Human Alveolar Epithelial Cells Research Articles

Recombinant Klotho protein protects pulmonary alveolar epithelial cells against sepsis-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway.

Inflammation-induced apoptosis of alveolar type II epithelial cells is a primary contributor to sepsis-induced acute respiratory distress syndrome (ARDS). Klotho is a single-pass transmembrane protein with anti-inflammatory and anti-apoptotic effects. However, the role and mechanism of Klotho in the development of ARDS remains unknown. This study aimed to investigate the effect of Klotho on sepsis-induced apoptosis in human pulmonary alveolar epithelial cells (HPAEpiCs) together with the potential mechanism. Cecal ligation and puncture (CLP) were performed to generate an in vivo sepsis model, and HPAEpiCs were treated with lipopolysaccharide (LPS) to mimic sepsis in vitro. Both models were administered recombinant Klotho protein. The morphology of the lung tissue was observed, and apoptotic cells and cell viability were detected. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA), while the expression of Bcl-2, Bax and cleaved caspase-3 was detected with western blotting. Klotho reversed the CLP-induced decrease in mouse survival in vivo (p < 0.001) and increased inflammatory cell infiltration and inflammatory substance exudation in the lung tissue of mice with sepsis (both p < 0.001). Klotho also suppressed apoptosis (p < 0.001) as demonstrated by IL-1β, IL-6 and TNF-α expression (all p < 0.001), and Bcl-2/Bax/caspase-3 pathway activation (p < 0.001). Klotho pretreatment significantly prevented LPS-induced apoptosis in vitro (p < 0.001), as demonstrated by IL-1β, IL-6 and TNF-α upregulation (all p < 0.001); and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs (p < 0.001). This study demonstrated that Klotho can ameliorate acute lung injury (ALI) induced by sepsis by inhibiting inflammatory responses and exerting anti-apoptotic effects by suppressing Bcl-2/Bax/caspase-3 pathway activation.

Volatile Organic Compounds in Cellular Headspace after Hyperbaric Oxygen Exposure: An In Vitro Pilot Study.

Volatile organic compounds (VOCs) might be associated with pulmonary oxygen toxicity (POT). This pilot study aims to identify VOCs linked to oxidative stress employing an in vitro model of alveolar basal epithelial cells exposed to hyperbaric and hyperoxic conditions. In addition, the feasibility of this in vitro model for POT biomarker research was evaluated. The hyperbaric exposure protocol, similar to the U.S. Navy Treatment Table 6, was conducted on human alveolar basal epithelial cells, and the headspace VOCs were analyzed using gas chromatography-mass spectrometry. Three compounds (nonane [p = 0.005], octanal [p = 0.009], and decane [p = 0.018]), of which nonane and decane were also identified in a previous in vivo study with similar hyperbaric exposure, varied significantly between the intervention group which was exposed to 100% oxygen and the control group which was exposed to compressed air. VOC signal intensities were lower in the intervention group, but cellular stress markers (IL8 and LDH) confirmed increased stress and injury in the intervention group. Despite the observed reductions in compound expression, the model holds promise for POT biomarker exploration, emphasizing the need for further investigation into the complex relationship between VOCs and oxidative stress.

LINC01002 functions as a ceRNA to regulate FRMD8 by sponging miR-4324 for the development of COVID-19

BackgroundSyndrome coronavirus-2 (SARS-CoV-2) has developed various strategies to evade the antiviral impact of type I IFN. Non-structural proteins and auxiliary proteins have been extensively researched on their role in immune escape. Nevertheless, the detailed mechanisms of structural protein-induced immune evasion have not been well elucidated.MethodsHuman alveolar basal epithelial carcinoma cell line (A549) was stimulated with polyinosinic-polycytidylic acid (PIC) and independently transfected with four structural proteins expression plasmids, including nucleocapsid (N), spike (S), membrane (M) and envelope (E) proteins. By RT-qPCR and ELISA, the structural protein with the most pronounced inhibitory effects on IFN-β induction was screened. RNA-sequencing (RNA-Seq) and two differential analysis strategies were used to obtain differentially expressed genes associated with N protein inhibition of IFN-β induction. Based on DIANA-LncBase and StarBase databases, the interactive competitive endogenous RNA (ceRNA) network for N protein-associated genes was constructed. By combining single-cell sequencing data (GSE158055), lncRNA-miRNA-mRNA axis was further determined. Finally, RT-qPCR was utilized to illustrate the regulatory functions among components of the ceRNA axis.ResultsSARS-CoV-2 N protein inhibited IFN-β induction in human alveolar epithelial cells most significantly compared with other structural proteins. RNA-Seq data analysis revealed genes related to N protein inhibiting IFNs induction. The obtained 858 differentially expressed genes formed the reliable ceRNA network. The function of LINC01002-miR-4324-FRMD8 axis in the IFN-dominated immune evasion was further demonstrated through integrating single-cell sequencing data. Moreover, we validated that N protein could reverse the effect of PIC on LINC01002, FRMD8 and miR-4324 expression, and subsequently on IFN-β expression level. And LINC01002 could regulate the production of FRMD8 by inhibiting miR-4324.ConclusionSARS-CoV-2 N protein suppressed the induction of IFN-β by regulating LINC01002 which was as a ceRNA, sponging miR-4324 and participating in the regulation of FRMD8 mRNA. Our discovery provides new insights into early intervention therapy and drug development on SARS-CoV-2 infection.

Spleen tyrosine kinase inhibitor R406 has both antiviral and anti-inflammatory effects on severe influenza A infection.

Death due to severe influenza is usually a fatal complication of a dysregulated immune response more than the acute virulence of an infectious agent. Although spleen tyrosine kinase (SYK) as a critical immune signaling molecule and therapeutic target plays roles in airway inflammation and acute lung injury, the role of SYK in influenza virus infection is not clear. Here, we investigated the antiviral and anti-inflammatory effects of SYK inhibitor R406 on influenza infection through a coculture model of human alveolar epithelial (A549) and macrophage (THP-1) cell lines and mouse model. The results showed that R406 treatment increased the viability of A549 and decreased the pathogenicity and mortality of lethal influenza virus in mice with influenza A infection, decreased levels of intracellular signaling molecules under the condition of inflammation during influenza virus infection. Combination therapy with oseltamivir further ameliorated histopathological damage in the lungs of mice and further delayed the initial time to death compared with R406 treatment alone. This study demonstrated that phosphorylation of SYK is involved in the pathogenesis of influenza, and R406 has antiviral and anti-inflammatory effects on the treatment of the disease, which may be realized through multiple pathways, including the already reported SYK/STAT/IFNs-mediated antiviral pathway, as well as TNF-α/SYK- and SYK/Akt-based immunomodulation pathway.

High-magnitude cyclic stretch induces protein sialylation and RNA de-sialylation at the alveolar epithelial membrane

Sialylated glycans such as sialoglycoproteins, sialoglycolipids and sialoglycoRNA are ubiquitous in the glycocalyx of cells where they play a key role in barrier function, mechanosensation, infection, immune cell traffcking, and inflammation. These functions are particularly relevant at the blood-gas barrier of the lung. The alveolar epithelial glycocalyx is furthermore unique in that it is constantly exposed to cyclic stretch because of normal breathing. Yet, the abundance, regulation and function of sialylated glycans in the alveolar epithelial glycocalyx remains unknown. Here, we focused on the effects of mechanical stretch on the abundance and de novo formation of sialoglyco-proteins and -RNA as major constituents of the alveolar epithelial sialome. Human primary alveolar epithelial cells (hPAEpiC) were exposed to high magnitude cyclic stretch (18% CS) at 0.25 Hz for 24-48 hours to mimic biomechanical forces at the alveolar epithelium during high tidal volume ventilation. Total sialic acids in sialoglycoproteins were labeled with biotin by periodate oxidation and aldehyde ligation (pAL), and total sialic acids in sialoglycoRNA were quantified by 1, 2-diamino-4,5-methylene dioxybenzene ( DMB) assay. De novo formed sialic acids were labeled with biotin using N-azidoacetyl-mannosamine (Ac4ManNAz). Biotinylated siaolglyco-proteins and -RNAs were detected with streptavidin by Western and Northern blots, respectively, and quantified densitometrically. Stretch-dependent changes in the genes regulating sialic acid biosynthesis were assessed by RNA sequencing. High magnitude cyclic stretch increased the sialylation of proteins of different sizes (35-190 kDa) in the hPAEpiC glycocalyx. MGE showed this increase to be attributable to an increased de novo synthesis of sialic acids. On the other hand, high magnitude cyclic stretch reduced the sialylation of hPAEpiC sialoglycoRNA with no alteration in de novo synthesis, suggesting shedding, release or loss of sialoglycoRNA. RNA seq of hPAEpiC revealed that cyclic stretch upregulated the gene GNE (which encodes the enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), the master regulator of sialic acid biosynthesis. Here, we demonstrate cyclic stretch-induced sialylation of proteins and de-sialylation of RNA in the alveolar epithelial glycocalyx. Given that sialic acids promote the adhesion of both infectious pathogens and immune cells, these effects may contribute to the pathogenesis of ventilator-associated pneumonia and ventilator-induced lung injury. This work is supported by the German Research Foundation, CRC 1449 subproject B01 to MO &amp; WMK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Secondary Organic Aerosol Generated from Biomass Burning Emitted Phenolic Compounds: Oxidative Potential, Reactive Oxygen Species, and Cytotoxicity.

Phenolic compounds are largely emitted from biomass burning (BB) and have a significant potential to form SOA (Phc-SOA). However, the toxicological properties of Phc-SOA remain unclear. In this study, phenol and guaiacol were chosen as two representative phenolic gases in BB plumes, and the toxicological properties of water-soluble components of their SOA generated under different photochemical ages and NOx levels were investigated. Phenolic compounds contribute greatly to the oxidative potential (OP) of biomass-burning SOA. OH-adducts of guaiacol (e.g., 2-methoxyhydroquinone) were identified as components of guaiacol SOA (GSOA) with high OP. The addition of nitro groups to 2,5-dimethyl-1,4-benzoquinone, a surrogate quinone compound in Phc-SOA, increased its OP. The toxicity of both phenol SOA (PSOA) and GSOA in vitro in human alveolar epithelial cells decreased with aging in terms of both cell death and cellular reactive oxygen species (ROS), possibly due to more ring-opening products with relatively low toxicity. The influence of NOx was consistent between cell death and cellular ROS for GSOA but not for PSOA, indicating that cellular ROS production does not necessarily represent all processes contributing to cell death caused by PSOA. Combining different acellular and cellular assays can provide a comprehensive understanding of aerosol toxicological properties.

New silver, rhodium and iridium complexes with anthracene-functionalized N-heterocyclic carbene ligands: Crystal structures, cytotoxicity and fluorescence studies

In the present study, we have synthesized anthracene-functionalized metal complexes to investigate their cytotoxicity and photophysical properties. In this context, firstly, an Ag–NHC (NHC = N-heterocyclic carbene) complex (2a) has been synthesized by the interaction of Ag2O and N-(2-methylbenzyl)-N-((anthracen-9-yl)methyl)benzimidazolium chloride (1a). Corresponding Rh– (3a) and Ir–NHC (4a) complexes have also been synthesized by the transmetalation reaction between 2a and corresponding metal compounds. Additionally, for comparison purposes, an ether-functionalized Ag–NHC complex (2b) with N-(methoxyethyl)-N-((anthracene-9-yl)methyl)benzimidazole-2-ylidene ligand, and [AgL2]NO3 type complex (2c) with a N-coordinated N-((anthracen-9-yl)methyl)benzimidazole ligand have been synthesized. All complexes have been characterized by the combination of NMR and mass spectroscopic techniques, and elemental analyses. Moreover, solid state structures of 2a, 2b and 4a have been determined by the single crystal X-ray diffraction analysis. Cytotoxic potentials of all compounds have been tested against human lung adenocarcinoma alveolar basal epithelial cell line (A549) using the MTT assay, and all complexes performed strong anti-proliferative activity. Stability studies have disclosed that only 1a and 2c are able to retain their structure in the culture medium (DMEM) that biological assays were carried out. Photophysical measurements revealed that all compounds have similar emission bands because of the conjugation of anthracene group. Finally, 1a and 2c have been visualized by fluorescence confocal microscopy to investigate cellular uptake and subcellular distribution of the complexes, and the findings indicated that both compounds dispersed along the cell membranes nuclear regions.

Co-delivery of paclitaxel and curcumin loaded solid lipid nanoparticles for improved targeting of lung cancer: In vitro and in vivo investigation

The objective of this study was to develop nanotechnology-mediated paclitaxel (PAC) and curcumin (CUR) co-loaded solid lipid nanoparticles (PAC-CUR-SLNs) for the treatment of lung cancer, which is a leading cause of death worldwide. Around 85 % cases of lungs cancer constitute non-small cell lung cancer (NSCLC). PAC-CUR-SLNs were prepared via high pressure homogenization. The in vitro drug release of PAC-CUR-SLNs was checked followed by their in vitro cytotoxic investigation using adenocarcinomic human alveolar basal epithelial cells (A549) cell lines. Anticancer effects along with side effects of the synergistic delivery of PAC-CUR-SLNs were studied in vivo, using BALB/c mice. PAC-CUR-SLNs were nano sized (190 nm), homogeneously disseminated particles with %IE of both PAC and CUR above 94 %. PAC-CUR-SLNs released PAC and CUR in a controlled fashion when compared with free drug suspensions. The cytotoxicity of PAC-CUR-SLNs was higher than individual drug-loaded SLNs and pure drugs. Moreover, the co-delivery displayed synergistic effect, indicating potential of PAC-CUR-SLNs in lung cancer treatment. In vivo tumor investigation of PAC-CUR-SLNs exhibited 12-fold reduced tumor volume and almost no change in body weight of BALB/c mice, when compared with the experimental groups including control group. The inhibition of tumor rate on day 28 was 82.7 % in the PAC-CUR-SLNs group, which was significantly higher than the pure drugs and monotherapies. It can be concluded that, encapsulating the co-loaded antitumor drugs like PAC-CUR in SLNs may help in improved targeting of the tumor with enhanced anticancer effect.

Mimosa pudica L. extract ameliorates pulmonary fibrosis via modulation of MAPK signaling pathways and FOXO3 stabilization

Ethnopharmacological relevanceIdiopathic pulmonary fibrosis (IPF) is a progressive fibrosing pulmonary disorder that has a poor prognosis and high mortality. Although there has been extensive effort to introduce several new anti-fibrotic agents in the past decade, IPF remains an incurable disease. Mimosa pudica L., an indigenous Vietnamese plant, has been empirically used to treat respiratory disorders. Nevertheless, the therapeutic effects of M. pudica (MP) on lung fibrosis and the mechanisms underlying those effects remain unclear. Aim of the studyThis study investigated the protective effect of a crude ethanol extract of the above-ground parts of MP against pulmonary fibrogenesis. Materials and methodsInflammatory responses triggered by TNFα in structural lung cells were examined in normal human lung fibroblasts and A549 alveolar epithelial cells using Western blot analysis, reverse transcription–quantitative polymerase chain reaction assays, and immunocytochemistry. The epithelial-to-mesenchymal transition (EMT) was examined via cell morphology observations, F-actin fluorescent staining, gene and protein expression measurements, and a wound-healing assay. Anti-fibrotic assays including collagen release, differentiation, and measurements of fibrosis-related gene and protein expression levels were performed on TGFβ-stimulated human lung fibroblasts and lung fibroblasts derived from mice with fibrotic lungs. Finally, in vitro anti-fibrotic activities were validated using a mouse model of bleomycin-induced pulmonary fibrosis. ResultsMP alleviated the inflammatory responses of A549 alveolar epithelial cells and lung fibroblasts, as revealed by inhibition of TNFα-induced chemotactic cytokine and chemokine expression, along with inactivation of the MAPK and NFκB signalling pathways. MP also partially reversed the TGFβ-promoted EMT via downregulation of mesenchymal markers in A549 cells. Importantly, MP decreased the expression levels of fibrosis-related genes/proteins including collagen I, fibronectin, and αSMA; moreover, it suppressed collagen secretion and prevented myofibroblast differentiation in lung fibroblasts. These effects were mediated by FOXO3 stabilization through suppression of TGFβ-induced ERK1/2 phosphorylation. MP consistently protected mice from the onset and progression of bleomycin-induced pulmonary fibrosis. ConclusionThis study explored the multifaceted roles of MP in counteracting the pathobiological processes of lung fibrosis. The results suggest that further evaluation of MP could yield candidate therapies for IPF.

Knockdown of KBTBD7 attenuates septic lung injury by inhibiting ferroptosis and improving mitochondrial dysfunction

Lung injury in sepsis is caused by an excessive inflammatory response caused by the entry of pathogenic microorganisms into the body. It is also accompanied by the production of large amounts of ROS. Ferroptosis and mitochondrial dysfunction have also been shown to be related to sepsis. Finding suitable sepsis therapeutic targets is crucial for sepsis research. BTB domain-containing protein 7 (KBTBD7) is involved in regulating inflammatory responses, but its role and mechanism in the treatment of septic lung injury are still unclear. In this study, we evaluated the role and related mechanisms of KBTBD7 in septic lung injury. In in vitro studies, we established an in vitro model by inducing human alveolar epithelial cells with lipopolysaccharide (LPS) and found that KBTBD7 was highly expressed in the in vitro model. KBTBD7 knockdown could reduce the inflammatory response by inhibiting the secretion of pro-inflammatory factors and inhibit the production of ROS, ferroptosis and mitochondrial dysfunction. Mechanistic studies show that KBTBD7 interacts with FOXA1, promotes FOXA1 expression, and indirectly inhibits SLC7A11 transcription. In vivo studies have shown that knocking down KBTBD7 improves lung tissue damage in septic lung injury mice, inhibits inflammatory factors, ROS production and ferroptosis. Taken together, knockdown of KBTBD7 shows an alleviating effect on septic lung injury in vitro and in vivo, providing a potential therapeutic target for the treatment of septic lung injury.

Assessment of Portuguese fitness centers: Bridging the knowledge gap on harmful microbial contamination with focus on fungi

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m−2.day−1) and Gram-negative bacteria (VRBA: 1.23 CFU m−2.day−1), while for fungi it was the open space area, with 1.86 × 101 CFU m−2.day−1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples’ contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati, as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination.

In vitro and in vivo evaluation of the anti-infective potential of the essential oil extracted from the leaves of Plectranthus amboinicus (lour.) spreng against Klebsiella pneumoniae and elucidation of its mechanism of action through proteomics approach

Ethnopharmacological relevanceMembers of Plectranthus genus such as Plectranthus amboinicus (Lour.) Spreng is a well-known folkloric medicine around the globe in treating several human ailments such as cardiovascular, respiratory, digestive, urinary tract, skin and infective diseases. Its therapeutic value is primarily attributed to its essential oil. Although several properties of Plectranthus amboinicus essential oil have been documented, its mechanism of action and safety has not been completely elucidated. Aim of the studyTo investigate the anti-infective potential of Plectranthus amboinicus essential oil against Klebsiella pneumoniae using in vitro and in vivo bioassays and identify its mode of action. The study was conducted to scientifically validate the traditional usage of Plectranthus amboinicus oil and propose it as a complementary and alternative medication to combat Klebsiella pneumoniae infections due to emerging antibiotic resistance problem. Materials and methodsPlectranthus amboinicus essential oil was extracted through steam distillation and was chemically characterized using Gas Chromatography Mass Spectrometry (GC-MS). The antibacterial activity was assessed using microbroth dilution assay, metabolic viability assay and growth curve analysis. The mode of action was elucidated by the proteomics approach using Nano-LC-MS/MS followed by in silico analysis. The results of proteomic analysis were further validated through several in vitro assays. The cytotoxic nature of the essential oil was also confirmed using adenocarcinomic human alveolar basal epithelial (A549) cells. Furthermore, the safety and in vivo anti-infective efficacy of Plectranthus amboinicus essential oil was evaluated through survival assay, CFU assay and histopathological analysis of vital organs using zebrafish as a model organism. ResultsThe chemical characterization of Plectranthus amboinicus essential oil revealed that it is predominantly composed of thymol. Thymol rich P. amboinicus essential oil demonstrated potent inhibitory effects on Klebsiella pneumoniae growth, achieving a significant reduction at a concentration of 400 μg/mL within 4 h of treatment The nano-LC-MS/MS approach unveiled that the essential oil exerted its impact by disrupting the antioxidant defense system and efflux pump system of the bacterium, resulting in elevated cellular oxidative stress and affect the biosynthesis of biofilm. The same was validated through several in vitro assays. Furthermore, the toxicity of Plectranthus amboinicus essential oil determined using A549 cells and zebrafish survival assay established a non-toxic concentration of 400 μg/mL and 12.5 μg/mL respectively. The results of anti-infective potential of the essential oil using Zebrafish as a model organism demonstrated significantly improved survival rates, reduced bacterial load, alleviated visible signs of inflammation and mitigated the adverse effects of infection on various organs, as evidenced by histopathological analysis ensuring its safety for potential therapeutic application. ConclusionThe executed in vitro and in vivo assays established the effectiveness of essential oil in inhibiting bacterial growth by targeting key proteins associated with the bacterial antioxidant defense system and disrupted the integrity of the cell membrane, highlighting its critical role in addressing the challenge posed by antibiotic-resistant Klebsiella pneumoniae.

Exploring the synthesis, structure and bioactivity of pyrimidine carboxylic acid-derived organic antimony (V) complexes: Cytostatic and antimicrobial evaluations

Eight new organoantimony complexes, R3SbL2 (R = Ph, p-MePh; L = L1, L2) and (R3Sb)2OL2 (R = pHPh, m-MePh; L = L1, L2), [Ph3Sb(C5H3N2O2)2] (1), [(Ph3Sb)2O(C5H3N2O2)2] (2), [(p-tol)3Sb(C5H3N2O2)2] (3), [(m-tol)6Sb2O(C5H3N2O2)2] (4), [Ph3Sb(C5H3N2O2)2] (5), [(Ph3Sb)2O(C5H3N2O2)2] (6), [(p-tol)3Sb(C5H3N2O2)2] (7) and [(m-tol)6Sb2O(C5H3N2O2)2] (8), were designed and synthesized by the reaction of 4-pyrimidine carboxylic acid (HL1) or 5-pyrimidine carboxylic acid (HL2) with organoantimony precursors. All the complexes were characterized by elemental analysis, FT-IR, NMR (1H and 13C), and single crystal X-ray diffraction analysis. Crystal structure results reveal that the central antimony atom of all the complexes displays a triangular biconical geometry. Among them, complexes 1, 3, 5 and 7 show a monomeric structure, while complexes 2, 4, 6 and 8 have a dimeric structure with oxygen bridges. The in vitro cytostatic activities of the eight complexes against a human alveolar basal epithelial lung cancer cell lines (A549) and hepatocellular carcinoma cell lines (HepG-2) were investigated, and the results showed that the organoantimony derivatives with the tolyl groups (3, 4, 7, 8) exhibited higher in vitro cytostatic activities compared with those with the phenyl groups (1, 2, 5, 6). At the same time, bacteriostatic tests were conducted to assess the inhibitory effects of complexes 1–8 against three prevalent plant pathogenic fungi and one bacterium. The results revealed that complex 4 exhibited significant fungicidal activities, on the other hand, complex 3 exhibited higher antibacterial activity against MRSA. These results will provide significant guidance for the further exploration of potential efficient biological activity complexes.

Knockdown of lncRNA NEAT1 suppresses streptococcus pneumoniae-induced ferroptosis in alveolar epithelial cells by regulating the Nrf2-GPX4 pathway

ObjectivesStreptococcus pneumoniae (SP) is a major cause of community-acquired pneumonia. Ferroptosis pitches in pneumonia. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) regulates ferroptosis in various cells. Therefore, this study probed the mechanism of lncRNA NEAT1 on SP-induced ferroptosis in AECs. MethodsSerum lncRNA NEAT1 level in 36 streptococcus pneumonia patients were retrospectively detected, with its correlations with inflammatory factor (TNF-α/IL-1β/IL-6) levels analyzed. Human pulmonary alveolar epithelial cells (HPAEpiC) were transfected with sh-NEAT1 and induced by SP. Cell viability was evaluated by CCK-8. Lactate dehydrogenase (LDH) activity was assessed. Iron content, and levels of TNF-α/IL-1β/IL-6/IL-10/lncRNA NEAT1/lipid peroxidation products [malondialdehyde (MDA)/glutathione (GSH)/reactive oxygen species/(ROS)]/ferroptosis-related proteins [Cyclooxgenase 2 (COX2)/recombinant solute carrier family 7 member 11 (SLC7A11)/total nuclear factor erythroid 2-related factor 2 (Nrf2)/cytoplasmic Nrf2 (C-Nrf2)/nuclear Nrf2 (N-Nrf2)/GPX4)] were determined by kit/ELISA/RT-qPCR/kits/Western blot. Nrf2 nuclear translocation was detected by immunofluorescence assay. On top of lncRNA NEAT1 knockdown, SP-induced HPAEpiC were treated with ML385. ResultsSerum lncRNA NEAT1 level was elevated in streptococcus pneumonia patients, and were positively interrelated with TNF-α/IL-1β/IL-6 levels. SP promoted cell HPAEpiC injury and inflammatory response, and up-regulated lncRNA NEAT1 level. LncRNA NEAT1 knockdown suppressed HPAEpiC injury/inflammatory response (reduced LDH activity and TNF-α/IL-1β/IL-6 levels, elevated IL-10) and suppressed ferroptosis (decreased iron/MDA/ROS contents and COX2 level, increased GSH/SLC7A11), facilitated Nrf2 nuclear translocation, and up-regulated GPX4. Nrf2-GPX4 pathway inhibition annulled NEAT1 knockdown-mediated improvement on SP-induced HPAEpiC ferroptosis/injury/inflammatory response. ConclusionsLncRNA NEAT1 knockdown suppressed SP-induced HPAEpiC ferroptosis by activating Nrf2-GPX4 pathway, thereby alleviating cell injury and inflammatory response.

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.

Metabolic landscape of human alveolar type II epithelial cells undergoing epithelial-mesenchymal transition induced directly by silica exposure

Epithelial-mesenchymal transition (EMT) plays an irreplaceable role in the development of silicosis. However, molecular mechanisms of EMT induced by silica exposure still remain to be addressed. Herein, metabolic profiles of human alveolar type II epithelial cells (A549 cells) exposed directly to silica were characterized using non-targeted metabolomic approaches. A total of 84 differential metabolites (DMs) were identified in silica-treated A549 cells undergoing EMT, which were mainly enriched in metabolisms of amino acids (e.g., glutamate, alanine, aspartate), purine metabolism, glycolysis, etc. The number of DMs identified in the A549 cells obviously increased with the elevated exposure concentration of silica. Remarkably, glutamine catabolism was significantly promoted in the silica-treated A549 cells, and the levels of related metabolites (e.g., succinate) and enzymes (e.g., α-ketoglutarate (α-KG) dehydrogenase) were substantially up-regulated, with a preference to α-KG pathway. Supplementation of glutamine into the cell culture could substantially enhance the expression levels of both EMT-related markers and Snail (zinc finger transcription factor). Our results suggest that the EMT of human alveolar epithelial cells directly induced by silica can be essential to the development of silicosis.

Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail

BackgroundThe alveolar epithelium is exposed to numerous stimuli, such as chemicals, viruses, and bacteria that cause a variety of pulmonary diseases through inhalation. Alveolar epithelial cells (AECs) cultured in vitro are a valuable tool for studying the impacts of these stimuli and developing therapies for associated diseases. However, maintaining the proliferative capacity of AECs in vitro is challenging. In this study, we used a cocktail of three small molecule inhibitors to cultivate AECs: Y-27632, A-83-01, and CHIR99021 (YAC). These inhibitors reportedly maintain the proliferative capacity of several types of stem/progenitor cells.ResultsPrimary human AECs cultured in medium containing YAC proliferated for more than 50 days (over nine passages) under submerged conditions. YAC-treated AECs were subsequently cultured at the air-liquid interface (ALI) to promote differentiation. YAC-treated AECs on ALI day 7 formed a monolayer of epithelial tissue with strong expression of the surfactant protein-encoding genes SFTPA1, SFTPB, SFTPC, and SFTPD, which are markers for type II AECs (AECIIs). Immunohistochemical analysis revealed that paraffin sections of YAC-treated AECs on ALI day 7 were mainly composed of cells expressing surfactant protein B and prosurfactant protein C.ConclusionsOur results indicate that YAC-containing medium could be useful for expansion of AECIIs, which are recognized as local stem/progenitor cells, in the alveoli.

Characteristics of Polycyclic Aromatic Hydrocarbons in Size-Resolved Particles in the Roadside Environment of Beijing: Seasonality, Source, and Toxicological Effects

The polycyclic aromatic hydrocarbons (PAHs) in size-resolved particles emitted from diverse sources are required for quantification to reduce the emissions in order to protect public health. Twenty-four PAHs in size-segregated particles in the roadside environment of Beijing were observed from 1 October 2021 to 30 September 2022. The size distributions of PAHs were bimodal, with peak concentrations ranging from size fractions of 0.43 to 0.65 μm and 4.7 to 5.8 μm in all four seasons, respectively. The highest concentration of PAHs in fine particles (PM2.1) was 35.3 ng m−3 in winter, followed by 16.0 ng m−3 in autumn, 15.3 ng m−3 in spring, and 6.5 ng m−3 in summer. Conversely, the concentration of PAHs in coarse particles (PM2.1–9) ranged from 6.8 ng m−3 (summer) to 20.5 ng m−3 (winter) from low to high. The size fractions of 0.43–2.1 μm PAHs increased most from clear to polluted days, which could be ascribed to the heterogeneous reactions. Source apportionment using positive matrix factorization showed that four sources, namely biomass combustion, coal combustion, diesel vehicles, and gasoline vehicles accounted for PAHs with the estimation of 17.4%, 22.1%, 26.4%, and 23.2% to PAHs in PM2.1; and 19.6%, 24.3%, 23.6%, and 20.1% in PM2.1–9, respectively. Furthermore, we used the human alveolar epithelial cell (BEAS-2B) to assess the toxicological effects of size-resolved atmospheric PAHs. The results showed that the cell survival rate caused by fine particles was lower than that of coarse particles with the same concentrations of PAHs, which is mainly related to the higher content of highly toxic PAHs in fine particles.

Circular RNA MKLN1 promotes epithelial-mesenchymal transition in pulmonary fibrosis by regulating the miR-26a/b-5p/CDK8 axis in human alveolar epithelial cells and mice models.

Pulmonary fibrosis involves destruction of the lung parenchyma and extracellular matrix deposition. Effective treatments for pulmonary fibrosis are lacking and its pathogenesis is still unclear. Studies have found that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) plays an important role in progression of pulmonary fibrosis. Thus, an in-depth exploration of its mechanism might identify new therapeutic targets. In this study, we revealed that a novel circular RNA, MKLN1 (circMKLN1), was significantly elevated in two pulmonary fibrosis models (intraperitoneally with PQ, 50mg/kg for 7days, and intratracheally with BLM, 5mg/kg for 28days). Additionally, circMKLN1 was positively correlated with the severity of pulmonary fibrosis. Inhibition of circMKLN1 expression significantly reduced collagen deposition and inhibited EMT in AECs. EMT was aggravated after circMKLN1 overexpression in AECs. MiR-26a-5p/miR-26b-5p (miR-26a/b), the targets of circMKLN1, were confirmed by luciferase reporter assays. CircMKLN1 inhibition elevated miR-26a/b expression. Significantly decreased expression of CDK8 (one of the miR-26a/b targets) was observed after inhibition of circMKLN1. EMT was exacerbated again, and CDK8 expression was significantly increased after circMKLN1 inhibition and cotransfection of miR-26a/b inhibitors in AECs. Our research indicated that circMKLN1 promoted CDK8 expression through sponge adsorption of miR-26a/b, which regulates EMT and pulmonary fibrosis. This study provides a theoretical basis for finding new targets or biomarkers in pulmonary fibrosis.

Effects of chitosan and TiO2 nanoparticles on the antibacterial property and ability to self-healing of cracks and retrieve mechanical characteristics of dental composites

The aim of this study was to improve the self-healing properties of dental nanocomposite using nanoparticles of TiO2 and chitosan. We evaluated flexural and compressive strength, crack-healing, and self-healing lifespan after 3 months of water aging. The effect of the developed composite on cell viability and toxicity was assessed by an MTT assay on human alveolar basal epithelial cells (A549 cell line). The nanocomposite included 7.5 wt% polyurea-formaldehyde (PUF) and 0, 0.5, and 1 wt% n-TiO2 and chitosan. After the fracture, the samples were put in a mold for 1–90 days to enable healing. Then, the fracture toughness of the healed nanocomposites and the healing yield were measured. The flexural strength of the nanocomposite improved by adding 0.5 wt% n-TiO2, while the compressive strength increased after adding 0.5 wt% chitosan (p > 0.1). When these two materials were used simultaneously, the flexural strength was improved by around 2%; however, the compressive strength was unaffected. Compared to the other sample, the nanocomposite with 0.5 wt% n-TiO2 and chitosan had higher KIC-healing and self-healing efficiency. Self-healing efficacy had no significant effect of water aging over 90 days compared to one day (p > 0.1), demonstrating that the PUF nanocapsules were not damaged.