BEAS-2B Cells Research Articles

Targeting Lung Cancer with Carvacrol-Triazole-Arylidenehydrazide Hybrids: In Vitro and In Silico Cytotoxicity Assessments.

In this study, a series of 16 arylidenehydrazide derivatives (7a-7p), hybridized with the natural product carvacrol, were successfully synthesized starting from anthranilic acid methyl ester. The cytotoxic effects of these compounds were examined against two different cell lines, A549 and BEAS-2B. Additionally, in silico studies were conducted to investigate the ligand-protein binding modes and their stabilities. Lastly, the predicted ADME properties were also explored. The compounds' structures were confirmed through meticulous NMR and MS spectral analyses. Biological assays indicated notable cytotoxic effects against human lung cancer (A549) and non-tumorigenic lung epithelial (BEAS-2B) cell lines, with compounds 7j, 7k, and 7l demonstrating high selectivity indices (SIs) and low IC50 values against A549 cells, signifying potent selective anticancer activity. Molecular docking and molecular dynamics (MD) simulations identified key binding interactions of these compounds with EGFR and BRAF proteins, emphasizing the importance of residues such as Lys-745 and Phe-856 in EGFR and Lys-483 in BRAF. Stability of these complexes was confirmed through 100 ns MD simulations. ADME analysis revealed favorable pharmacokinetic properties for the prominent compounds, particularly 7k. These results suggest that arylidenehydrazide derivatives, especially 7k, are promising selective anticancer agents with potential for further development.

Robust peptide/RNA complexes prepared with microfluidic mixing for pulmonary delivery by nebulisation.

Small interfering RNA (siRNA) and messengerRNA (mRNA) have drawn considerable attention in recent years due to their ability to modulate the expression of specific disease-related proteins. However, it is difficult to find safe, robust, and effective RNA delivery systems suitable for pulmonary delivery to treat lung diseases. In this study, two cationic peptides, namely LAH4-L1 and PEG12KL4, were employed as non-viral vectors for siRNA and mRNA delivery. Four formulations (i.e. LAH4-L1/siRNA; PEG12KL4/siRNA; LAH4-L1/mRNA and PEG12KL4/mRNA) were investigated. Microfluidic mixing method was utilised to fabricate RNA complexes in a controllable and reproducible manner. Upon optimisation of the microfluidic mixing protocol, a vibrating mesh nebuliser was employed to aerosolise the RNA complexes, and their transfection efficiency was evaluated on A549 and BEAS-2B cells. Following nebulisation, inhalable mist was generated for all RNA formulations with mass median aerodynamic diameter below 5μm. Although the hydrodynamic particle sizes of the RNA complexes were significantly reduced to around 100nm after nebulisation regardless of the original size of the complexes prior to nebulisation, the RNA binding efficiency and the in vitro RNA transfection ability of all the peptide formulations were successfully preserved with no significant differences compared to the same system before nebulisation. The current result indicates that both LAH4-L1 and PEG12KL4 hold significant potential for future clinical application for pulmonary siRNA and mRNA delivery through nebulisation.

Efficacy of Spray Cryotherapy on Airway Secretions in Canine Models of Chronic Bronchitis and Mechanism Insights.

Introduction Bronchoscopic spray cryotherapy (SCT) is a novel treatment showing promise for chronic bronchitis (CB), characterized by excessive mucus secretion and productive cough. A large animal model for preclinical research of SCT is lacking, and its treatment's efficacy and mechanisms for CB are not well understood. Methods Eight Labradors were exposed to 200 ppm SO2 for 6 months to develop a CB model. Evaluations included pulmonary resistance, bronchoalveolar lavage fluid (BALF), CT images, and pathology. After model validation, six dogs received SCT and were observed for short-term (7 days) and long-term (30 days) outcomes. Metrics included pulmonary resistance, bronchoscopy findings, and BALF analysis for inflammatory factors, acetylcholine, and mucins. Bronchial tissue was assessed via HE staining, electron microscopy, and IHC staining. BEAS-2B cells were used to study MUC5AC expression in response to LPS and acetylcholine. Results SO2 exposure led to persistent cough, increased pulmonary resistance, goblet cell hyperplasia, and inflammation. Mucin, MUC5AC, and MUC5B levels in BALF increased over time, which validated the CB model. SCT treatment reduced mucus and pulmonary resistance, improved bronchial structure, and decreased goblet cells. SCT significantly reduced BALF mucin, MUC5AC, MUC5B, acetylcholine, IL-6, INF-γ, TNF-α, and IL-10, and bronchial MUC5AC and CHRM3. In the LPS treatment BEAS-2B cells, MUC5AC expression increased when acetylcholine pretreatment concentration increased. Conclusion The SO2 inhalation protocol effectively establishes a CB model in dogs. SCT effectively treats CB by reducing mucin levels and may lower MUC5AC expression by decreasing acetylcholine and CHRM3.

Moxifloxacin-Loaded Polymeric Nanoparticles for Overcoming Multidrug Resistance in Chronic Pulmonary Infections Caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) infections are increasingly challenging due to their propensity to form biofilms and low outer membrane permeability, especially in chronically infected patients with thick mucus. P. aeruginosa exhibits multiple drug resistance mechanisms, making it one of the most significant global public health threats. In this study, we found that moxifloxacin (MXC) and antibacterial peptides (ε-poly-l-lysine, ε-PLL) exhibited a synergistic effect against multidrug-resistant P. aeruginosa (MDR-P. aeruginosa). MXC was combined with ε-PLL to prepare lipase-responsive nanoparticles (MCIP/(PEG-PCL)/PLL NPs) with a weakly negative charge. The weakly negatively charged MCIP/(PEG-PCL)/PLL NPs demonstrated remarkable mucus and biofilm penetration capabilities, thereby overcoming one of the adaptive drug resistance mechanisms. MCIP/(PEG-PCL)/PLL NPs improved the outer and inner membrane permeability and inhibited the expression of the efflux pump MexAB-OprM gene in MDR-P. aeruginosa, thereby overcoming mechanisms of both intrinsic and acquired drug resistance. Meanwhile, the nanoparticles demonstrated an ability to reduce repeated infections with MDR-P. aeruginosa. Additionally, the bacterial burden in the lungs of mice treated with MCIP/(PEG-PCL)/PLL NPs was significantly lower than that in the MXC group, resulting in a 99% clearance rate. Notably, MCIP/(PEG-PCL)/PLL NPs showed no toxicity toward BEAS-2B cells or RAW 267.4 cells, nor did they adversely affect pulmonary function or major organs. This study demonstrated the potential of the nanodrug delivery system composed of the antibiotic moxifloxacin and the antibacterial peptide ε-PLL in addressing the clinical challenges of treating chronic pulmonary infections caused by MDR-P. aeruginosa.

RNA m6A involves in regulation of oxidative stress and apoptosis may via NF-kB pathway in cadmium-induced lung cells

Cadmium has been identified as an environmental pollutant and a carcinogen. N6-methyladenosine (m6A) plays a crucial role in the development of lung tumors, but the mechanisms remain incompletely clarified. In present study, our data demonstrated that prolonged treatment of 1 μmol/L CdSO4 for 40 passages in bronchial epithelial cells (Beas-2B cells) resulted in the development of a malignant phenotype, which manifested as boosted proliferation, migration and invasion capacity as well as apoptosis reduction. Proteomic assay revealed that in passage 40 cells, 350 proteins showed differentially expressed in comparison to control, and these proteins were primarily enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of “pathways in cancer” and “Chemical carcinoma-reactive oxygen species”. Moreover, the mRNAs of Nuclear factor kappa B (NF-κB) p65 and NAD(P)H: quinone oxidoreductase 1 (NQO1), the key signaling molecules in these two signaling pathways, were predicted to contain m6A modification sites with high confidence. The subsequent experimental results indicated that levels of m6A and Fat mass and obesity associated protein (FTO) elevated, while Alkylated DNA repair protein alkB homolog 5 (ALKBH5) and YTH Domain Containing Protein 2 (YTHDC2) reduced with the increasing of cadmium treatment generations. Furthermore, the reduction of m6A levels by 3-deazide adenosine (DAA, m6A inhibitor) was found to significantly inhibit malignant characteristics of cadmium-induced cells, activate molecules involved in the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway, and inhibit the activity of NF-κB. It is also noteworthy that the results based on animals indicate that the relevant indicators and biological changes are partially similar to cell experiments. In detail, m6A modification levels in lung tissue were observed to increase while the expressions of FTO, ALKBH5 and YTHDC2 were found to drop. Additionally, immunofluorescence examination illustrated the co-localization of the m6A regulatory proteins FTO and YTHDC2 with NF-κB. The presented data collectively suggest that chronic cadmium treatment may impact the m6A level through influencing regulatory proteins, which could potentially trigger oxidative stress and apoptosis by regulating transcription factors such as NF-κB and NRF2. In conclusion, our study provides a scientific foundation for understanding cadmium toxicity and offers novel insights for treating cadmium-induced lung diseases.

Echinacoside attenuates Klebsiella pneumoniae-induced pneumonia via inhibition of the TLR4/NF-κB signaling.

The Gram-negative bacterium Klebsiella pneumoniae (K. pneumoniae) is one major causative agent of community- and hospital-acquired pneumonia. Echinacoside (ECH) is a phenylethanoid glycoside isolated from Cistanche deserticola that possesses anti-inflammatory activity. Our research aimed to confirm whether ECH alleviates K. pneumoniae-induced pneumonia and explore the underlying regulatory mechanisms. BEAS-2B cells and BALB/c mice were infected by K. pneumoniae to establish the cellular and animal models, respectively, followed by ECH treatment. Inflammatory cytokine levels were detected by RT-qPCR and ELISA. The lung wet/dry (W/D) weight ratio and the myeloperoxidase (MPO) activity in lung tissues were examined. The pulmonary histopathologic changes were observed through hematoxylin and eosin (H&E) staining. The levels of TLR4/NF-κB pathway-associated molecules were estimated through western blotting, immunohistochemical, and immunohistochemical staining. K. pneumoniae infection caused lung histopathologic damage, enhanced MPO activity, elevated lung W/D weight ratio, and upregulated inflammatory cytokine levels in mice and promoted inflammatory cytokine expression in BEAS-2B cells, which were reversed by ECH treatment. K. pneumoniae infection-induced upregulation in TLR4, phosphorylated (p)-p65, and p-IκBα levels, and downregulation in IκBα levels in BEAS-2B cells and pneumonia mice were overturned by ECH treatment. ECH ameliorates K. pneumoniae-induced pneumonia through suppressing the TLR4/NF-κB pathway.

LC3B-regulated autophagy mitigates zinc oxide nanoparticle-induced epithelial cell dysfunction and acute lung injury.

Zinc oxide nanoparticles (ZnONPs) are widely utilized across various industries, raising concerns about their potential toxicity, especially in the respiratory system. This study explores the role of autophagy, regulated by microtubule-associated protein 1A/1B-light chain 3B (LC3B), in ZnONPs-induced toxicity using both in vivo (LC3B knockout mice) and in vitro (BEAS-2B cells) models. Our findings demonstrate that LC3B-regulated autophagy mitigates ZnONPs-induced epithelial cell dysfunction and acute lung injury. In the absence of LC3B, oxidative stress, inflammation, and intracellular zinc accumulation are exacerbated, resulting in mitochondrial dysfunction and epithelial cell death. In vitro, LC3B knockdown disrupted zinc ion transporter expression and impaired mitophagic flux in BEAS-2B cells. Treatment with zinc ion chelators alleviated these toxic effects, confirming that free zinc ions play a critical role in driving ZnONPs toxicity. These findings highlight that targeting autophagy and maintaining zinc homeostasis could offer therapeutic strategies to reduce ZnONPs-induced lung damage.

Cigarette and e-cigarette reversely regulated autophagy via distinct level of ROS in vitro

With the increasing prevalence of e-cigarettes, a systematic evaluation of the cytotoxicity among the components of e-cigarettes is needed. This study aimed to compare the cytotoxicity of conventional cigarettes and e-cigarettes, identify the toxic components of e-cigarettes, and explore the mechanisms of cellular injury. In this study, the conventional cigarette and seven flavored e-cigarettes, along with their constituents, including solvents, nicotine, and seven flavoring agents were tested. The Cambridge filter pad method was employed to collect conventional cigarettes smoke condensate (CSC) and e-cigarettes aerosol condensate (EAC). CCK-8 assays were used to assess their cytotoxicity on respiratory system-related cells and cardiovascular-related cells. The findings revealed significantly higher cytotoxicity in CSC compared to EACs, with EACs exhibiting IC50 values at least 12 times higher across various flavors and cell lines. Notably, among the seven flavors, the Cola-flavored EAC exhibited the most pronounced cytotoxicity, with flavoring agents identified as the primary contributors to the cytotoxicity of e-cigarettes. Furthermore, noteworthy differences in cellular mechanisms were observed between CSC and EAC-Cola in BEAS-2B cells, particularly in terms of ROS level and autophagy regulation. CSC induced approximately 10 times higher levels of ROS compared to EAC. Due to the distinct level of ROS, CSC significantly inhibited autophagy while EAC induced autophagy. Scavenge of ROS clearly eliminates the autophagy modulated by both CSC and EAC. These findings contribute to a comprehensive understanding of the cytotoxicity of e-cigarettes and conventional cigarettes, providing a scientific basis for health risk assessment and regulatory mechanisms.

Neochlorogenic acid ameliorates allergic airway inflammation by suppressing type 2 immunity and upregulating HO-1 expression.

Neochlorogenic acid is a natural compound isolated from various fruits and vegetables that has anti-inflammation and anti-oxidative effects in macrophages. Inflammatory immune cells and tracheal epithelial cells can stimulate airway hyperresponsiveness, inflammation, and reactive oxygen species. In this study, we investigated the effect of neochlorogenic acid in ameliorating inflammatory and oxidative responses in asthmatic mice. We used an ovalbumin (OVA)-induced mouse model, treating mice with neochlorogenic acid by intraperitoneal injection. We also treated inflammatory human tracheal epithelial (BEAS-2B) cells with neochlorogenic acid to evaluate inflammatory cytokine levels and oxidative responses. The results demonstrate that neochlorogenic acid attenuated airway hyperresponsiveness, eosinophil infiltration, and goblet cell hyperplasia in the lungs of asthmatic mice. Neochlorogenic acid also reduced type 2 cytokine expression in bronchoalveolar lavage fluid and improved oxidative stress in the lung. Neochlorogenic acid effectively blocked monocyte attachment to adherent BEAS-2B cells, and reduced pro-inflammatory cytokine and reactive oxygen species production in inflammatory BEAS-2B cells. These findings suggest that neochlorogenic acid is a potential immunomodulator that can ameliorate airway hyperresponsiveness and airway inflammation in asthmatic mice.

Azithromycin inhibits the intracellular persistence of Acinetobacter baumannii by inducing host cell autophagy in human bronchial epithelial cells

The invasion of host cells by bacteria, leading to intracellular infections, is a major cause of infection recurrence. Drug-resistant Acinetobacter baumannii (A. baumannii) is one of the most challenging public health issues worldwide, with very limited clinical treatment options available. A. baumannii has been found to be able to invade host cells and proliferate within them in recent studies. In addition to the direct antimicrobial effect of antibiotics, the activation of host autophagic flux also plays an important role in eliminating intracellular pathogens. Herein, this study aimes to evaluate the clearance effect of antibiotics on intracellular A. baumannii both in vivo and in vitro, and explore the relationship between this effect and autophagy. The results showed that intracellular pathogens resulted in a significant increase in the minimum bactericidal concentration, while azithromycin can significantly eliminate intracellular A. baumannii in vitro and in vivo. Notably, 60μg/mL azithromycin demonstrated intracellular clearance against multidrug-resistant A. baumannii and markedly induced autophagosomes in BEAS-2B cells with a mild stimulation of autophagosomes degradation. These findings indicated that azithromycin can significantly clear intracellular A. baumannii and its ability to clear intracellular A. baumannii may be related to the stimulation of autophagosome formation and the induction of host autophagy, which has important implications for the clinical treatment of A. baumannii infections, especially when intracellular infections are present.

New ent-cleistanthanes and ent-pimaras diterpenoids with potential cytotoxicity from Phyllanthus franchetianus H. Lév.

Seven diterpenoids including five ent-cleistanthanes (1, 2, 4–6) and two ent-pimaranes (3, 7) were isolated for the first time from the aerial part of Phyllanthus franchetianus H. Lév. Their structures were elucidated on the basis of the extensive spectroscopic analyses, single-crystal X-ray diffraction and ECD analysis. Phyllanthanes A-C (1–3) are new compounds. Notably, the ent-cleistanthanes 1 and 4–5 exhibited moderate cytotoxicity against five human cancer (HL-60, A549, HepG2, MDA-MB-231, SW480) (IC50 = 5.01–32.41 μM) and one normal BEAS-2B (IC50 = 20.45–24.33 μM) cell lines.

Arsenic-Induced Inflammatory Response via ROS-Dependent Activation of ERK/NF-kB Signaling Pathways: Protective Role of Natural Polyphenol Tannic Acid.

Arsenic (As), a highly toxic metalloid, is present throughout our environment as a result of both natural and human-related activities. Furthermore, As exposure could lead to a persistent inflammatory response, which may facilitate the pathogenesis of several diseases in various organs. This study was performed to investigate the As-induced inflammatory response and the underlying molecular mechanisms invitro. Further, the anti-inflammatory effects of a natural dietary polyphenol tannic acid (TA) were also explored. In human normal bronchial (BEAS-2B), adenocarcinoma alveolar basal (A549), and murine macrophages (J774) cell lines, a trivalent form of As (as As3+) exposure markedly induced the expression of various pro-inflammatory mediators (cytokines and chemokines). Additionally, it was found that As3+ exposure induced reactive oxygen species (ROS) generation and activation of the nuclear factor-kappa B (NF-kB) p65 and extracellular signal-regulated kinase (ERK)1/2 pathways in BEAS-2B cells. As expected, the blockade of either ERK1/2 (PD98059) or NF-kB p65 (IMD0354), or both pathways attenuated As3+-induced pro-inflammatory mediators release. Interestingly, pre-treatment with ROS inhibitor N-acetylcysteine (NAC) attenuated activation of ERK/NF-kB pathways, suggesting that ROS have a critical role in pathway's activation and subsequent inflammatory response. Further, TA pre-treatment effectively attenuated As3+-induced inflammatory response by suppressing ROS production and ERK/NF-kB signaling pathways activation. Therefore, this study provides scientific evidence for the anti-inflammatory activities of TA and the underlying molecular mechanisms.

Bioactive secondary metabolites from Talaromyces rugulosus GIZ45-A37 and their anti-inflammatory activity

As part of our plan to discover new bioactive ingredients from entomophagous fungi, a new macrolide compound, talarolide (1), and a new bioactive natural product, talarenal (2), with two known compounds, WF-3681 methy ester (3) and aspergillumarin A (4) were isolated from an insect derived strain Talaromyces rugulosus GIZ45-A37. The structures were determined based on extensive comprehensive spectroscopic (NMR and HR-ESI-MS) analyses and compared with literature values. The relative configuration of 1 was defined by DFT-NMR chemical shift calculations and subsequent DP4/DP4+ probability methods. Compounds 1 and 2 showed inhibitory effect on IL-6 production at 10 and 20 μM in LPS-stimulated beas-2b cells, indicating their potential anti-inflammatory activity.

MET Exon 14 Skipping and Novel Actionable Variants: Diagnostic and Therapeutic Implications in Latin American Non-Small-Cell Lung Cancer Patients.

Targeted therapy indications for actionable variants in non-small-cell lung cancer (NSCLC) have primarily been studied in Caucasian populations, with limited data on Latin American patients. This study utilized a 52-genes next-generation sequencing (NGS) panel to analyze 1560 tumor biopsies from NSCLC patients in Chile, Brazil, and Peru. The RNA sequencing reads and DNA coverage were correlated to improve the detection of the actionable MET exon 14 skipping variant (METex14). The pathogenicity of MET variants of uncertain significance (VUSs) was assessed using bioinformatic methods, based on their predicted driver potential. The effects of the predicted drivers VUS T992I and H1094Y on c-MET signaling activation, proliferation, and migration were evaluated in HEK293T, BEAS-2B, and H1993 cell lines. Subsequently, c-Met inhibitors were tested in 2D and 3D cell cultures, and drug affinity was determined using 3D structure simulations. The prevalence of MET variants in the South American cohort was 8%, and RNA-based diagnosis detected 27% more cases of METex14 than DNA-based methods. Notably, 20% of METex14 cases with RNA reads below the detection threshold were confirmed using DNA analysis. The novel actionable T992I and H1094Y variants induced proliferation and migration through c-Met/Akt signaling. Both variants showed sensitivity to crizotinib and savolitinib, but the H1094Y variant exhibited reduced sensitivity to capmatinib. These findings highlight the importance of RNA-based METex14 diagnosis and reveal the drug sensitivity profiles of novel actionable MET variants from an understudied patient population.

Triphenylphosphine-Modified IridiumIII, RhodiumIII, and RutheniumII Complexes to Achieve Enhanced Anticancer Selectivity by Targeting Mitochondria.

The incorporation of an organelle-targeting moiety into compounds has proven to be an effective strategy in the development of targeted anticancer drugs. We herein report the synthesis, characterization, and biological evaluation of novel triphenylphosphine-modified half-sandwich iridiumIII, rhodiumIII, and rutheniumII complexes. The primary goal was to enhance anticancer selectivity through mitochondrial targeting. All these triphenylphosphine-modified complexes exhibited promising cytotoxicity in the micromolar range (5.13-23.22) against A549 and HeLa cancer cell lines, surpassing the activity of comparative complexes that lack the triphenylphosphine moiety. Noteworthy is their good selectivity toward cancer cells compared to normal BEAS-2B cells, underscored by selectivity index ranging from 7.3 to >19.5. Mechanistically, these complexes primarily target mitochondria rather than interacting with DNA. The targeting of mitochondria and triggering mitochondrial dysfunction were confirmed using both confocal microscopy and flow cytometry. Their ability to depolarize mitochondrial membrane potential (MMP) and enhance reactive oxygen species (ROS) was observed, thereby leading to intrinsic apoptotic pathways. Moreover, these complexes lead to cell cycle arrest in the G2/M phase and demonstrated antimigration effects, significantly inhibiting the migration of A549 cells in wound-healing assays.

Machine Learning-Assisted "Shrink-Restricted" SERS Strategy for Classification of Environmental Nanoplastic-Induced Cell Death.

The biotoxicity of nanoplastics (NPs), especially from environmental sources, and "NPs carrier effect" are in the early stages of research. This study presents a machine learning-assisted "shrink-restricted" SERS strategy (SRSS) to monitor molecular changes in the cellular secretome exposure to six types of NPs. Utilizing three-dimensional (3D) Ag@hydrogel-based SRSS, active targeting of molecules within adjustable nanogaps was achieved to track information. Machine learning was employed to analyze the overall spectral profiles, biochemical signatures, and time-dependent changes. Results indicate that environmentally derived NPs exhibited higher toxicity to BEAS-2B and L02 cells. Notably, the "NPs carrier effect," resulting from pollutant adsorption, proved to be more harmful. This effect altered the death pathway of BEAS-2B cells from a combination of apoptosis and ferroptosis to primarily ferroptosis. Furthermore, L02 cells demonstrated greater metabolic vulnerability to NPs exposure than that of BEAS-2B cells, especially concerning the "NPs carrier effect." Traditional detection methods for cell death often rely on end point assays, which limit temporal resolution and focus on single or multiple markers. In contrast, our study pioneers a machine learning-assisted SERS approach for monitoring overall metabolic levels post-NPs exposure at both cellular and molecular levels. This endeavor has significantly advanced our understanding of the risks associated with plastic pollution.

Inhibitory Effects of Compounds Isolated from Morinda citrifolia L. (Noni) Seeds against Particulate Matter-Induced Injury.

Morinda citrifolia L. (noni) is native to the tropical and semitropical areas and has been commercially available in health food stores and chain grocery stores specializing in natural foods, recently. Noni seeds are discarded as waste products through the industrial production of noni juice even though their bioactivity components might be a potential source of functional foods. Not many studies of phytochemistry and biological activity have been investigated on noni seeds until now. In this study, the phytochemical investigation of M. citrifolia seeds led to the isolation of eight compounds (1-8) including four lignans (5-8). Their chemical structures were elucidated based on extensive spectroscopic analysis as well as the comparison with those reported in the literature. The isolated lignans were then evaluated for their anti-inflammatory activity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphynyltetrazolium bromide (MTT) assay in human bronchial epithelium BEAS-2B cells stimulated by 1-nitropyrene. As results, both four isolated lignans displayed high effects on the viability of BEAS-2B cells, indicating promising anti-inflammatory role in the airway disease.

Production of recombinant protein of Tyr p 32 from Tyrophagus putrescentiae and identifying its immunoreactivity

The present study was aimed to produce the recombinant protein of Tyrophagus putrescentiae allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of T. putrescentiae and inserted into pET-28a (+) vector. The constructed plasmid pET-28a (+)-Tyr p 32 was transformed into BL21 (DE3) receptor cells. After being induced with IPTG, the recombinant protein was purified with Ni column, and then identified on SDS-PAGE and Western blotting. Serum of children with allergic asthma and(or) rhinitis was collected, IgE-ELISA and IgE-Western blotting were used to detect the binding rate of rTyr p 32 to human T.putrescentiae-positive serum. After human bronchial epithelial cells BEAS-2B being cultured with rTyr p 32, the expression levels of IL-6 and IL-8 cytokines was detected by ELISA and qRT-PCR, t-test was used for pairwise comparison between groups. The results showed that the cDNA length of Tyr p 32 was 885 bp. The sequence identity between Tyr p 32 and Der p 32, Der f 32 was 70.21% and 68.03%, respectively. According to SDS-PAGE and Western blotting, the molecular weight of rTyr p 32 was about 35 000 Da, which was consistent with the theoretical value. IgE-ELISA results showed that the positive rate of rTyr p 32 was 41.38% (12/29) against T. putrescentiae-positive serum. When BEAS-2B cells were cultured with rTyr p 32, the expression of IL-6 and IL-8 increased in the cell supernatant in a dose-dependent manner (t=-29.10,P=0.001 2;t=-33.69,P=0.000 9), which also significantly increased the mRNA expression levels of IL-6 and IL-8 (t=-9.15,P=0.011 7;t=-17.16,P=0.003 4). In conclusion, the recombinant protein rTyr p 32 was successfully prepared, which provides raw materials for component diagnosis and specific immunotherapy of allergic diseases.

Silencing SMAD4 inhibits inflammation and ferroptosis in asthma by blocking the IL-17A signaling pathway

BackgroundThe TGF-β/SMAD signaling pathway is crucial in the pathogenesis of asthma. However, SMAD family member 4 (SMAD4), a key mediator of TGF-β, its roles and underlying mechanisms in asthma remain unclear.MethodsThe in vivo and in vitro roles of SMAD4 in asthma were investigated through an ovalbumin (OVA)-induced mouse model and an interleukin-13 (IL-13)-induced cell model. The molecular mechanism of SMAD4 influenced asthma was examined using transcriptome sequencing, followed by feedback experiments involving recombinant human interleukin 17 A (rhIL-17 A), an IL-17 A signaling pathway activator.ResultsSMAD4 was highly expressed in the asthma models. SMAD4 silencing alleviated damage to lung tissue and decreased inflammatory infiltration. Expression levels of Caspase-3, IgG, and inflammatory factors were reduced after silencing SMAD4. Silencing SMAD4 suppressed ferroptosis. Silencing SMAD4 also enhanced IL-13-induced BEAS-2B cell proliferation and suppressed apoptosis. Furthermore. IL-17 A signaling pathway was promoted in the asthma models, as evidenced by elevated IL-17RA, IL-17 A, and Act1 protein levels. SMAD4 silencing inhibited the expression levels of these IL-17 A pathway-associated proteins. Moreover, rhIL-17 A treatment notably reversed the impacts of SMAD4 silencing on asthma in the IL-13-induced cell model and OVA-induced mouse model, indicating that silencing SMAD4 inhibited inflammation and ferroptosis in asthma by blocking the IL-17 A signaling pathway.ConclusionSilencing SMAD4 prevents inflammation and ferroptosis in asthma by inhibiting the IL-17 pathway, which provides a novel potential approach for asthma therapy.

Baicalin Targeting ALOX15 Inhibits Ferroptosis of Lung Epithelial Cells and Alleviates Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a progressive lung disorder characterized by chronic inflammation and irreversible airflow limitation. Ferroptosis has been implicated in the pathogenesis of COPD. In this study, we investigated the potential of baicalin, a natural compound with anti-inflammatory properties, to target ALOX15 and inhibit ferroptosis in lung epithelial cells, thereby alleviating COPD. Overexpression ALOX15 of BEAS-2B cells was induced by RSL-3, an activator of ferroptosis, to mimic the inflammatory environment in COPD. The effects of baicalin on ferroptosis were assessed by measuring cell viability, lipid peroxidation, lipid ROS, and ferrous ion (Fe2+). The expression of OH-1, ALOX15, and GPX4 was evaluated using Western blotting. Baicalin treatment significantly increased cell viability in RSL-3 induced epithelial cells. Our findings also demonstrate that baicalin significantly increases cell viability and reduces lipid ROS and Fe2+ levels, thereby inhibiting ferroptosis and improving cell viability, irrespective of ALOX15 expression Furthermore, baicalin upregulated the expression of GPX4, a key regulator of ferroptosis. Importantly, baicalin treatment inhibited ALOX15 expression, a critical enzyme involved in lipid peroxidation. Our study demonstrates that baicalin targeting ALOX15 inhibits ferroptosis of lung epithelial cells and alleviates COPD. These findings provide a basis for further research and development of baicalin-based therapies for COPD patients, with the potential to improve their clinical outcomes and quality of life.