Lung Fibroblast MRC-5 Research Articles

Chlorine containing tetrahydropyrimidines: Synthesis, characterization, anticancer activity and mechanism of action

The aim of the presented research was to explore anticancer potential of eleven newly synthesized tetrahydropyrimidine derivatives. The compounds were synthesized via Biginelli multicomponent one-pot reaction using different derivatives of vanillin, ethyl 4-chloroacetoacetate and (N-methyl)urea. The cytotoxic effects of the compounds were examined on three human malignant cell lines (HeLa, K562, and MCF7), and normal lung fibroblasts MRC-5. The mechanisms of anticancer activity were examined for two compounds 4a and 4b which showed the strongest and selective cytotoxicity against chronic myelogenous leukaemia K562 cells (IC50 = 1.76 ± 0.09, and 1.66 ± 0.05, respectively). The changes of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and vascular endothelial growth factor A (VEGFA) were investigated in the K562 cell line, as well as oncomiRNA miR-10b, miR-23a described to have both features, depending on a specific type of malignancy, and miR-34a with mostly described as a tumour suppressor.

Inhalable nano-structured microparticles for extracellular matrix modulation as a potential delivery system for lung cancer

The use of inhalable nanoparticulate-based systems in the treatment of lung cancer allows for efficient localized delivery to the lungs with less undesirable systemic exposure. For this to be attained, the inhaled particles should have optimum properties for deposition and at the same time avoid pulmonary clearance mechanisms. Drug delivery to solid tumors is furthermore challenging, due to dense extracellular matrix (ECM) formation, which hinders the penetration and diffusion of therapeutic agents. To this end, the aim of the current work is to develop an ECM-modulating nano-structured microparticulate carrier, that not only enables the delivery of therapeutic nanoparticles (NPs) to the lungs, but also enhances their intratumoral penetration. The system is composed of acetalated maltodextrin (AcMD) NPs embedded into a water-soluble trehalose/leucine matrix, in which collagenase was loaded with different mass concentrations (10 %, 30 % and 50 %). The collagenase-containing AcMD nano-structured microparticles (MPs) exhibited suitable median volume diameters (2.58 ± 1.35 to 3.01 ± 0.68 µm), hollow corrugated morphology, sufficient redispersibility, low residual moisture content (2.71 ± 0.17 % to 3.10 ± 0.20 %), and favorable aerodynamic properties (Mass median aerodynamic diameter (MMAD): 1.93 ± 0.06 to 2.80 ± 0.10 µm and fine particle fraction (FPF): 68.02 ± 6.86 % to 69.62 ± 2.01 %). Importantly, collagenase retained as high as 89.5 ± 6.7 % of its enzymatic activity after spray drying. MPs containing 10 % mass content of collagenase did not show signs of cytotoxicity on either human lung adenocarcinoma A549 cells or lung MRC-5 fibroblasts. The nanoparticle penetration was tested using adenocarcinoma A549/MRC-5 co-culture spheroid model, where the inclusion of collagenase resulted in deeper penetration depth of AcMD-NPs.

Differentiation and Growth-Arrest-Related lncRNA (DAGAR): Initial Characterization in Human Smooth Muscle and Fibroblast Cells.

Vascular smooth muscle cells (SMCs) can transition between a quiescent contractile or "differentiated" phenotype and a "proliferative-dedifferentiated" phenotype in response to environmental cues, similar to what in occurs in the wound healing process observed in fibroblasts. When dysregulated, these processes contribute to the development of various lung and cardiovascular diseases such as Chronic Obstructive Pulmonary Disease (COPD). Long non-coding RNAs (lncRNAs) have emerged as key modulators of SMC differentiation and phenotypic changes. In this study, we examined the expression of lncRNAs in primary human pulmonary artery SMCs (hPASMCs) during cell-to-cell contact-induced SMC differentiation. We discovered a novel lncRNA, which we named Differentiation And Growth Arrest-Related lncRNA (DAGAR) that was significantly upregulated in the quiescent phenotype with respect to proliferative SMCs and in cell-cycle-arrested MRC5 lung fibroblasts. We demonstrated that DAGAR expression is essential for SMC quiescence and its knockdown hinders SMC differentiation. The treatment of quiescent SMCs with the pro-inflammatory cytokine Tumor Necrosis Factor (TNF), a known inducer of SMC dedifferentiation and proliferation, elicited DAGAR downregulation. Consistent with this, we observed diminished DAGAR expression in pulmonary arteries from COPD patients compared to non-smoker controls. Through pulldown experiments followed by mass spectrometry analysis, we identified several proteins that interact with DAGAR that are related to cell differentiation, the cell cycle, cytoskeleton organization, iron metabolism, and the N-6-Methyladenosine (m6A) machinery. In conclusion, our findings highlight DAGAR as a novel lncRNA that plays a crucial role in the regulation of cell proliferation and SMC differentiation. This paper underscores the potential significance of DAGAR in SMC and fibroblast physiology in health and disease.

In silico and in vitro chemometrics, cell toxicity and permeability of naringenin 8-sulphonate and derivatives.

Sulphur containing natural compounds are among the most biologically relevant metabolites in vivo. Naringenin 8-sulphonate from Parinari excelsa Sabine was evaluated in a previous work, demonstrating ability to act as a natural anti-inflammatory. Although the interference of this molecule against different inflammatory mediators was described, there is no information regarding its potential toxicity and pharmacokinetics, which are essential for its capacity to reach its therapeutic targets. In fact, despite the existence of reports on naringenin ADMET properties, the influence of sulphation patterns on them remains unknown. This work aims to assess the in vitro pharmacokinetic and toxicological behavior of naringenin 8-sulphonate, as well as to understand the importance of the presence and position of the sulphur containing group for that. Naringenin 8-sulphonate physicochemical and ADMET properties were investigated using in silico tools and cell-based in vitro models. At the same time, naringenin and naringenin 4'-O-sulphate were investigated to evaluate the impact of the sulphonate group on the results. ADMETlab 2.0 in silico tool was used to predict the compounds' physicochemical descriptors. Pharmacokinetic properties were determined experimentally in vitro. While MRC-5 lung fibroblasts and HaCaT keratinocytes were used to evaluate the cytotoxicity of samples through MTT and LDH assays, Caco-2 human intestinal epithelial cells were used for the determination of genotoxicity, through alkaline comet assay, and as a permeability model to assess the ability of compounds to cross biological barriers. Experimental determinations showed that none of the compounds was cytotoxic. In terms of genotoxicity, naringenin 8-sulphonate and naringenin caused significant DNA fragmentation, whereas naringenin 4'-O-sulphate did not. When it comes to permeability, the two sulphur-containing compounds with a sulphur containing group were clearly less capable to cross the Caco-2 cell barrier than naringenin. In this study, we conclude that the sulphur containing group from naringenin 8-sulphonate is disadvantageous for the molecule in terms of ADMET properties, being particularly impactful in the permeability in intestinal barrier models. Thus, this work provides important insights regarding the role of flavonoids sulphation and sulphonation upon pharmacokinetics and toxicity.

Synthesis, crystal structure and cytotoxic activity of 2,2′-bipyridine / 1,10-Phenanthroline based Copper(II) complexes with diphenylphosphinic acid

Mixed-ligand 2,2′-bipyridine, 1,10-phenanthroline based copper(II) complexes are often considered as potential antitumor agents. This research was aimed at synthesis and finding the cytotoxic potential of copper(II) complexes with diphenylphosphinic acid (HL) and 2,2′-bipyridine, 1,10-phenanthroline derivatives – [Cu(phen)(H2O)L2]·H2O (1), [Cu(dmphen)(H2O)L2] (2), [Cu(Cl-phen)(H2O)2L]L·EtOH (3), [Cu(bipy)(H2O)L2] (4) and Cu(dmbipy)(H2O)L2 (5) (phen – 1,10-phenanthroline, dmphen – 4,7-dimethyl-1,10-phenanthroline, Cl-phen – 5-chloro-1,10-phenanthroline, bipy – 2,2′-bipyridine, dmbipy – 4,4′-dimethyl-2,2′-bipyridine). Obtained mononuclear compounds have been characterized by EPR and IR-spectroscopy, elemental, thermogravimetric, single-crystal and powder X-ray diffraction analyses. According to single-crystal X-ray diffraction data, complexes possess distorted trigonal bipyramidal or square pyramidal geometry. 2,2′-Bipyridine and 1,10-phenanthroline derivatives have been shown to be chelating agents in these complexes, while anion of diphenylphosphinic acid acts as a monodentate ligand. Complexes 1–5 have been monitored for their solution stability using UV–vis spectroscopy. In the present study, cytotoxic activity of the complexes has been investigated on 2D human cell culture models (larynx carcinoma Hep2, hepatocellular carcinoma HepG2, breast carcinoma MCF-7 and non-tumor lung fibroblasts MRC5) and 3D HepG2 human cell model. 1,10-Phenanthroline based complexes have demonstrated the highest cytotoxicity superior to the reference drug cisplatin. The level of reactive oxygen species generation in Hep2 cells has been shown to increase after incubation with complexes.

Addition of Polyphenols to Drugs: The Potential of Controlling "Inflammaging" and Fibrosis in Human Senescent Lung Fibroblasts In Vitro.

The combination of a polyphenol, quercetin, with dasatinib initiated clinical trials to evaluate the safety and efficacy of senolytics in idiopathic pulmonary fibrosis, a lung disease associated with the presence of senescent cells. Another approach to senotherapeutics consists of controlling inflammation related to cellular senescence or "inflammaging", which participates, among other processes, in establishing pulmonary fibrosis. We evaluate whether polyphenols such as caffeic acid, chlorogenic acid, epicatechin, gallic acid, quercetin, or resveratrol combined with different senotherapeutics such as metformin or rapamycin, and antifibrotic drugs such as nintedanib or pirfenidone, could present beneficial actions in an in vitro model of senescent MRC-5 lung fibroblasts. A senescent-associated secretory phenotype (SASP) was evaluated by the measurement of interleukin (IL)-6, IL-8, and IL-1β. The senescent-associated β-galactosidase (SA-β-gal) activity and cellular proliferation were assessed. Fibrosis was evaluated using a Picrosirius red assay and the gene expression of fibrosis-related genes. Epithelial-mesenchymal transition (EMT) was assayed in the A549 cell line exposed to Transforming Growth Factor (TGF)-β in vitro. The combination that demonstrated the best results was metformin and caffeic acid, by inhibiting IL-6 and IL-8 in senescent MRC-5 cells. Metformin and caffeic acid also restore cellular proliferation and reduce SA-β-gal activity during senescence induction. The collagen production by senescent MRC-5 cells was inhibited by epicatechin alone or combined with drugs. Epicatechin and nintedanib were able to control EMT in A549 cells. In conclusion, caffeic acid and epicatechin can potentially increase the effectiveness of senotherapeutic drugs in controlling lung diseases whose pathophysiological component is the presence of senescent cells and fibrosis.

Synthesis of selenophene-containing flavonols and 2-styrylchromones: Evaluation of their activities compared with selenophene-containing chalcones as potential anticancer agents.

Previously, we documented the synthesis and assessed the biological effects of chalcones containing selenium against HT-29 human colorectal adenocarcinoma cells, demonstrating their significant potential. As research on selenium-containing flavonoids remains limited, this article outlines our design and synthesis of three selenium-based flavonols and three 2-styrylchromones. We conducted evaluations of these compounds to determine their impact on human lung cancer cells (A549, H1975, CL1-0, and CL1-5) and their influence on normal lung fibroblast MRC5 cells. Additionally, we included selenium-based chalcones in our testing for comparative purposes. Our findings highlight that the simplest compound, designated as compound 1, exhibited the most promising performance among the tested molecules.

Lung fibroblast-derived extracellular vesicles and soluble factors alleviate elastase-induced lung injury

One of the main pathological features of chronic obstructive pulmonary disease (COPD) is the loss of functional alveolar tissue as a consequence of impaired regenerative capacities (emphysema). Recent research suggests that the secretome from mesenchymal cells, particularly extracellular vesicles (EVs), may possess regenerative properties beneficial for lung repair. However, the regenerative potential of the soluble factors (SFs) within the secretome remains largely unexplored in COPD. To this extent, we purified EVs and SFs secreted by lung fibroblasts to generate EV-enriched and SF-enriched fractions, and evaluated their effects on elastase-induced lung injury in both precision-cut lung slices (PCLS) and a mouse model. EV- and SF-enriched fractions were concentrated and purified from the conditioned medium of cultured MRC-5 lung fibroblasts using a combination of ultrafiltration and size exclusion chromatography, and were subsequently characterized according to the MISEV guidelines. Treatment with EV- or SF-enriched concentrates prevented and improved elastase-induced emphysema in PCLS, leading to reduced lung injury and upregulated markers of alveolar epithelial cells (aquaporin 5 and surfactant protein C), indicating potential parenchymal regeneration. Accordingly, prophylactic intratracheal treatment with lung fibroblast-derived EV- and SF-enriched concentrates in vivo attenuated elastase-induced lung tissue destruction, improved lung function, and enhanced gene expression of alveolar epithelial cell markers. Here, alveolar repair not only serves the purpose of facilitating gas exchange, but also by reinstating the essential parenchymal tethering required for optimal airway mechanics. In conclusion, this study highlights the therapeutic potential of both lung fibroblast-derived EV- and SF-enriched concentrates for the treatment of lung injury and emphysema.

The Effect of Substrate Properties on Cellular Behavior and Nanoparticle Uptake in Human Fibroblasts and Epithelial Cells.

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell-nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell-substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell-matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment.

Freshwater algae Cladophora glomerata and Vaucheria sp. from Serbia as sources of bioactive compounds: Chemical analysis and biological activities

We examined potential biological activities of two taxa of freshwater algae, Cladophora glomerata and Vaucheria sp., from Serbia. The total phenolic and pigment contents, antioxidant potential, antimicrobial, antibiofilm activities, and cytotoxicity of the ethanol and acetone extracts were evaluated. The extracts were also subjected to Fourier transform infrared spectroscopy (FTIR) analysis. The levels of total phenolic compounds, chlorophylls a and b, and carotenoids varied based on both the algal taxa and the type of extracts. FTIR analysis showed the presence of lipids, unsaturated fatty acids, protein, carbohydrates, and phenols in the algal extracts. The extracts had moderate DPPH radical scavenging activity and lower reducing power compared with ascorbic acid. The antimicrobial activity expressed as minimum inhibitory concentrations ranged from 0.31 mg/mL to 10 mg/mL. The strains of Staphylococcus aureus and Bacillus cereus isolated from food samples, as well as S. aureus ATCC 25923, were the most sensitive. For the first time, the antibiofilm activity test revealed 98.7% inhibition of S. aureus biofilm formation. The extracts exhibited cytotoxic effects on choriocarcinoma JAR cells but without selectivity on normal fetal lung fibroblast MRC-5 cells. This is the first report on the biological activities of freshwater macroalgae from Serbia.

The cytotoxic and antiproliferative properties of ruthenium nitrosyl complexes and their modulation effect on cytochrome P450 in the HepG2 cell line.

Ruthenium nitrosyl complexes are actively investigated as antitumor agents. Evaluation of potential interactions between cytochromes P450 (CYPs) with new compounds is carried out regularly during early drug development. In this study we have investigated the cytotoxic and antiproliferative activities of ruthenium nitrosyl complexes with methyl/ethyl esters of nicotinic and isonicotinic acids and γ-picoline against 2D and 3D cultures of human hepatocellular carcinoma HepG2 and non-cancer human lung fibroblasts MRC-5, assessed their photoinduced activity at λrad = 445 nm, and also evaluated their modulating effect on CYP3A4, CYP2C9, and CYP2C19. The study of cytotoxic and antiproliferative activities against 2D and 3D cell models was performed using phenotypic-based high content screening (HCS). The expression of CYP3A4, CYP2C9, and CYP2C19 mRNAs and CYP3A4 protein was examined using target-based HCS. The results of CYP3A4 mRNA expression were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The ruthenium nitrosyl complexes exhibited a dose-dependent cytotoxic effect against HepG2 and MRC-5 cells. The cytotoxic activity of complexes with ethyl isonicotinate (1) and nicotinate (3, 4) was significantly lower for MRC-5 than for HepG2, for a complex with methyl isonicotinate (2) it was higher for MRC-5 than for HepG2, for a complex with γ-picoline (5) it was comparable for both lines. The antiproliferative effect of complexes 2 and 5 was one order of magnitude higher for MRC-5; for complexes 1, 3, and 4 it was comparable for both lines. The cytotoxic activity of all compounds for 3D HepG2 was lower than for 2D HepG2, with the exception of 4. Photoactivation affected the activity of complex 1 only. Its cytotoxic activity decreased, while the antiproliferative activity increased. The ruthenium nitrosyl complexes 1-4 acted as inducers of CYP3A4 and CYP2C19, while the complex with γ-picoline (5) induced of CYP3A4. Among the studied ruthenium nitrosyl complexes, the most promising potential antitumor compound is the ruthenium compound with methyl nicotinate (4).

Curcumin ameliorates pulmonary fibrosis progression by inhibiting the fibrotic process of lung fibroblasts.

The objective of this study was to analyze the effect of curcumin (Cur) on pulmonary fibrosis (PF), so as to provide new clinical evidence for future PF treatment. To achieve these goals, the researchers set up bought human lung fibroblasts MRC-5 as a control group without treatment, a model group for PF cell modeling, and an intervention group for Cur intervention after PF modeling. Cell proliferation capacity and cellular TGF-β1, α-SMA, Collagen I, Collagen III, Bax, N-cadherin and E-cadherin protein expression were determined. The results show that markedly enhanced cell proliferation capacity and TGF-β1, α-SMA, Collagen I and Collagen III protein levels were observed in the model group, while the cell activity and fibrosis degree in the intervention group were significantly decreased compared with the model group (P<0.05). In addition, the intervention group exhibited lower N-cadherin and Bax with higher E-cadherin than the model group (P<0.05). In addition, the team found that the inflammatory response and oxidative stress were also more significantly improved in the intervention group (P<0.05). These experimental results tell us that Cur can ameliorate the fibrotic process of PF by inhibiting the activity of MRC-5.

Comprehensive Stereoselectivity Assessment of Toxicokinetics, Tissue Distribution, Cytotoxicity, and Environmental Fate of Chiral Pesticide Propiconazole.

Propiconazole (PRO) has been widely used in the treatment of fungal infection in fruits, vegetables, cereals, and seeds. In this study, a newly established chiral liquid chromatography tandem mass spectrometry method was applied to the systemic stereoselectivity evaluation of PRO enantiomers, including toxicokinetics, tissue distributions, cytotoxicity, accumulation, and degradation. Our results showed that both trans (+)-2S,4S-PRO and cis (-)-2S,4R-PRO had lower Cmax and AUC0-∞ and higher CLz/F values in plasma and lower accumulation concentrations in the liver, heart, and brain. In cytotoxic assays, cis (-)-2S,4R-PRO exhibited the lowest cytotoxicity in PC12 neuronal, N9 microglia, SH-SY5Y neuroblastoma, and MRC5 lung fibroblast cell lines. Moreover, the Eisenia fetida incubation experiment revealed that the accumulations of both trans (+)-2S,4S-PRO and cis (-)-2S,4R-PRO were higher than those of their antipodes in E. fetida. In summary, our findings first suggested that the application of cis (-)-2S,4R-PRO for agriculture would hugely reduce the environmental risk.

Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism

The pharmacological interest in mitochondria is very relevant since these crucial organelles are involved in the pathogenesis of multiple diseases, such as cancer. In order to modulate cellular redox/oxidative balance and enhance mitochondrial function, numerous polyphenolic derivatives targeting mitochondria have been developed. Still, due to the drug resistance emergence in several cancer therapies, significant efforts are being made to develop drugs that combine the induction of mitochondrial metabolic reprogramming with the ability to generate reactive oxygen species, taking into consideration the varying metabolic profiles of different cell types. We previously developed a mitochondria-targeted antioxidant (AntiOxCIN6) by linking caffeic acid to lipophilic triphenylphosphonium cation through a 10-carbon aliphatic chain. The antioxidant activity of AntiOxCIN6 has been documented but how the mitochondriotropic compound impact energy metabolism of both normal and cancer cells remains unknown. We demonstrated that AntiOxCIN6 increased antioxidant defense system in HepG2 cells, although ROS clearance was ineffective. Consequently, AntiOxCIN6 significantly decreased mitochondrial function and morphology, culminating in a decreased capacity in complex I-driven ATP production without affecting cell viability. These alterations were accompanied by an increase in glycolytic fluxes. Additionally, we demonstrate that AntiOxCIN6 sensitized A549 adenocarcinoma cells for CIS-induced apoptotic cell death, while AntiOxCIN6 appears to cause metabolic changes or a redox pre-conditioning on lung MRC-5 fibroblasts, conferring protection against cisplatin. We propose that length and hydrophobicity of the C10-TPP+ alkyl linker play a significant role in inducing mitochondrial and cellular toxicity, while the presence of the antioxidant caffeic acid appears to be responsible for activating cytoprotective pathways.

In vitro antitumor activity of carboplatin and analogues

In order to obtain compounds with better biological activity and lower toxicity, numerous modifications have been made to the cisplatin and carboplatin molecules. The aim of the study was to examine the in vitro antiproliferative activity of two new carboplatin analogues and their effect on both the cell cycle and the induction of apoptosis. Carboplatin and analogues were tested on four human cell lines: myelogenous leukemia K562, colon adenocarcinoma HT-29, breast adenocarcinoma MCF-7 and lung fibroblasts MRC-5. Cell lines were exposed to commercial and synthesized carboplatin and two carboplatin analogues. Their growth inhibition was measured by the MTT test after 24 h incubation. The cell cycle modulation and induction of apoptosis were analyzed by flow cytometry on MCF-7 cell line. After 24 h exposure, both carboplatin analogs induced dose-dependent cytotoxicity in all tested cell lines. The most sensitive was MCF-7 cell line. Tested substances showed moderate cytotoxicity against healthy MRC-5 cells. Flow cytometry analysis showed that both analogues modulated the cell cycle, increasing cell population in sub-G1 phase. The same response in the form of the apoptosis induction was also obtained with the Annexin-V test. Apoptosis induction was confirmed with double-fluorescence staining method. Two new carboplatin analogues strongly inhibit the growth of MCF-7 breast cancer cells, inducing apoptosis and changes in cell cycle distribution.

CIRC_0033530 KNOCKDOWN ALLEVIATES LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY MODEL OF HUMAN LUNG FIBROBLASTS BY MIR-1184/TLR4 AXIS.

Background: Circular RNAs have been reported to be involved in regulating the progression of sepsis and sepsis-associated damage. Herein, this work investigated whether circ_0033530 had roles in the process of septic acute lung injury (sepsis-ALI) and its associated mechanism. Methods: Lipopolysaccharide (LPS)-stimulated human lung fibroblasts MRC-5 were used to mimic the cell model of sepsis-ALI in vitro . Levels of genes and proteins were detected by quantitative real-time polymerase chain reaction and Western blotting. Functional experiments were conducted using 5-ethynyl-2'-deoxyuridine assay, Cell Counting Kit-8 assay, flow cytometry, and enzyme-linked immunosorbent assay. The interaction between miR-1184 and circ_0033530 or toll-like receptor 4 (TLR4) was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. Results: Circ_0033530 expression was lower in sepsis patients and LPS-induced fibroblasts than those in healthy control and untreated cells. Functionally, knockdown of circ_0033530 protected fibroblasts against LPS-induced proliferation arrest, apoptosis, and inflammatory response. Mechanistically, circ_0033530 acted as a sponge for miR-1184, and TLR4 RNA was targeted by miR-1184, indicating the circ_0033530/miR-1184/TLR4 axis. Further rescue experiments showed that circ_0033530 silencing-mediated growth inhibition and inflammation on fibroblasts were attenuated by miR-1184 downregulation or TLR4 upregulation. Conclusion: Circ_0033530 knockdown alleviated LPS-induced proliferation arrest, apoptosis, and inflammation in lung fibroblasts by miR-1184/TLR4 axis, and provided molecular theoretical basis for circ_0033530 on the pathogenesis of sepsis-ALI.

The Toxicological Assessment of Anoectochilus burmannicus Ethanolic-Extract-Synthesized Selenium Nanoparticles Using Cell Culture, Bacteria, and Drosophila melanogaster as Suitable Models.

Selenium nanoparticles (SeNPs) are worthy of attention and development for nutritional supplementation due to their health benefits in both animals and humans with low toxicity, improved bioavailability, and controlled release, being greater than the Se inorganic and organic forms. Our previous study reported that Anoectochilus burmannicus extract (ABE)-synthesized SeNPs (ABE-SeNPs) exerted antioxidant and anti-inflammatory activities. Furthermore, ABE could stabilize and preserve the biological activities of SeNPs. To promote the ABE-SeNPs as supplementary and functional foods, it was necessary to carry out a safety assessment. Cytotoxicity testing showed that SeNPs and ABE-SeNPs were harmless with no killing effect on Caco2 (intestinal epithelial cells), MRC-5 (lung fibroblasts), HEK293 (kidney cells), LX-2 (hepatic stellate cells), and 3T3-L1 (adipocytes), and were not toxic to isolated human PBMCs and RBCs. Genotoxicity assessments found that SeNPs and ABE-SeNPs did not induce mutations in Salmonella typhimurium TA98 and TA100 (Ames test) as well as in Drosophila melanogaster (somatic mutation and recombination test). Noticeably, ABE-SeNPs inhibited mutation in TA98 and TA100 induced by AF-2, and in Drosophila induced by urethane, ethyl methanesulfonate, and mitomycin c, suggesting their anti-mutagenicity ability. This study provides data that support the safety and anti-genotoxicity properties of ABE-SeNPs for the further development of SeNPs-based food supplements.

Intermittent hypoxia induces myofibroblast differentiation and extracellular matrix production of MRC5s via HIF-1α-TGF-β/Smad pathway.

To investigate whether or notintermittent hypoxia (IH), the main characteristic of obstructive sleep apnea (OSA) may affect the myofibroblast differentiation and extracellular matrix (ECM) production of lung fibroblast through the HIF-1α-TGF-β/Smad pathway and assess the interventional role of a HIF-1α inhibitor, 2-methoxyestradiol (2-ME2). The human lung fibroblast MRC5 cells were exposed to normoxia or IH conditions, and the expression of myofibroblast differentiation marker α-smooth muscle actin (α-SMA) and ECM protein collagen I were evaluated. To clarify the underlying mechanism, the expression level of HIF-1α, TGF-β, and p-Smads/Smads were measured and the effects of inhibiting HIF-1α with 2-ME2 on the α-SMA expression level and ECM production through the TGF-β/Smad pathway were assessed. Si HIF-1α was applied to genetically inhibit HIF-1α in MRC5 cells, and the related proteins were assessed. IH increased the protein and mRNA expression of Collagen I and α-SMA of MRC5 cells in a time-dependent manner. IH activated the protein and mRNA level of HIF-1α and TGF-β and increased the phosphorylation of Smad2/Smad3 of MRC5 cells in a time-dependent manner. 2-ME2 inhibited the activation of HIF-1α induced by IH and decreased overexpression of TGF-β, p-Smad2/Smad2, and p-Smad3/Smad3, which in turn partially reversed the upregulation of α-SMA and Collagen I induced by IH in MRC5 cells. When HIF-1α was successfully silenced by si-HIF-1α, upregulation of TGF-β induced by intermittent hypoxia was partially decreased. This study showed that IH contributes to myofibroblast differentiation and excessive ECM production of MRC5 cells through activation of the HIF-1α-TGF-β/Smad pathway. 2-ME2 partially attenuated myofibroblast differentiation induced by IH by inhibiting theHIF-1α-TGF-β/Smad pathway.

Discovery of N-Arylsulfonyl-Indole-2-Carboxamide Derivatives as Galectin-3 and Galectin-8 C-Terminal Domain Inhibitors.

Both galectin-3 and galectin-8 are involved in cell adhesion, migration, apoptosis, angiogenesis, and inflammatory processes by recognizing galactose-containing glycoproteins. Inhibiting galectin-3/8 activities is a potential treatment for cancer and tissue fibrosis. Herein, a series of novel N-arylsulfonyl-5-aryloxy-indole-2-carboxamide derivatives was disclosed as dual inhibitors toward galectin-3 and galectin-8 C-terminal domain with Kd values of low micromolar level (Cpd53, gal-3: Kd= 4.12 μM, gal-8C: Kd= 6.04 μM; Cpd57, gal-3: Kd= 12.8 μM, gal-8C: Kd= 2.06 μM), which are the most potent and selective noncarbohydrate-based inhibitors toward gal-3/8 isoforms to date. The molecular docking investigations suggested that the unique amino acids Arg144 in galectin-3 and Ser213 in galectin-8C could contribute to their potency and selectivity. The scratch wound assay demonstrated that Cpd53 and Cpd57 were able to inhibit the MRC-5 lung fibroblast cells migration as well. This class of inhibitors could serve as a new starting point for further discovering structurally distinct gal-3 and gal-8C inhibitors to be used in cancer and tissue fibrosis treatment.

LKB1 depletion-mediated epithelial–mesenchymal transition induces fibroblast activation in lung fibrosis

The factors that determine fibrosis progression or normal tissue repair are largely unknown. We previously demonstrated that autophagy inhibition-mediated epithelial–mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments local myofibroblast differentiation in pulmonary fibrosis by paracrine signaling. Here, we report that liver kinase B1 (LKB1) inactivation in ATII cells inhibits autophagy and induces EMT as a consequence. In IPF lungs, this is caused by the down-regulation of CAB39L, a key subunit within the LKB1 complex. 3D co-cultures of ATII cells and MRC5 lung fibroblasts coupled with RNA sequencing (RNA-seq) confirmed that paracrine signaling between LKB1-depleted ATII cells and fibroblasts augmented myofibroblast differentiation. Together, these data suggest that reduced autophagy caused by LKB1 inhibition can induce EMT in ATII cells and contribute to fibrosis via aberrant epithelial–fibroblast crosstalk.