechanistically, RUT alleviated ER stress by inhibiting the production of CHOP, glucose-regulated protein-78, caspase-12 and activating transcription factor 6. Additionally, western blotting demonstrated that RUT activated the phosphorylation of AMPK and SIRT1, which indicated the involvement of the AMPK/SIRT1 signaling pathway in the protective effect of RUT against LPS-induced lung injury. In conclusion, these results demonstrated that RUT mitigated LPS-induced lung cell injury by inhibiting ER stress via the activation of the AMPK/SIRT1 signaling pathway.

Methylprednisolone up-regulates annexin A1 (ANXA1) to inhibit the inflammation, apoptosis and oxidative stress of cigarette smoke extract (CSE)-induced bronchial epithelial cells, a chronic obstructive pulmonary disease in vitro model, through the formyl peptide receptor 2 (FPR2) receptors and the adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) pathway

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a progressive degenerative disease, of which smoking is the main causer. We carried out this study with the aim of exploring the underlying mechanism of methylprednisolone (MP) treating the COPD. To stimulate COPD in vitro, cigarette smoke extract (CSE)was employed to induce human bronchial epithelial cells BEAS-2B. With the help of MTT and Tunel assays, the viability and apoptosis of BEAS-2B cells after indicated treatment were assessed. The levels of inflammatory response and oxidative stress were determined by the changes of markers basing on their commercial kits. Additionally, annexin A1 (ANXA1) expressions at both protein and mRNA levels were assessed with Western blot and Reverse transcription‑quantitative PCR (RT-qPCR). Moreover, the expressions of apoptosis- and formyl peptide receptor 2 (FPR2) receptors and the adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) pathway-related proteins were determined with Western blot., related proteins and proteins. As a result, MP up-regulated the ANXA1 expression in CSE-induced BEAS-2B cells. MP enhanced the viability but suppressed the apoptosis, inflammatory response and oxidative stress of CSE-induced BEAS-2B cells via regulating FPR2/AMPK pathway, while ANXA1 knockdown exhibited oppositive effects on them. In conclusion, MP up-regulated ANXA1 to inhibit the inflammation, apoptosis and oxidative stress of BEAS-2B cells induced by CSE, alleviating COPD through suppressing the FPR2/AMPK pathway.

Glycyrrhizin Attenuates c-Src-Mediated Lipopolysaccharide-Induced Inflammatory Response and Apoptosis in Bronchial Epithelial Cells by Upregulating miR-146b-5p

Background: Bronchial asthma is a common chronic inflammatory disease of the respiratory tract, whose pathogenesis involves a variety of factors. The purpose of this study was to explore the effect of traditional Chinese medicine Glycyrrhizin (Gly) on lipopolysaccharide (LPS)-induced inflammation and apoptosis of bronchial epithelial cells and its action mechanism. Methods: Gly (20 µM) was used to treat bronchial epithelial BEAS-2B cells stimulated with LPS. The expression of SRC and miR-146b-5p in BEAS-2B cells was modified by the respective transfections with pcDNA-SRC, miR-146b-5p mimic and miR-146b-5p inhibitor. STRING and Starbase online databases were used to predict the relationship between Gly, miR-146b-5p and SRC. Luciferase reporter assays were performed to verify the binding of miR-146b-5p to SRC. The viability, inflammatory response and apoptosis of BEAS-2B cells were examined by CCK-8, ELISA and Tunel assays respectively. The expressions of apoptosis-related proteins (Bcl-2, Bax, caspase3 and Cleaved-caspase3), SRC and miR-146b-5p were detected by qRT-PCR or western blotting. Results: Gly inhibited LPS-induced inflammation and apoptosis in BEAS-2B cells. The interaction between Gly and SRC was predicted by STRING. SRC expression was high in BEAS-2B cells stimulated with LPS and could be negatively regulated by Gly. Overexpression of SRC effectively alleviated the inhibitory effect of Gly on LPS-induced damages in BEAS-2B cells. In addition, results of luciferase reporter assays verified SRC as a direct target gene of miR-146b-5p. The expression level of miR-146b-5p was downregulated by LPS stimulation in BEAS-2B cells. Gly decreased the expression of SRC in LPS-stimulated BEAS-2B cells. These results could all be reversed by miR-146b-5p knockdown. Conclusion: Gly decreases the expression of SRC by upregulating the level of miR-146b-5p, thus alleviating the inflammation and apoptosis of bronchial epithelial cells treated with LPS. Our results provide a new theoretical basis for applying Gly to the clinical management of asthma.

The toxicity of cooking oil fumes on human bronchial epithelial cells through ROS-mediated MAPK, NF-κB signaling pathways and NLRP3 inflammasome.

Cooking oil fumes (COFs) are the main pollutants in kitchen and indoor air, which threaten human health. Exposure to COFs may lead to respiratory diseases and impair pulmonary function. To investigate the toxicity of COFs on human bronchial epithelial cells (Beas-2B) and explore the underlying mechanisms, MTT assay was conducted to detect the viability of Beas-2B. Intracellular reactive oxygen species (ROS) levels and nitric oxide (NO) levels were determined with DCFH-DA assay and DAF-FM assay. The expression of genes involved in inflammation were measured with quantitative real-time PCR (qRT-PCR). The phosphorylation and the expression of proteins related to Mitogen-activated protein kinase (MAPK), NF-κB signaling pathways were measured with western blot. Our results revealed that COFs decreased cell viability, increased the ROS levels and NO levels and induced apoptosis in Beas-2B cells. The results of qRT-PCR and western blot showed that the expression of NLRP3, p65, iNOS, IL-1β, and the factors related to oxidative stress and inflammation increased, NF-κB signaling pathway and MAPK signaling pathway were activated. This study provided some useful information to evaluate the toxicity of COFs and revealed the possible mechanism for the damage on respiratory system induced by COFs.