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

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

Respiratory Safety Evaluation in Mice and Inhibition of Adenoviral Amplification in Human Bronchial Endothelial Cells Using a Novel Type of Chlorine Dioxide Gas Reactor

Since the onset of the COVID-19 pandemic, there has been a growing demand for effective and safe disinfectants. A novel use of chlorine dioxide (ClO2) gas, which can satisfy such demand, has been reported. However, its efficacy and safety remain unclear. For the safe use of this gas, the stable release of specific concentrations is a must. A new type of ClO2 generator called Dr.CLOTM has recently been introduced. This study aimed to investigate: (1) the effects of Dr.CLOTM on inhibiting adenoviral amplification on human bronchial epithelial (HBE) cells; and (2) the acute inhalation safety of using Dr.CLOTM in animal models. After infecting HBE cells with a recombinant adenovirus, the inhibitory power of Dr.CLOTM on the virus was expressed as IFU/mL in comparison with the control group. The safety of ClO2 gas was indirectly predicted using mice by measuring single-dose inhalation toxicity in specially designed chambers. Dr.CLOTM was found to evaporate in a very constant concentration range at 0–0.011 ppm/m3 for 42 days. In addition, 36–100% of adenoviral amplification was suppressed by Dr.CLOTM, depending on the conditions. The LC50 of ClO2 gas to mice was approximately 68 ppm for males and 141 ppm for females. Histopathological evaluation showed that the lungs of female mice were more resistant to the toxicity from higher ClO2 gas concentrations than those of male mice. Taken together, these results indicate that Dr.CLOTM can be used to provide a safe indoor environment due to its technology that maintains the stable concentration and release of ClO2 gas, which could suppress viral amplification and may prevent viral infections.

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site–targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon–containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

SLC6A14 impacts cystic fibrosis lung disease severity via mTOR and epithelial repair modulation

Cystic fibrosis (CF), due to variants in CFTR gene, is associated with chronic infection/inflammation responsible for airway epithelium alteration and lung function decline. Modifier genes induce phenotype variability between people with CF (pwCF) carrying the same CFTR variants. Among these, the gene encoding for the amino acid transporter SLC6A14 has been associated with lung disease severity and age of primary airway infection by the bacteria Pseudomonas aeruginosa. In this study, we investigated whether the single nucleotide polymorphism (SNP) rs3788766, located within SLC6A14 promoter, is associated with lung disease severity in a large French cohort of pwCF. We also studied the consequences of this SNP on SLC6A14 promoter activity using a luciferase reporter and the role of SLC6A14 in mammalian target of rapamycin (mTOR) signaling pathway and airway epithelial repair. We confirm that SLC6A14 rs3788766 SNP is associated with lung disease severity in pwCF (p=0.020; n=3,257, pancreatic insufficient, aged 6 to 40 years old), with the minor allele G being deleterious. In bronchial epithelial cell lines deficient for CFTR, SLC6A14 promoter activity is reduced in the presence of the rs3788766 G allele. SLC6A14 inhibition with a specific pharmacological blocker reduced 3H-arginine transport, mTOR phosphorylation and bronchial epithelial repair rates in wound healing assays. To conclude, our study highlights that SLC6A14 genotype might affect lung disease severity of people with cystic fibrosis via mTOR and epithelial repair mechanisms modulation in the lung.

Promotor Hypomethylation Mediated Upregulation of miR-23b-3p Targets PTEN to Promote Bronchial Epithelial-Mesenchymal Transition in Chronic Asthma

Chronic asthma is characterized by airway inflammation and irreversible airway remodeling. Epithelial-mesenchymal transition (EMT) is a typical pathological change of airway remodeling. Our previous research demonstrated miR-23b inhibited airway smooth muscle proliferation while the function of miR-23b-3p has not been reported yet. Besides, miRNA is regulated by many factors, including DNA methylation. The function of miR-23b-3p and whether it is regulated by DNA methylation are worth exploring. Balb/c mice were given OVA sensitization to develop the asthmatic model. Expression of miR-23b-3p and EMT markers were measured by RT-qPCR, WB and immunohistochemistry (IHC). DNA methylation was detected by methylation-specific PCR (MSP) and the MassARRAY System. Asthmatic mice and TGF-β1-stimulated bronchial epithelial cells (BEAS-2B) showed EMT with increased miR-23b-3p. Overexpression of miR-23b-3p promoted EMT and migration, while inhibition of miR-23b-3p reversed these transitions. DNA methyltransferases were decreased in asthmatic mice. MSP and MassARRAY System detected the promotor of miR-23b showed DNA hypomethylation. DNA methyltransferase inhibitor 5’-AZA-CdZ increased the expression of miR-23b-3p. Meanwhile, PTEN was identified as a target gene of miR-23b-3p. Our results indicated that promotor hypomethylation mediated upregulation of miR-23b-3p targets PTEN to promote EMT in chronic asthma. miR-23b-3p and DNA methylation might be potential therapeutic targets for irreversible airway remodeling.

The PPAR-γ agonist pioglitazone exerts proinflammatory effects in bronchial epithelial cells during acute Pseudomonas aeruginosa pneumonia

Pseudomonas (P.) aeruginosa is a common respiratory pathogen that causes injurious airway inflammation during acute pneumonia. PPAR (peroxisome proliferator-activated receptor)-γ is involved in the regulation of metabolic and inflammatory responses in different cell types and synthetic agonists of PPAR-γ exert anti-inflammatory effects on myeloid cells in vitro and in models of inflammation in vivo. We sought to determine the effect of the PPAR-γ agonist pioglitazone on airway inflammation induced by acute P. aeruginosa pneumonia, focusing on bronchial epithelial cells. Mice pretreated with pioglitazone or vehicle (-24 and -1 hour) were infected with P. aeruginosa via the airways. Pioglitazone treatment was associated with increased expression of chemokine (Cxcl1, Cxcl2, Ccl20) and cytokine genes (Tnfa, Il6, Cfs3) in bronchial brushes obtained 6 hours after infection. This proinflammatory effect was accompanied by increased expression of Hk2 and Pfkfb3, genes encoding rate limiting enzymes of glycolysis; concurrently, the expression of Sdha, important for maintaining metabolite flux in the tricarboxylic acid cycle, was reduced in bronchial epithelial cells of pioglitazone treated-mice. Pioglitazone inhibited bronchoalveolar inflammatory responses measured in lavage fluid. These results suggest that pioglitazone exerts a selective proinflammatory effect on bronchial epithelial cells during acute P. aeruginosa pneumonia, possibly by enhancing intracellular glycolysis.