BEAS-2B Cells Research Articles

Mitochondrial Reactive Oxygen Species in TRIF-Dependent Toll-like Receptor 3 Signaling in Bronchial Epithelial Cells against Viral Infection.

Toll-like receptor 3 (TLR3) plays an important role in double-stranded RNA recognition and triggers the innate immune response by acting as a key receptor against viral infections. Intracellular reactive oxygen species (ROS) are involved in TLR3-induced inflammatory responses during viral infections; however, their relationship with mitochondrial ROS (mtROS) remains largely unknown. In this study, we show that polyinosinic-polycytidylic acid (poly(I:C)), a mimic of viral RNA, induced TLR3-mediated nuclear factor-kappa B (NF-κB) signaling pathway activation and enhanced mtROS generation, leading to inflammatory cytokine production. TLR3-targeted small interfering RNA (siRNA) and Mito-TEMPO inhibited inflammatory cytokine production in poly(I:C)-treated BEAS-2B cells. Poly(I:C) recruited the TLR3 adaptor molecule Toll/IL-1R domain-containing adaptor, inducing IFN (TRIF) and activated NF-κB signaling. Additionally, TLR3-induced mtROS generation suppression and siRNA-mediated TRIF downregulation attenuated mitochondrial antiviral signaling protein (MAVS) degradation. Our findings provide insights into the TLR3-TRIF signaling pathway and MAVS in viral infections, and suggest TLR3-mtROS as a therapeutic target for the treatment of airway inflammatory and viral infectious diseases.

Neutrophil infiltration associated genes on the prognosis and tumor immune microenvironment of lung adenocarcinoma.

The neutrophils exhibit both anti-tumor and pro-tumor effects in cancers. The correlation between neutrophils and tumor development in lung adenocarcinoma (LUAD) is still uncertain, possibly due to a lack of specific neutrophil infiltration evaluation methods. In this study, we identified 30 hub genes that were significantly associated with neutrophil infiltration in LUAD through data mining, survival analysis, and multiple tumor-infiltrating immune cells (TICs) analysis, including TIMER, CIBERSORT, QUANTISEQ, XCELL, and MCPCOUNTER. Consensus clustering analysis showed that these 30 hub genes were correlated with clinical features in LUAD. We further developed a neutrophil scoring system based on these hub genes. The neutrophil score was significantly correlated with prognosis and tumor immune microenvironment (TIME) in LUAD. It was also positively associated with PD-L1 expression and negatively associated with tumor mutational burden (TMB). When combined with the neutrophil score, the predictive capacity of PD-L1 and TMB for prognosis was significantly improved. Thus, the 30 hub genes might play an essential role in the interaction of neutrophils and LUAD, and the neutrophil scoring system might effectually assess the infiltration of neutrophils. Furthermore, we verified the expression of these 30 genes in the LUAD tumor tissues collected from our department. We further found that overexpressed TNFAIP6 and TLR6 and downregulated P2RY13, SCARF1, DPEP2, PRAM1, CYP27A1, CFP, GPX3, and NCF1 in LUAD tissue might be potentially associated with neutrophils pro-tumor effects. The following in vitro experiments demonstrated that TNFAIP6 and TLR6 were significantly overexpressed, and P2RY13 and CYP27A1 were significantly downregulated in LUAD cell lines, compared to BEAS-2B cells. Knocking down TNFAIP6 in A549 and PC9 resulted in the upregulation of FAS, CCL3, and ICAM-1, and the downregulation of CCL2, CXCR4, and VEGF-A in neutrophils when co-culturing with the conditioned medium (CM) from LUAD cells. Knocking down TNFAIP6 in LUAD also led to an elevated early apoptosis rate of neutrophils. Therefore, overexpressed TNFAIP6 in LUAD cancer cells might lead to neutrophils "N2" polarization, which exhibited pro-tumor effects. Further research based on the genes identified in this pilot study might shed light on neutrophils' effects on LUAD in the future.

The Genotoxicity of Organic Extracts from Particulate Emissions Produced by Neat Gasoline (E0) and a Gasoline-Ethanol Blend (E15) in BEAS-2B Cells.

Emissions from modern gasoline engines represent an environmental and health risk. In this study, we aimed to compare the toxicity of organic compound mixtures extracted from particulate matter (PM extracts) produced by neat gasoline (E0) and a blend containing 15% ethanol (E15), which is offered as an alternative to non-renewable fossil fuels. Human lung BEAS-2B cells were exposed to PM extracts, and biomarkers of genotoxicity, such as DNA damage evaluated by comet assay, micronuclei formation, levels of phosphorylated histone H2AX, the expression of genes relevant to the DNA damage response, and exposure to polycyclic aromatic hydrocarbons (PAHs), were determined. Results showed that both PM extracts significantly increased the level of oxidized DNA lesions. The E0 extract exhibited a more pronounced effect, possibly due to the higher content of nitrated PAHs. Other endpoints were not substantially affected by any of the PM extracts. Gene expression analysis revealed mild but coordinated induction of genes related to DNA damage response, and a strong induction of PAH-inducible genes, indicating activation of the aryl hydrocarbon receptor (AhR). Our data suggest that the addition of ethanol into the gasoline diminished the oxidative DNA damage, but no effect on other genotoxicity biomarkers was observed. Activated AhR may play an important role in the toxicity of gasoline PM emissions.

Multiplexed transcriptional profiling of Dermatophagoides house dust mites allergens in human epithelium cells.

Allergic asthma, a chronic disease characterized by airway inflammation, poses a significant public health concern. It is well-established that house dust mites (HDMs) are common inducers of allergic responses in individuals, particularly children. In central Taiwan, our research team observed that over 80% of allergic children exhibited sensitization to various HDMs species. This investigation aims to bridge the gap between these observations and a better understanding of the early fundamental mechanisms for preventing allergic diseases. Specifically, our study delves into the impact of crude extracts of HDMs on human epithelial BEAS-2B cells. Our findings, based on RNA sequencing (RNA-seq) analysis, shed light on how three major Dermatophagoides HDMs allergens activate a common Toll-like receptor signaling pathway in human epithelial cells within a 4-h treatment. During this process, the nuclear transcription factor NF-κB translocated into the cell nucleus within 30 min of allergen stimulation, triggering the expression of pro-inflammatory genes such as IL-6 and IL-8 over 4 h. Additionally, when the cells were treated with specific Dermatophagoides microceras (Der m) allergens, it resulted in the upregulation of genes that regulate type 1 diabetes mellitus (T1DM) signaling pathways. This led to the mediation of IL-12A inflammation. Furthermore, there was an increase in gene sets associated with cilia function and the microtubule cytoskeleton in human epithelial cells after treatment with a combination of Der m allergens and Dexamethasone. Additionally, OMICs analysis was conducted to examine the effects of HDMs allergenic stimulation on human epidermal cells. We aimed to improve our understanding of the molecular mechanisms within cells and identify potential targets and natural products in the treatment of asthma caused by HDMs allergens.

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).

Long non-coding RNA SNHG4 aggravates cigarette smoke-induced COPD by regulating miR-144-3p/EZH2 axis

ObjectiveThe purpose of this study was to explore the expression level of SNHG4 in patients with COPD and its diagnostic value in COPD, to probe the biological function of SNHG4 in COPD at the cellular level, and to reveal the interaction between SNHG4 and miR-144-3p/EZH2 axis.MethodsThe serum levels of SNHG4, miR-144-3p and EZH2 in healthy people and patients with COPD were detected by RT-qPCR. The diagnostic value of SNHG4 in COPD was evaluated by ROC curve. Pearson method was chosen to estimate the correlation between SNHG4 and clinical indicators in patients with COPD. Cigarette smoke extract (CSE) was obtained, and Beas-2B cells were exposed with 2% CSE to establish an inflammatory cell model of COPD in vitro. MTT assay was used to detect cell viability, flow cytometry was used to evaluate cell apoptosis, and ELISA was performed to detect inflammatory cytokines. Dual-luciferase reporting assay was carried out to verify the targeting of lncRNA-miRNA or miRNA-mRNA.Results(1) The expression of SNHG4 is decreased in patients with COPD, and the expression level in acute exacerbation COPD was lower than that in stable COPD. SNHG4 demonstrated high diagnostic accuracy in distinguishing between stable and acute exacerbation COPD. (2) The expression of SNHG4 was decreased in CSE-induced Beas-2B cells, and overexpression of SNHG4 was beneficial to alleviate CSE-induced apoptosis and inflammation. (3) The expression of miR-144-3p is up-regulated in patients with COPD and CSE-induced Beas-2B cells. MiR-144-3p has a targeting relationship with SNHG4, which is negatively regulated by SNHG4. Overexpression of miR-144-3p could counteract the beneficial effects of increased SNHG4 on CSE-induced cells. (4) The expression of EZH2 is reduced in patients with COPD and CSE-induced Beas-2B cells. Bioinformatics analysis and luciferase reporter gene confirmed that EZH2 is the downstream target gene of miR-144-3p and is negatively regulated by miR-144-3p.ConclusionThe expression of SNHG4 decreased in patients with COPD, and it may promote the progression of COPD by inhibiting the viability, promoting apoptosis and inflammatory response of bronchial epithelial cells via regulating the miR-144-3p/EZH2 axis.

Yeni Sentezlenen Potansiyel HIF İnhibitörlerinin Hipoksik Koşullarda Küçük Hücre Dışı Akciğer Kanseri Üzerindeki İnhibitör Etkilerinin Belirlenmesi

According to global estimates, there are 2.3 million new cases and 1.8 million fatalities due to lung cancer each year. Despite recent progress in diagnosis and treatment, persistent challenges highlight the urgent need for novel therapeutics and innovative approaches to combat lung cancer effectively. Accordingly, in the present study, we aimed to investigate the anticancer properties of potential inhibitors of HIF-1α, compound 7a and 7b. In the study, HTB-54 and BEAS-2B cell lines were used. MTT cell viability experiments were performed to determine the effect of newly synthesized HIF inhibitors 7a and 7b on cell viability under normoxic and hypoxic conditions. Quantitative expression levels of HIF1A were determined by real-time PCR approach. While the half maximum inhibitory concentration (IC50) of compound 7a in HTB-54 cells was 10.37 µM under normoxic conditions, it was found to be 10.63 µM under hypoxic conditions. The IC50 value of another HIF inhibitor 7b in HTB-54 cells was found to be 8.80 µM under normoxic conditions and 9.54 µM under hypoxic conditions. The expression level of HIF1A was found to be lower in cells exposed to compounds 7a and 7b under hypoxia compared to the control group. Conversely, in normoxia, HIF1A expression level in cells exposed to compound 7a increased 6.5-fold (p<0.0001) compared to the control group, while it was found to increase approximately 9-fold (p<0.0001) when exposed to 7b. Consequently, both compound 7a and 7b holds great promise for future therapeutic interventions to lung cancer.

Long non-coding RNA (LncRNA) non-coding RNA activated by DNA damage (NORAD) knockdown alleviates airway remodeling in asthma via regulating miR-410-3p/RCC2 and inhibiting Wnt/β-catenin pathway

BackgroundAsthma is a chronic inflammatory disorder with high prevalence in childhood. Airway remodeling, an important structural change of the airways, is resulted from epithelial-mesenchymal transition. Long non-coding RNA non-coding RNA activated by DNA damage (NORAD) has been found to promote epithelial-mesenchymal transition in multiple cancers. This study aimed to analyze the role of NORAD in asthma, mainly focusing on epithelial-mesenchymal transition-mediated airway remodeling, and further explored the NORAD-miRNA-mRNA network. MethodsNORAD expression was analyzed in transforming growth factor-β1-induced BEAS-2B human bronchial epithelial cells and ovalbumin-challenged asthmatic mice. The influences of NORAD on the epithelial-mesenchymal transition characteristics and Wnt/β-catenin pathway activation were analyzed in vitro. The interactions between NORAD and miR-410-3p as well as miR-410-3p and regulator of chromosome condensation 2 were detected by dual-luciferase reporter assay and RNA pull-down assay. Rescue experiments using miR-410-3p antagonist and chromosome condensation 2 overexpression were used to confirm the mechanism of NORAD. Additionally, the role and mechanism of NORAD were further evaluated in asthmatic mice. ResultsNORAD expression was elevated in both asthmatic models. Knockdown of NORAD impeded spindle-like morphology changes, elevated E-cadherin expression, decreased N-cadherin expression, suppressed cell migration, and inactivated the Wnt/β-catenin pathway in transforming growth factor-β1-stimulated BEAS-2B cells. NORAD acted as a sponge of miR-410-3p to regulate chromosome condensation 2 expression. Rescue assays demonstrated that silencing of NORAD ameliorated transforming growth factor-β1-induced EMT via miR-410-3p/chromosome condensation 2/Wnt/β-catenin axis. In vivo, knockdown of NORAD led to the reduction of inflammatory cell infiltration and collagen deposition, suppression of IL-4, IL-13, transforming growth factor-β1 and immunoglobulin E production, decreasing of N-cadherin, chromosome condensation 2, β-catenin and c-Myc expression, but increasing of E-cadherin and miR-410-3p expression. ConclusionsSilencing of NORAD alleviated epithelial-mesenchymal transition-mediated airway remodeling in asthma via mediating miR-410-3p/chromosome condensation 2/Wnt/β-catenin pathway.

Proteomic analysis reveals the molecular mechanism of Astragaloside in the treatment of non-small cell lung cancer by inducing apoptosis

BackgroundAstragaloside III (AS III), a saponin-like metabolite derived from the traditional Chinese medicine Astragali Radix, has been shown to be effective in the treatment of cancer and heart failure, and a variety of digestive disorders. However, its molecular mechanism in the treatment of non-small cell lung cancer (NSCLC) is unknown.MethodsHuman lung cancer A549 cells and NCI-H460 cells and a normal human lung epithelial cell BEAS-2B were treated with different concentrations of AS III. CCK-8 and EdU staining were used to determine the anti-proliferative effects of AS III in vitro. Quantitative proteomic analysis was performed on A549 cells treated with the indicated concentrations of AS III, and the expression levels of apoptosis-related proteins were examined by Western blotting.ResultsAS III treatment significantly inhibited proliferation and increased apoptosis in A549 and H460 cells and modulated functional signaling pathways associated with apoptosis and metabolism. At the molecular level, AS III promoted a reduction in the expression of ANXA1 (p < 0.01), with increased levels of cleaved Caspase 3 and PARP 1. In addition, AS III treatment significantly decreased the LC3-I/LC3-II ratio. The results of experiment in vitro showed that AS III promoted NSCLC apoptosis by down-regulating the phosphorylation levels of P38, JNK, and AKT (p < 0.01), inhibiting the expression of Bcl-2 (p < 0.01), and up-regulating the expression of Bax (p < 0.01).ConclusionThese findings provide a mechanism whereby AS III treatment induces apoptosis in NSCLC cells, which may be achieved in part via modulation of the P38, ERK and mTOR signaling pathways.

TRPM2 exacerbates airway inflammation by regulating oxidized-CaMKⅡ in allergic asthma

BackgroundAirway epithelial cells play important roles in allergic asthma. Transient receptor potential melastatin-related 2 (TRPM2) and oxidized Ca2+/calmodulin-dependent protein kinase Ⅱ (ox-CaMKⅡ) participate in the airway inflammation. This study aimed to analyze the effects of TRPM2 on ox-CaMKⅡ in the airway epithelial cells during allergic asthma. MethodsBEAS-2B cells were treated with different dose of IL-13 (0, 5, 10, 20 ng/mL) for 24 h to analyze the changes of TRPM2 and ox-CaMKⅡ protein. Cells expressing different level of TRPM2 were obtained by transfection of TRPM2 siRNA or TRPM2-short cDNA. The transfected cells were treated with 10 ng/mL of IL-13 to analyze the effects of TRPM2 on the ox-CaMKⅡ. A CaMKⅡ inhibitor KN-93 was used to confirm the effects of TRPM2 on levels of ox-CaMKⅡ, p-MEK and p-ERK in the IL-13-treated BEAS-2B cells. Wild-type (WT) mice and TRPM2-knockout (TRPM2−/−) mice were induced by ovalbumin (OVA) to compare the differences of inflammation, levels of ox-CaMKII, p-MEK and p-ERK in airways. ResultsCell viability was clearly decreased by the 20 ng/mL of IL-13. The levels of TRPM2 and ox-CaMKII protein in cells were increased with increasing doses of IL-13. Transfection of TRPM2 siRNA or TRPM2-short cDNA respectively decreased or increased the levels of ox-CaMKⅡ in the IL-13-stimulated cells. The results of KN-93 treatment were similar to the results of TRPM2 siRNA transfection, that the levels of ox-CaMKⅡ, p-MEK and p-ERK were significantly decreased in the IL-13-treated cells. Compared with the OVA-induced WT mice, levels of inflammation, ox-CaMKⅡ, p-MEK and p-ERK in the airways were significantly weakened in the OVA-induced TRPM2−/− mice. ConclusionsTRPM2 plays a vital role in regulating ox-CaMKⅡ in airway epithelial cells during allergic asthma.

Silk Fibroin-Modified Liposome/Gene Editing System Knocks out the PLK1 Gene to Suppress the Growth of Lung Cancer Cells.

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR-Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR-Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy.

Polystyrene nanoplastics-induced lung apoptosis and ferroptosis via ROS-dependent endoplasmic reticulum stress

It has been shown that exposure to nanoplastics (MNPs) through inhalation can induce pulmonary toxicity, but the toxicological mechanism of MNPs on the respiratory system remains unclear. Therefore, we explored the toxicological mechanism of exposure to polystyrene nanoplastics (PS-NPs) (0.05, 0.15, 0.2 mg/mL) on BEAS-2B cells. Results revealed that PS-NPs induce oxidative stress, increased apoptosis rate measured by flow cytometry, the key ferroptosis protein (GPX4 and FTH1) reduction, increased iron content, mitochondrial alterations, and increased malondialdehyde (MDA) level. Besides, consistent results were observed in mice exposed to PS-NPs (5 mg/kg/2d, 10 mg/kg/2d). Thus, we proved that PS-NPs induced cell death and lung damage through apoptosis and ferroptosis. In terms of mechanism, the elevation of the endoplasmic reticulum (ER) stress protein expression (IRE1α, PERK, XBP1S, and CHOP) revealed that PS-NPs induce lung damage by activating the two main ER stress pathways. Furthermore, the toxicological effects of PS-NPs observed in this study are attenuated by the ROS inhibitor N-acetylcysteine (NAC). Collectively, NPs-induced apoptosis and ferroptosis are attenuated by NAC via inhibiting the ROS-dependent ER stress in vitro and in vivo. This improves our understanding of the mechanism by which PS-NPs exposure leads to pulmonary injury and the potential protective effects of NAC.

Daphnetin Alleviates Bleomycin-Induced Pulmonary Fibrosis through Inhibition of Epithelial-to-Mesenchymal Transition and IL-17A.

Idiopathic pulmonary fibrosis (IPF) is a chronic and refractory interstitial lung disease. Although there is no cure for IPF, the development of drugs with improved efficacy in the treatment of IPF is required. Daphnetin, a natural coumarin derivative, has immunosuppressive, anti-inflammatory, and antioxidant activities. However, its antifibrotic effects have not yet been elucidated. In this study, we investigated the antifibrotic effects of daphnetin on pulmonary fibrosis and the associated molecular mechanism. We examined the effects of daphnetin on splenocytes cultured in Th17 conditions, lung epithelial cells, and a mouse model of bleomycin (BLM)-induced pulmonary fibrosis. We identified that daphnetin inhibited IL-17A production in developing Th17 cells. We also found that daphnetin suppressed epithelial-to-mesenchymal transition (EMT) in TGF-β-treated BEAS2B cells through the regulation of AKT phosphorylation. In BLM-treated mice, the oral administration of daphnetin attenuated lung histopathology and improved lung mechanical functions. Our findings clearly demonstrated that daphnetin inhibited IL-17A and EMT both in vitro and in vivo, thereby protecting against BLM-induced pulmonary fibrosis. Taken together, these results suggest that daphnetin has potent therapeutic effects on lung fibrosis by modulating both Th17 differentiation and the TGF-β signaling pathway, and we thus expect daphnetin to be a drug candidate for the treatment of IPF.

Apoptosis as a mechanism of human respiratory cell death upon exposure to carbon nanotubes

Introduction. Carbon nanotubes (CNTs) are a group of promising nanomaterials for industrial and biomedical applications. There has been shown influence of the physicochemical characteristics of CNTs on the toxic effects, including the ability to cause DNA damage and induce apoptosis. In this study, there was carried out a comparative assessment of pro-apoptotic effects under exposure to single-walled and multi-walled CNTs produced in Russia on human respiratory cells.&#x0D; Materials and methods. Human bronchial epithelial cells BEAS-2B, alveolar epithelial cells A549, and lung fibroblasts MRC5-SV40 were exposed to pristine and purified TUBALLTM SWCNTs and Taunit-M MWCNTs. In cells exposed to 4 concentrations (100, 50, 0.03, 0.0006 μg/ml) of all types of CNTs for 72 hours, the level of mRNA of the P53, BAX and BCL2 genes, as well as the level of reactive oxygen species were assessed.&#x0D; Results. All types of CNTs initiated apoptosis in human respiratory epithelial cells BEAS-2B and A549, but not in MRC5-SV40 lung fibroblasts. BEAS-2B were more sensitive to the effects of MWCNTs, while A549 were more sensitive to pristine SWCNTs. Apoptosis was initiated at low concentrations, including those corresponding to industrial exposures. The mechanism of oxidative stress could act as a factor in triggering apoptosis in lung epithelial cells.&#x0D; Limitations. Relatively short (72 hours) cell incubation time and the use of 2D cell models that do not consider real cell interactions.&#x0D; Conclusion. There were revealed differences in the mechanisms of initiation of the internal pathway of apoptosis and sensitivity to different types of CNTs depending on the type of epithelial cells. Comparative analysis of the initiation of apoptosis by different types of CNTs has shown that there are differences in potential target cells and toxic mechanisms, which should be considered in further studies.

Cold-inducible RNA-binding protein induces inflammatory responses via NF-κB signaling pathway in normal human bronchial epithelial cells infected with streptococcus pneumoniae

BackgroundCommunity-acquired pneumonia causes significant illness and death worldwide, requiring further investigation and intervention. The invasion of Streptococcus pneumoniae (S. pneumoniae, S.p) can lead to serious conditions like meningitis, sepsis, or pneumonia. Extracellular Cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern that triggers inflammatory responses and plays an important role in both acute and chronic inflammatory diseases. It remains unclear whether CIRP is involved in the process of S. pneumoniae infection in normal human bronchial epithelial cells (BEAS-2B). MethodsCell counting kit (CCK)-8 assay was used to detect the activity of BEAS-2B cells. The subcellular localization of CIRP was detected by immunofluorescence. The mRNA and protein levels of CIRP, nuclear factor kappa-B (NF-κB) p65, toll like receptor-4 (TLR4), interleukin-6 (IL-6) were detected using quantitative real-time PCR (PCR) and Western Blot (WB). The protein expressions of CIRP, IL-1β, IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were assessed by enzyme-linked immunosorbent assay (ELISA). ResultsCIRP affects the activity of BEAS-2B cells induced by S. pneumoniae infection. After infection, CIRP translocates from the nucleus to the cytoplasm, thereby inducing the production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and MCP-1). Additionally, the NF-κB p65 protein increases in infected cells but decreases with si-CIRP interference. Treatment with TLR4 neutralizing antibodies or NF-κB inhibitor effectively reduces the expressions of IL-1β, IL-6, TNF-α, and MCP-1. ConclusionsThe infection with S. pneumoniae upregulates CIRP expression and translocates it from the nucleus to the cytoplasm in BEAS-2B cells, leading to the release of proinflammatory factors via activation of NF-κB signaling pathway. CIRP as a key mediator in S. pneumoniae-induced inflammation offers potential targets for therapeutic intervention against community-acquired pneumonia.

Comparative studies on the cytotoxic effects induced by iron oxide nanoparticles in cancerous and noncancerous human lung cells subjected to an alternating magnetic field

The cytotoxic effects of water-based ferrofluids composed of iron oxide nanoparticles, including magnetite (Fe3O4) and maghemite (γ-Fe2O3), ranging from 15 to 100 nm, were examined on various lung cancer cells including adenocarcinomic human alveolar basal epithelial cells (A549), nonsmall lung squamous cell carcinoma (H1703), small cell lung cancer cells (DMS 114), and normal bronchial epithelial cells (BEAS-2B). The cytotoxic effect was evaluated both with and without exposure to an alternating magnetic field (AMF). The studies revealed that neither AMF nor iron oxide nanoparticles when tested individually, produced cytotoxic effects on either cancerous or noncancerous cells. However, when applied together, they led to a significant decrease in cell viability and proliferative capacity due to the enhanced effects of magnetic fluid hyperthermia (MFH). The most pronounced effects were found for maghemite (<50 nm) when subjected to an AMF. Notably, A549 cells exhibited the highest resistance to the proposed hyperthermia treatment. BEAS-2B cells demonstrated susceptibility to magnetized iron oxide nanoparticles, similar to the response observed in lung cancer cells. The studies provide evidence that MFH is a promising strategy as a standalone treatment for different types of lung cancer cells. Nevertheless, to prevent any MFH-triggered adverse effects on normal lung cells, targeted magnetic ferrofluids should be designed.

Oncostatin M's Involvement in the Pathogenesis of Chronic Rhinosinusitis: Focus on Type 1 and 2 Inflammation.

The cytokine oncostatin M (OSM) elicits pathogenic effects involving disruption of the epithelial barrier function as a part of immunological response networks. It is unclear how these integrated cytokine signals influence inflammation and other physiological processes in the pathology of chronic rhinosinusitis (CRS). We investigated the expression and distribution of OSM and OSM receptor (OSMR) in CRS patients' sinonasal specimens, and we compared the results with a panel of inflammatory cytokine levels and clinical features. We classified CRS patients as eosinophilic (ECRS, n = 36) or non-eosinophilic (non-ECRS, n = 35) based on the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis phenotypic criteria and compared their cases with those of 20 control subjects. We also examined OSM's stimulatory effects on cytokine receptor expression levels using the human bronchial epithelium cell line BEAS-2B. RT-PCR showed that the OSM mRNA levels were significantly increased in the CRS patients' ethmoid sinus mucosa. The OSM mRNA levels were positively correlated with those of TNF-α, IL-1β, IL-13, and OSMR-β. In BEAS-2B cells, OSM treatment induced significant increases in the OSMRβ, IL-1R1, and IL-13Ra mRNA levels. OSM is involved in the pathogenesis of CRS in both type 1 and type 2 inflammation, suggesting the OSM signaling pathway as a potential therapeutic target for modulating epithelial stromal interactions.

β-aminoisobutyrics acid, a metabolite of BCAA, activates the AMPK/Nrf-2 pathway to prevent ferroptosis and ameliorates lung ischemia-reperfusion injury

BackgroundLung ischemia-reperfusion (I/R) injury is a serious clinical problem without effective treatment. Enhancing branched-chain amino acids (BCAA) metabolism can protect against cardiac I/R injury, which may be related to bioactive molecules generated by BCAA metabolites. L-β-aminoisobutyric acid (L-BAIBA), a metabolite of BCAA, has multi-organ protective effects, but whether it protects against lung I/R injury is unclear.MethodsTo assess the protective effect of L-BAIBA against lung I/R injury, an animal model was generated by clamping the hilum of the left lung, followed by releasing the clamp in C57BL/6 mice. Mice with lung I/R injury were pre-treated or post-treated with L-BAIBA (150 mg/kg/day), given by gavage or intraperitoneal injection. Lung injury was assessed by measuring lung edema and analyzing blood gases. Inflammation was assessed by measuring proinflammatory cytokines in bronchoalveolar lavage fluid (BALF), and neutrophil infiltration of the lung was measured by myeloperoxidase activity. Molecular biological methods, including western blot and immunofluorescence, were used to detect potential signaling mechanisms in A549 and BEAS-2B cells.ResultsWe found that L-BAIBA can protect the lung from I/R injury by inhibiting ferroptosis, which depends on the up-regulation of the expressions of GPX4 and SLC7A11 in C57BL/6 mice. Additionally, we demonstrated that the Nrf-2 signaling pathway is key to the inhibitory effect of L-BAIBA on ferroptosis in A549 and BEAS-2B cells. L-BAIBA can induce the nuclear translocation of Nrf-2. Interfering with the expression of Nrf-2 eliminated the protective effect of L-BAIBA on ferroptosis. A screening of potential signaling pathways revealed that L-BAIBA can increase the phosphorylation of AMPK, and compound C can block the Nrf-2 nuclear translocation induced by L-BAIBA. The presence of compound C also blocked the protective effects of L-BAIBA on lung I/R injury in C57BL/6 mice.ConclusionsOur study showed that L-BAIBA protects against lung I/R injury via the AMPK/Nrf-2 signaling pathway, which could be a therapeutic target.

A cancer cell and lysosome dual-targeted photosensitizer for fluorescence imaging and photodynamic therapy

A novel cancer cell and lysosome dual-targeted photosensitizer (PS) termed by BMP was developed for fluorescence imaging and photodynamic therapy (PDT). BMP consists of a porphyrin(Zn) core with appropriate singlet-triplet state distribution enables it to function as an imaging agent as well as a PS, a biotin moiety that can target cancer cells, and a morpholine group specific to lysosomes. Intracellular experiments demonstrated that the dual-targeted photosensitizer BMP could preferably accumulate in cancer cells and subsequently locate within lysosome organelles. Upon irradiation, BMP could generate red fluorescence emission (Φem = 6.3%) and singlet oxygen (1O2, ΦΔ = 55%) for the dual functionality of cell imaging and PDT, respectively. Notably, BMP exhibited much higher photocytotoxicity toward cancer cells (A549 cells) compared to normal cells (BEAS-2B cells). Importantly, 1O2 produced by BMP induced oxidative damage of lysosomes, and subsequently triggered cell death of A549 cells, as evidenced by the loss of morphological integrity and cell rupture.

Increased Lipogenesis Is Important for Hexavalent Chromium-Transformed Lung Cells and Xenograft Tumor Growth

Hexavalent chromium, Cr(VI), is a known carcinogen and environmental health concern. It has been established that reactive oxygen species, genomic instability, and DNA damage repair deficiency are important contributors to the Cr(VI)-induced carcinogenesis mechanism. However, some hallmarks of cancer remain under-researched regarding the mechanism behind Cr(VI)-induced carcinogenesis. Increased lipogenesis is important to carcinogenesis and tumorigenesis in multiple types of cancers, yet the role increased lipogenesis has in Cr(VI) carcinogenesis is unclear. We report here that Cr(VI)-induced transformation of three human lung cell lines (BEAS-2B, BEP2D, and WTHBF-6) resulted in increased lipogenesis (palmitic acid levels), and Cr(VI)-transformed cells had an increased expression of key lipogenesis proteins (ATP citrate lyase [ACLY], acetyl-CoA carboxylase [ACC1], and fatty acid synthase [FASN]). We also determined that the Cr(VI)-transformed cells did not exhibit an increase in fatty acid oxidation or lipid droplets compared to their passage-matched control cells. Additionally, we observed increases in ACLY, ACC1, and FASN in lung tumor tissue compared with normal-adjacent lung tissue (in chromate workers that died of chromate-induced tumors). Next, using a known FASN inhibitor (C75), we treated Cr(VI)-transformed BEAS-2B with this inhibitor and measured cell growth, FASN protein expression, and growth in soft agar. We observed that FASN inhibition results in a decreased protein expression, decreased cell growth, and the inhibition of colony growth in soft agar. Next, using shRNA to knock down the FASN protein in Cr(VI)-transformed BEAS-2B cells, we saw a decrease in FASN protein expression and a loss of the xenograft tumor development of Cr(VI)-transformed BEAS-2B cells. These results demonstrate that FASN is important for Cr(VI)-transformed cell growth and cancer properties. In conclusion, these data show that Cr(VI)-transformation in vitro caused an increase in lipogenesis, and that this increase is vital for Cr(VI)-transformed cells.