DNMT3A-mediated high expression of circ_0057504 promotes benzo[a]pyrene-induced DNA damage via the NONO-SFPQ complex in human bronchial epithelial cells.

Benzo[a]pyrene (B[a]P) is a class I carcinogen and hazardous environmental pollutant with genetic toxicity. Understanding the molecular mechanisms underlying genetic deterioration and epigenetic alterations induced by environmental contaminants may contribute to the early detection and prevention of cancer. However, the role and regulatory mechanisms of circular RNAs (circRNAs) in the B[a]P-induced DNA damage response (DDR) have not been elucidated. In this study, human bronchial epithelial cell lines (16HBE and BEAS-2B) were exposed to various concentrations of B[a]P, and BALB/c mice were treated with B[a]P intranasally. B[a]P exposure was found to induce DNA damage and upregulate circular RNA hsa_circ_0057504 (circ_0057504) expression in vitro and in vivo. In addition, B[a]P upregulated TMEM194B mRNA and circ_0057504 expression through inhibition of DNA methyltransferase 3 alpha (DNMT3A) expression in vitro. Modulation (overexpression or knockdown) of circ_0057504 expression levels using a lentiviral system in human bronchial epithelial cells revealed that circ_0057504 promoted B[a]P-induced DNA damage. RNA pull-down and western blot assays showed that circ_0057504 interacted with non-POU domain-containing octamer-binding (NONO) and splicing factor proline and glutamine rich (SFPQ) proteins and regulated formation of the NONO-SFPQ protein complex. Thus, our findings indicate that circ_0057504 acts as a novel regulator of DNA damage in human bronchial epithelial cells exposed to B[a]P. The current study reveals novel insights into the role of circRNAs in the regulation of genetic damage, and describes the effect and regulatory mechanisms of circ_0057504 on B[a]P genotoxicity.

Potent necrosis effect of methanethiol mediated by METTL7B enzyme bioactivation mechanism in 16HBE cell.

Methanethiol is a widely existing malodorous pollutant with health effects on the human population. However, the cytotoxicity mechanism of methanethiol in vitro and its metabolic transformation (bioactivation or detoxification) have not been fully elucidated. Herein, the metabolites of methanethiol during cell culture and the cytotoxicity of methanethiol in human bronchial epithelial (16HBE) cells were investigated. Results indicate that methanethiol (10-50 µM) was partially converted into dimethyl sulfide, mainly catalyzed by thiol S-methyltransferase in the 16HBE cells, and then it induced potent cytotoxicity and cell membrane permeability. Moreover, methanethiol induced intracellular reactive oxygen species (ROS) up to 50 µM and further activated the tumor necrosis factor (TNF) signaling pathway, which eventually led to the decline in the mitochondrial membrane potential (MMP) and cell necrosis. However, all these effects were significantly alleviated with gene silencing of the methyltransferase-like protein 7B (METTL7B). These results indicate that methanethiol may induce cell necrosis in human respiratory tract cells mainly mediated by S-methyltransferase with interfering TNF and ROS induction. Non-target metabolomics results suggest that methanethiol potently affects expression of endogenous small molecule metabolites in 16HBE cells. To some extent, this work shows the possible conversion path and potential injury mechanism of human respiratory tract cells exposed to methanethiol.

Cellular and Molecular Signatures of Oxidative Stress in Bronchial Epithelial Cell Models Injured by Cigarette Smoke Extract.

Exposure of the airways epithelium to environmental insults, including cigarette smoke, results in increased oxidative stress due to unbalance between oxidants and antioxidants in favor of oxidants. Oxidative stress is a feature of inflammation and promotes the progression of chronic lung diseases, including Chronic Obstructive Pulmonary Disease (COPD). Increased oxidative stress leads to exhaustion of antioxidant defenses, alterations in autophagy/mitophagy and cell survival regulatory mechanisms, thus promoting cell senescence. All these events are amplified by the increase of inflammation driven by oxidative stress. Several models of bronchial epithelial cells are used to study the molecular mechanisms and the cellular functions altered by cigarette smoke extract (CSE) exposure, and to test the efficacy of molecules with antioxidant properties. This review offers a comprehensive synthesis of human in-vitro and ex-vivo studies published from 2011 to 2021 describing the molecular and cellular mechanisms evoked by CSE exposure in bronchial epithelial cells, the most used experimental models and the mechanisms of action of cellular antioxidants systems as well as natural and synthetic antioxidant compounds.

Involvement of m6A regulatory factor IGF2BP1 in malignant transformation of human bronchial epithelial Beas-2B cells induced by tobacco carcinogen NNK.

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a Group 1 human carcinogen, as classified by the International Agency for Research of Cancer (IARC), and plays a significant role in lung carcinogenesis. However, its carcinogenic mechanism has not yet been fully elucidated. In this study, we performed colony formation assays, soft-agar assays, and tumor growth in nude mice to show that 100 mg/L NNK facilitates the malignant transformation of human bronchial epithelial Beas-2B cells. Transcriptome sequencing showed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a post-transcriptional regulator, was differentially expressed in NNK-induced malignant transformed Beas-2B cells (2B-NNK cells). Small interfering RNA (SiRNA) was used to downregulate the expression of the IGF2BP1 gene. The reduction in protein expression, cell proliferation rate, and colony-forming ability and the increase in the apoptosis rate of Beas-2B cells transfected with the SiRNA indicated a role for IGF2BP1 in NNK-induced malignant transformation. IGF2BP1 is an N6-methyladenosine (m6A) regulatory factor, but it is not known whether its association with m6A mediates the malignant transformation of cells. Therefore, we measured the overall levels of m6A in Beas-2B cells. We found that the overall m6A level was lower in 2B-NNK cells, and knocking down IGF2BP1, the overall level of m6A was restored. Hence, we concluded that IGF2BP1 is involved in the NNK-induced malignant transformation of Beas-2B cells, possibly via m6A modification. This study therefore contributes novel insights into the environmental pathogenesis of lung cancer and the gene regulatory mechanisms of chemical carcinogenesis.

Taxifolin improves inflammatory injury of human bronchial epithelial cells by inhibiting matrix metalloproteinase (MMP) 10 via Wnt/ß-catenin pathway.

Taxifolin (TXL), also known as dihydroquercetin, is one of the most important flavonoids prevalent across the plant kingdom. Increasing evidence has demonstrated its critical role in respiratory diseases. The present study aims to reveal the detailed mechanism in TNF-α-stimulated BEAS-2B cells by which TXL might exert effects on the development of asthma. Cell viability detection of BEAS-2B treated with TXL before and after TNF-α induction employed MMT. The expressions of inflammatory cytokines, MUC5AC and ICAM-1 were determined by quantitative reverse transcription PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and Western blot after TXL was exposed to an in vitro asthma model. Then, light transmittance and apoptosis were then measured employing fluorescein transmittance, TUNEL and Western blot. After overexpressing MMP10, the abovementioned assays were performed again. Finally, the association between Wnt/ß-catenin pathway and MMP10 was confirmed by detecting the proteins in this pathway. TXL increases the cell viability of TNF-induced BEAS-2B cells. TXL suppressed the inflammation, mucus formation, and apoptosis in TNF-α-induced BEAS-2B cells. Furthermore, after the prediction of binding sites between TXL and MMP10, it was found that overexpression of MMP10 reversed the effects of TXL on suppressing the progression of TNF-α-induced BEAS-2B cells. Finally, TXL blocked Wnt/ß-catenin pathway by inhibiting MMP10 expression.TXL can be a promising drug for the treatment of asthma due to its inhibition of MMP10 expression by blocking Wnt/ß-catenin pathway. Future experimental in vivo studies of asthma on this commonly used bioactive flavonoid could open new avenues for the therapies of asthma.

Repeated radon exposure induced lung damage via oxidative stress-mediated mitophagy in human bronchial epithelial cells and mice.

This study aimed to investigate the potential molecular mechanism underlying radon-induced lung damage. Our results showed that long-term radon exposure induced mitochondrial damage and redox imbalance in BEAS-2B cells and a time-dependent lung pathological injury in mice. The activation of Nrf-2 and its down-stream antioxidants, and the gene expression of the indicated markers at different stages of autophagy were found to be induced with the increasing of radon exposure time. Changes in the gene expression of PINK-1, Parkin, and p62 induced by radon showed differences in mechanisms of mitophagy activation and profiles of autophagic flux between BEAS-2B cells and mice. Our findings not only demonstrated that long-term radon exposure induced damages to bronchial epithelial cells and the mice lung through increasing oxidative stress, decreasing mitochondrial function and activating mitophagy with different profiles of autophagic flux, but also revealed Nrf-2 as a central regulator of mitochondrial homeostasis and lung damage.

Arsenic activates STAT3 signaling during the transformation of the human bronchial epithelial cells.

Arsenic (As3+), a metalloid abundant in environment, is classified as a group I carcinogen associated with several common human cancers, including cancers in lung, skin, bladder, liver, and prostate (Wei et al., 2019). The mechanisms of As3+-induced carcinogenesis had been extensively studied, and different mechanisms might be involved in different types of cancer (Wei et al., 2019). Recent studies showed that exposure to a high dose of arsenic is able to induce lung cancer. Meanwhile, prolonged exposure to a low concentration of arsenic can increase the risk of lung cancer also (Liao et al., 2009; Fernández et al., 2012). Emerging evidence indicated that prolonged exposure to arsenic promotes malignant transformation and some of the transformed cells have cancer-stem-like properties (Ngalame et al., 2014). In the present report, we revealed that exposure to As3+ for short time period inhibited tyrosine-705 phosphorylation of signal transducer and activator of transcription 3 (pSTAT3Y705) and induced Src homology region 2 domain-containing phosphatase-1 (SHP-1) in bronchial epithelial cell line, BEAS-2B. In addition, we found that long term exposure of the cells to As3+ activates phosphorylation of STAT3 at serine 727 (pSTAT3S727) as well as pSTAT3Y705. Moreover, As3+ is able to induce the expression of miRNA-21 (miR-21) and decrease the expression of PDCD4. Taken together, our data suggest that activation of STAT3 and induction of miR-21 are important contributing factors to the reduced expression of PDCD4, which may play significant role in As3+-induced transformation of BEAS-2B cells.

Exploring the molecular mechanisms of the inhibition of acrolein-induced BEAS-2B cytotoxicity by luteolin using network pharmacology and cell biology technology.

Acrolein is a highly reactive unsaturated hazardous air pollutant, which is extremely irritating to the respiratory tract. Luteolin, an active flavonoid compound, possesses multiple biological activities. The purpose of this study was to evaluate the mechanism of the inhibition of acrolein-induced human bronchial epithelial (BEAS-2B) cells cytotoxicity by luteolin using network pharmacology and cell biology technology. Firstly, network pharmacology results indicated that oxidative stress processes might play an important role in luteolin inhibiting lung injury. Next, it was verified at the cellular level. Reactive oxygen species (ROS) generation increased, glutathione (GSH) level decreased after exposure to acrolein. MAPK signaling pathways were activated, which activated downstream IκBα/NF-κB signaling pathways. Meanwhile, acrolein caused oxidative DNA damage and double-strand breaks, induced DNA damage response (DDR) and apoptosis. These adverse effects were significantly reversed by luteolin, which inhibited the activation of MAPK/IκBα/NF-κB and DDR pathways, and reduced the ratio of Bax/Bcl-2. Moreover, luteolin also had a similar effect to antioxidant N-acetyl cysteine (NAC) in the regulation of signaling transduction mechanisms, which indicated that the regulation of oxidative stress played an important role in the process. These results provide an experimental basis for elucidating the molecular mechanisms of the inhibition of acrolein-induced BEAS-2B cytotoxicity with luteolin.

The protective effects of Omarigliptin against Lipopolysaccharide (LPS)- induced inflammatory response and expression of mucin 5AC (MUC5AC) in human bronchial epithelial cells.

The epidemic of chronic inflammatory lung diseases such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD) has become a global public health problem. Oxidative stress, inflammation, and overproduction of airway mucus play critical roles in the progression of these diseases. Omarigliptin, an oral dipeptidyl peptidase 4 (DPP-4) inhibitor, has been demonstrated to have anti-inflammatory effects in patients with type II diabetes. However, its role in chronic inflammatory lung diseases remains enigmatic. This study is to investigate whether Omarigliptin possesses a beneficial effect against Lipopolysaccharide (LPS)-induced injuries in human BEAS-2B bronchial epithelial cells. Our results show that Omarigliptin suppressed LPS-induced oxidative stress by attenuating the generation of mitochondrial reactive oxygen species (ROS) and decrease in reduced glutathione (GSH) in BEAS-2B cells. Additionally, Omarigliptin mitigated inflammatory response by inhibiting the expression of pro-inflammatory mediators, including interleukin-1ß (IL-1ß), interleukin-12 (IL-12), and macrophage chemoattractant protein-1 (MCP-1) in LPS-challenged BEAS-2B cells. Moreover, Omarigliptin mitigated the LPS-induced overproduction of MUC5AC by rescuing the expression of the suppressor of cytokine signaling 1(SOCS1). Importantly, we found that this process is mediated by the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway. Based on these findings, we conclude that Omarigliptin might be a promising agent for the treatment of chronic inflammatory lung diseases.

Vitamin D3 protects against nitrogen mustard-induced apoptosis of the bronchial epithelial cells via activating the VDR/Nrf2/Sirt3 pathway.

Respiratory system injury is the main cause of mortality for nitrogen mustard (NM)-induced damage. Previous studies indicate that reactive oxygen species (ROS) participates in NM-mediated respiratory injuries, but the detailed mechanism is not quite clear. Human bronchial epithelial cell lines 16HBE and BEAS-2B were treated with HN2, a type of NM. In detail, it was shown that HN2 treatment induced impaired cell viability, excessive mitochondrial ROS production and enhanced cellular apoptosis in bronchial epithelial cells. Moreover, impaired Sirt3/SOD2 axis was observed upon HN2 treatment, with decreased Sirt3 and increased acetylated SOD2 expression levels. Sirt3 overexpression partially ameliorated HN2-induced cell injury. Meanwhile, vitamin D3 treatment partially attenuated HN2-induced apoptosis and improved the mitochondrial functions upon HN2 intervention. In addition, HN2 exposure decreased VDR expression, thus inhibiting the Nrf2 phosphorylation and Sirt3 activation. Inhibition of Nrf2 or Sirt3 could decrease the protective effects of vitamin D3 and enhance mitochondrial ROS production via modulating mitochondrial redox balance. In conclusion, impaired VDR/Nrf2/Sirt3 axis contributed to NM-induced apoptosis, while vitamin D3 supplementation provides protective effects via the activation of VDR and the improvement of mitochondrial functions. This study provides novel mechanism and strategy for NM exposure-induced pulmonary injuries.

Identification of the function and regulatory network of circ_009773 in DNA damage induced by nanoparticles of neodymium oxide.

The health hazards of nanoparticles of neodymium oxide (NPs-Nd2O3) have aroused public concern in recent years. Exposure to NPs-Nd2O3 can change the level of reactive oxygen species (ROS) that cause DNA damage and alter whole transcriptome expression profiles for micro (mi)RNA, circular (circ)RNA, long noncoding (lnc)RNA, and mRNA. However, there have been no reports to our knowledge about the role of circRNAs in DNA damage caused by NPs-Nd2O3. In our study, we analyzed the circRNA expression profile of human bronchial epithelial cells(16HBE)exposed to 40 µg/ml NPs-Nd2O3. Our results indicated that exposure produced 1025 up-regulated and 890 down-regulated circRNAs. Real-time quantitative polymerase chain reaction (qRT-PCR) was applied to verify some of the significantly changed circRNAs and demonstrated that circ_009773 was apparently down-regulated. Through exploration of its host gene function, we found that circ_009773 may be related to DNA damage. Functional experiments found that circ_009773 regulated NPs-Nd2O3-induced DNA damage in 16HBE cells. A circ_009773-associated competing endogenous (ce)RNA network was constructed based on one differentially expressed (DE) circRNA, 74 DE miRNAs and 208 DE mRNAs. Module analysis identified hub genes related to DNA damage and repair and a protein-protein interaction (PPI) network was created.

Budesonide promotes airway epithelial barrier integrity following double-stranded RNA challenge.

Airway epithelial barrier dysfunction is increasingly recognized as a key feature of asthma and other lung diseases. Respiratory viruses are responsible for a large fraction of asthma exacerbations, and are particularly potent at disrupting epithelial barrier function through pattern recognition receptor engagement leading to tight junction dysfunction. Although different mechanisms of barrier dysfunction have been described, relatively little is known about whether barrier integrity can be promoted to limit disease. Here, we tested three classes of drugs commonly prescribed to treat asthma for their ability to promote barrier function using a cell culture model of virus-induced airway epithelial barrier disruption. Specifically, we studied the corticosteroid budesonide, the long acting beta-agonist formoterol, and the leukotriene receptor antagonist montelukast for their ability to promote barrier integrity of a monolayer of human bronchial epithelial cells (16HBE) before exposure to the viral mimetic double-stranded RNA. Of the three, only budesonide treatment limited transepithelial electrical resistance and small molecule permeability (4 kDa FITC-dextran flux). Next, we used a mouse model of acute dsRNA challenge that induces transient epithelial barrier disruption in vivo, and studied the effects budesonide when administered prophylactically or therapeutically. We found that budesonide similarly protected against dsRNA-induced airway barrier disruption in the lung, independently of its effects on airway inflammation. Taken together, these data suggest that an under-appreciated effect of inhaled budesonide is to maintain or promote airway epithelial barrier integrity during respiratory viral infections.

SB203580-A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma.

Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-ß1 (TGF-ß1), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-ß1 also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-ß1-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-ß/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma.

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity. Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients. Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses. Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-ß expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections. Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-ß expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene.

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

Dosing intact birch pollen grains at the air-liquid interface (ALI) to the immortalized human bronchial epithelial cell line BEAS-2B.

In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.

Effect of EGCG on bronchial epithelial cell premalignant lesions induced by cigarette smoke and on its CYP1A1 expression.

Epigallocatechin­3­gallate (EGCG) has been demonstrated to exhibit anticancer effects; however, the mechanisms behind these are not yet clear. The objective of the present study was to assess the effect of EGCG on smoking­induced, precancerous, bronchial epithelial cell lesions and determine a potential protective mechanism. Human bronchial epithelial (HBE) cells were treated with cigarette smoke extract (CSE). Benzopyrene­DNA adducts were detected by immunofluorescence cytochemistry. Changes to microRNA (miRNA) expression levels were detected via microarray. The effects of EGCG on smoke­induced benzopyrene­DNA adduct formation and the subsequent change in miRNA expression were analyzed. Subsequently, the protective effect of EGCG on smoke inhalation­induced precancerous lesions was investigated. The expression levels of miRNA target genes were also analyzed. After CSE treatment, benzopyrene­DNA adducts appeared in HBE cells, along with a resultant change in miRNA expression. EGCG inhibited the effects of CSE exposure; benzopyrene­DNA adduct formation was reduced and miRNA expression changes were suppressed. In vivo, EGCG significantly reduced benzopyrene­DNA adduct formation and the subsequent development of precancerous lesions in rat lungs induced by cigarette smoke inhalation. Moreover, EGCG downregulated CYP1A1 overexpression, a target gene of multiple smoking­induced miRNAs, in rat lungs. EGCG may reduce the risk of lung cancer by downregulating the expression of the key gene CYP1A1, preventing the formation of smoking­induced benzopyrene­DNA adducts and alleviating smoking­induced bronchial epithelial dysplasia and heterogeneity.

Tetramethylpyrazine Alleviates Tight Junction Disruption of Bronchial Mucosal Epithelial Cells Caused by Interleukin-17 via Inhibiting Nuclear Factor-κB-p65/Tumor Necrosis Factor-α Signaling Pathway.

Bronchial mucosal epithelial dysregulation and barrier disruption are involved in the initiation and development of acute lung injury (ALI). Some inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17) contribute to the pathological changes of ALI. However, the roles and relationship between TNF-α and IL-17 during the disruption of bronchial epithelial tight junction remain unclear. Tetramethylpyrazine (TMP) is confirmed to have beneficial functions in hemostasis, inflammation, and cell growth. Here, we demonstrated the protective effects of TMP on bronchial mucosal epithelial injury induced by IL-17. We showed that IL-17 stimulation in vitro markedly reduced occludin and zonula occludens-1 (ZO-1) expression in bronchial mucosal epithelial cells via the nuclear factor-κB-p65/TNF-α signaling pathway, including NF-κB-p65-triggered TNF-α gene transcription and expression. TMP obviously rescued IL-17-induced occludin and ZO-1 downregulation. Mechanically, TMP substantially suppressed NF-κB-p65 activation and NF-κB-p65-induced TNF-α production in bronchial mucosal epithelial cells caused by IL-17. Taken together, this study indicates that TMP has a protective effect on bronchial mucosal epithelial cell injury due to IL-17 induction by inhibiting the NF-κB-p65/TNF-α signaling pathway.

Role of nitric oxide, bradykinin B2 receptor, and TRPV1 in the airway alterations caused by simvastatin in rats.

Dry cough has been reported in patients receiving statin therapy. However, the underlying mechanism or other possible alterations in the airways induced by statins remain unknown. Thus, the aim of this study was to evaluate whether simvastatin promotes alterations in airways, such as bronchoconstriction and plasma extravasation, as well as the mechanism involved in these events. Using methods to detect alterations in airway resistance and plasma extravasation, we demonstrated that simvastatin [20 mg/kg, intravenous (i.v.)] caused plasma extravasation in the trachea (79.8 + 14.8 µg/g/tissue) and bronchi (73.3 + 8.8 µg/g/tissue) of rats, compared to the vehicle (34.2 + 3.6 µg/g/tissue and 29.3 + 5.3 µg/g/tissue, respectively). NG-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, intraperitoneal), a nitric oxide (NO) synthase inhibitor, Icatibant [HOE 140, 10 nmol/50 µl, intratracheal (i.t.)], a bradykinin B2 antagonist, and capsazepine (100 nmol/50 µl, i.t.), a TRPV1 antagonist, attenuated simvastatin-induced plasma extravasation. Simvastatin (5, 10 and 20 mg/kg) did not cause bronchoconstriction per se, but exacerbated the bronchoconstrictive response to bradykinin (30 nmol/kg, i.v.), a B2 agonist (0.7 + 0.1 ml/H2O), or capsaicin (30 nmol/kg, i.v.), a TRPV1 agonist (0.8 + 0.1 ml/H2O), compared to the vehicle (0.1 + 0.04 ml/H2O and 0.04 + 0.01 ml/H2O, respectively). The bronchoconstriction elicited by bradykinin (100 nmol/kg, i.v.) in simvastatin non-treated rats was inhibited by L-NAME. The exacerbation of bronchoconstriction induced by bradykinin or capsaicin in simvastatin-treated rats was inhibited by L-NAME, HOE 140 or capsazepine. These results suggest that treatment with simvastatin promotes the release of bradykinin, which, via B2 receptors, releases NO that can then activate the TRPV1 to promote plasma extravasation and bronchoconstriction.