Association of Interactions between Metabolic 'Caretaker' Genes, p53, MDM2, and Tobacco Use with the Risk of Oral Cancer: A Multifactor Dimensionality Reduction Approach.

BACKGROUND: The present study investigated the association of interactions between gene polymorphisms in metabolic 'caretaker' genes (Phase I: CYP1A1, CYP2E1; Phase II: GSTM1, GSTT1), the cell cycle regulatory gene, p53, along with its negative controller, MDM-2, and the environment variable (tobacco). A nonparametric model, multifactor dimensionality reduction (MDR), was applied to analyse these interactions. MATERIALS AND METHODS: This case-control study was carried out on 242 subjects. Genomic DNA was extracted from peripheral blood lymphocytes.11 gene variants with an exposure variable (tobacco use) were analysed using MDR to identify the best locus model for gene-gene and gene-environment interactions. Statistical significance was evaluated using a 1000-fold permutation test using MDR permutation testing software (version 1.0 beta 2). The value of p<0.05 was considered statistically significant. RESULTS: The best three-locus model for gene-gene interaction included two of the p53 gene polymorphisms; rs17878362 (intron 3) and rs1042522 (exon 4) and rs6413432 in the Phase I gene, CYP2E1(DraI). The three-locus model to evaluate the gene-environment interaction included two intronic polymorphisms of the p53 gene, that is, rs17878362 (intron 3) and rs1625895 (intron 6), and rs4646903 in the Phase I gene CYP1A1*2C. The interaction graphs revealed independent main effects of the tobacco and p53 polymorphism, rs1042522 (exon 4), and a significant additive interaction effect between rs17878362 (intron 3) and rs1042522 (exon 4). CONCLUSIONS: The nonparametric approach highlighted the potential role of tobacco use and variations in the p53 gene as significant contributors to oral cancer risk. The findings of the present study will help implement preventive strategies in both tobacco use and screening using a molecular pathology approach.

Tobacco Alkaloid Assessment in a DSS-Induced Colitis Mouse Model with a Fully Humanized Immune System.

Inflammatory bowel disease (IBD) refers to chronic intestinal immune-mediated diseases including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease (CD). Epidemiological, clinical, and preclinical evidence has highlighted the potential anti-inflammatory properties of naturally occurring alkaloids. In the present study, we investigated the potential anti-inflammatory activities of the tobacco alkaloids nicotine and anatabine in a dextran sulfate sodium (DSS)-induced UC mouse model with a fully humanized immune system. Our results show that nicotine significantly reduced all acute colitis symptoms and improved colitis-specific endpoints, including histopathologically assessed colon inflammation, tissue damage, and mononuclear cell infiltration. The tobacco alkaloid anatabine showed similar effectiveness trends, although they were generally weaker or not significant. Gene expression analysis in the context of biological network models of IBD further pinpointed a possible mechanism by which nicotine attenuated DSS-induced colitis in humanized mice. The current study enables further investigation of possible molecular mechanisms by which tobacco alkaloids attenuate UC symptoms.

Thirdhand tobacco smoke exposure increases the genetic background-dependent risk of pan-tumor development in Collaborative Cross mice.

Increasing evidence has shown that thirdhand smoke (THS) exposure is likely to induce adverse health effects. An important knowledge gap remains in our understanding of THS exposure related to cancer risk in the human population. Population-based animal models are useful and powerful in investigating the interplay between host genetics and THS exposure on cancer risk. Here, we used the Collaborative Cross (CC) mouse population-based model system, which recapitulates the genetic and phenotypic diversity observed in the human population, to assess cancer risk after a short period of exposure, between 4 and 9 weeks of age. Eight CC strains (CC001, CC019, CC026, CC036, CC037, CC041, CC042 and CC051) were included in our study. We quantified pan-tumor incidence, tumor burden per mouse, organ tumor spectrum and tumor-free survival until 18 months of age. At the population level, we observed a significantly increased pan-tumor incidence and tumor burden per mouse in THS-treated mice as compared to the control (p = 3.04E-06). Lung and liver tissues exhibited the largest risk of undergoing tumorigenesis after THS exposure. Tumor-free survival was significantly reduced in THS-treated mice compared to control (p = 0.044). At the individual strain level, we observed a large variation in tumor incidence across the 8 CC strains. CC036 and CC041 exhibited a significant increase in pan-tumor incidence (p = 0.0084 and p = 0.000066, respectively) after THS exposure compared to control. We conclude that early-life THS exposure increases tumor development in CC mice and that host genetic background plays an important role in individual susceptibility to THS-induced tumorigenesis. Genetic background is an important factor that should be taken into account when determining human cancer risk of THS exposure.

Cigarette Smoke Enhances the Malignant Phenotype of Esophageal Adenocarcinoma Cells by Disrupting a Repressive Regulatory Interaction Between miR-145 and LOXL2.

Although linked to esophageal carcinogenesis, the mechanisms by which cigarette smoke mediates initiation and progression of esophageal adenocarcinomas (EAC) have not been fully elucidated. In this study, immortalized esophageal epithelial cells and EAC cells (EACCs) were cultured with or without cigarette smoke condensate (CSC) under relevant exposure conditions. Endogenous levels of microRNA (miR)-145 and lysyl-likeoxidase 2 (LOXL2) were inversely correlated in EAC lines/tumors compared with that in immortalized cells/normal mucosa. The CSC repressed miR-145 and upregulated LOXL2 in immortalized esophageal epithelial cells and EACCs. Knockdown or constitutive overexpression of miR-145 activated or depleted LOXL2, respectively, which enhanced or reduced proliferation, invasion, and tumorigenicity of EACC, respectively. LOXL2 was identified as a novel target of miR-145 as well as a negative regulator of this miR in EAC lines/Barrett's epithelia. Mechanistically, CSC induced recruitment of SP1 to the LOXL2 promoter; LOXL2 upregulation coincided with LOXL2 enrichment and concomitant reduction of H3K4me3 levels within the promoter of miR143HG (host gene for miR-145). Mithramycin downregulated LOXL2 and restored miR-145 expression in EACC and abrogated LOXL2-mediated repression of miR-145 by CSC. These findings implicate cigarette smoke in the pathogenesis of EAC and demonstrate that oncogenic miR-145-LOXL2 axis dysregulation is potentially druggable for the treatment and possible prevention of these malignancies.

Cigarette smoke induces the ROS accumulation and iNOS activation through deactivation of Nrf-2/SIRT3 axis to mediate the human bronchial epithelium ferroptosis.

Cigarette smoke (CS)-induced oxidative stress drives the pathogenesis of respiratory diseases, in which the activation and accumulation of reactive oxygen species (ROS) play an important role. Ferroptosis, a regulated cell death induced by Fe2+-dependent, lipid peroxidation, and ROS, is closely related to CS-induced airway injury disease, but its mechanism remains unclear. We found that bronchial epithelial ferroptosis and expression of iNOS in smoking patients were significantly higher than that in non-smokers. The iNOS, induced by CS exposure, was involved in bronchial epithelial cell ferroptosis, whereas genetic depletion or pharmacologic inactivation of iNOS attenuated the CS-induced ferroptosis and mitochondrial dysfunction. Our mechanistic studies found that SIRT3 directly bound to and negatively regulated iNOS to mediate ferroptosis. Moreover, we found that the Nrf-2/SIRT3 signal was deactivated by cigarette smoke extract (CSE)-induced ROS. Collectively, these results linked CS to human bronchial epithelial cell ferroptosis through ROS deactivation of the Nrf-2/SIRT3 signal to promote iNOS expression. Our study provides new insights into the pathogenesis of CS-induced tracheal injury diseases such as chronic bronchitis, emphysema, and chronic obstructive pulmonary disease.

The role of transforming growth factor-ß2 in cigarette smoke-induced lung inflammation and injury.

AIMS: Transforming growth factor-ß2 (TGF-ß2) plays an important role in pleiotropic functions and has been reported to be involved in the pathogenesis of chronic obstructive lung disease. The role of TGF-ß2 in regulating cigarette smoke (CS)-induced lung inflammation and injury has not been investigated, and its underlying mechanism remains unclear. MAIN METHODS: Primary bronchial epithelial cells (PBECs) were treated with cigarette smoke extract (CSE), and the signaling pathway of TGF-ß2 regulating lung inflammation was investigated. Mice were exposed to CS and treated with TGF-ß2 i.p. or bovine whey protein extract containing TGF-ß2 p.o., and the role of TGF-ß2 in alleviating lung inflammation/injury was studied. KEY FINDINGS: In vitro, we demonstrated that TGF-ß2 attenuated CSE-induced IL-8 production from PBECs through the TGF-ß receptor I (TGF-ßRI), Smad3, and mitogen-activated protein kinase signaling pathways. Selective TGF-ßRI inhibitor (LY364947) and antagonist of Smad3 (SIS3) abolished the effect of TGF-ß2 on alleviating CSE-induced IL-8 production. In vivo, CS exposure for 4 weeks in mice increased the levels of total protein, inflammatory cell counts, and monocyte chemoattractant protein-1 in bronchoalveolar fluid and induced lung inflammation/injury, as revealed by immunohistochemistry. Administration of TGF-ß2 through intraperitoneal injection or oral feeding with bovine whey protein extract containing TGF-ß2 significantly reduced CS-induced lung inflammation and injury. SIGNIFICANCE: We concluded that TGF-ß2 reduced CSE-induced IL-8 production through the Smad3 signaling pathway in PBECs and alleviated lung inflammation/injury in CS-exposed mice. The anti-inflammatory effect of TGF-ß2 on CS-induced lung inflammation in humans deserves further clinical study.

Effects of prolonged stimulation with heated tobacco products (Ploom TECH+ ) on gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Heated tobacco products have recently become commercially available. These products, as well as combustible cigarettes, produce aerosols; the risk of various diseases associated with heated tobacco products may be the same or higher than that with combustible cigarettes. In this study, we examined the effect of Ploom TECH+ extract on gingival epithelial cells. METHODS: Tobacco leaves from Ploom TECH+ tobacco capsules and water were mixed and heated; the supernatant subsequently collected was the heated tobacco product (HTP; control: HTP not added). Normal human gingival epithelial progenitors were cultured alternately with or without HTP for a total of 1 month. Subsequently, RNA, DNA, and proteins were isolated from these samples and comprehensively analyzed using RNA sequencing (RNA-seq), reduced representation bisulfite sequencing (RRBS), and western blotting, respectively. RESULTS: RNA-seq revealed that 284 genes showed a twofold increase and 145 genes showed a twofold decrease in gene expression. A heat map showed genetic differences between the control and HTP groups. A principal component analysis plot showed a clear genetic distribution between the control and HTP. Gene Ontology (GO) analysis showed that genes related to seven GO terms, including cornification and keratinization, were induced by long-term HTP stimulation. By contrast, GO pathways with a significant decrease in component expression were not detected. RRBS revealed that CpG island methylation increased more than twofold in 158 genes and decreased to less than twofold in 171 genes. Methylation of these CpG islands was not correlated with changes in gene expression levels. HTP treatment increased S100A7 expression. CONCLUSION: Long-term HTP stimulation affected epithelial differentiation and keratinization of gingival epithelial cells. Thus, habitual use of Ploom TECH+ may be a risk factor for tobacco-related oral mucosal diseases.

Dry tobacco leaves: an in vivo and in silico approach to the consequences of occupational exposure.

Exposure of tobacco workers handling dried tobacco leaves has been linked to an increased risk of toxicity and respiratory illness due to the presence of nicotine and other chemicals. This study aimed to evaluate the DNA damage caused by the exposure of tobacco growers during the dry leaf classification process and the relation to cellular mechanisms. A total of 86 individuals participated in the study, divided into a group exposed to dry tobacco (n = 44) and a control group (n = 42). Genotoxicity was evaluated using the alkaline comet assay and lymphocyte micronucleus (MN) assay (CBMN-Cyt), and measurement of telomere length. The levels of oxidative and nitrosative stress were evaluated through the formation of thiobarbituric acid reactive species, and nitric oxide levels, respectively. The inorganic elements were measured in the samples using particle-induced X-ray emission method. The combination of variables was demonstrated through principal component analysis and the interactions were expanded through systems biology. Comet assay, MN, death cells, thiobarbituric acid reactive species, and nitrosative stress showed a significant increase for all exposed groups in relation to the control. Telomere length showed a significant decrease for exposed women and total exposed group in relation to men and control groups, respectively. Bromine (Br) and rubidium (Rb) in the exposed group presented higher levels than control groups. Correlations between nitrate and apoptosis; Br and MN and necrosis; and Rb and telomeres; besides age and DNA damage and death cells were observed. The systems biology analysis demonstrated that tobacco elements can increase the nuclear translocation of NFKB dimers inducing HDAC2 expression, which, associated with BRCA1 protein, can potentially repress transcription of genes that promote DNA repair. Dry tobacco workers exposed to dry leaves and their different agents showed DNA damage by different mechanisms, including redox imbalance.

Association of combustible cigarettes and heated tobacco products use with SARS-CoV-2 infection and severe COVID-19 in Japan: a JASTIS 2022 cross-sectional study.

Insufficient evidence has been accumulated regarding associations of heated tobacco products (HTPs) use with coronavirus infection and severity of coronavirus disease 2019 (COVID-19), an ongoing pandemic. We conducted a cross-sectional study using data from an internet questionnaire administered in February 2022 to 30,130 individuals from the general Japanese population (age range, 16-81 years). Single users of HTPs and dual users of combustible cigarettes and HTPs comprised 5.2% and 7.3% of respondents, and 6.7% and 38.0% of those infected (n = 1117). Approximately 70% of infected dual users experienced severe disease. Single users of HTPs and dual users were more likely to be infected with coronavirus than never-users (adjusted odds ratio [aOR] = 1.65/4.66; 95% confidence interval [CI] 1.26-2.15/3.89-5.58). Regarding severity, former and current tobacco users (former/combustible cigarettes/HTPs: aOR = 1.88/3.17/1.90; 95%CI 1.11-3.19/1.77-5.67/1.01-3.59) were more likely to be administered oxygen than never-users, and dual users required oxygen administration the most (aOR = 4.15, 95%CI 2.70-6.36). Use of HTPs may increase risks of coronavirus infection and severe COVID-19. Our results provide an opportunity to consider the safety of tobacco products use, including HTPs, during the COVID-19 pandemic.

Prenatal tobacco and cannabis co-exposure and offspring obesity development from birth to mid-childhood.

BACKGROUND: Although the association between prenatal tobacco exposure and child obesity risk is well-established, less is known about co-exposure to tobacco and cannabis. OBJECTIVE: Determine the relation between prenatal substance co-exposure and obesity risk. METHODS: In a diverse sample of pregnant women, we examined the association between prenatal substance exposure (tobacco-only and co-exposure) and child BMI (kg/m2 ) trajectories from birth to mid-childhood (n = 262), overweight/obese status based on BMI percentiles from toddlerhood (24 months) to mid-childhood (9-12 years), and adiposity outcomes at mid-childhood (fat mass [kg], fat mass [%] and fat free mass [kg]; n = 128). Given that the major goal of this study was to examine the associations between prenatal substance exposure and child outcomes, we oversampled pregnant women for substance use (with tobacco as the primary focus). RESULTS: Multilevel models demonstrated that children in both exposure groups had a steeper increase in BMI trajectory from birth to mid-childhood and among co-exposed children, girls had a steeper increase than boys. Odds ratio of having obesity by mid-childhood was 12 times higher among those co-exposed than non-exposed. Co-exposure led to significantly greater fat mass and fat mass % compared with no exposure, but exposure to only tobacco was no different than no exposure. CONCLUSIONS: Results highlight potentiating effects of cannabis exposure in the context of maternal tobacco use in pregnancy on obesity risk and the importance of multi-method assessments of obesity.

Dietary Phytochemicals as Potential Chemopreventive Agents against Tobacco-Induced Lung Carcinogenesis.

Lung cancer is the second most common cancer in the world. Cigarette smoking is strongly connected with lung cancer. Benzo[a]pyrene (BaP) and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-butanone (NNK) are the main carcinogens in cigarette smoking. Evidence has supported the correlation between these two carcinogens and lung cancer. Epidemiology analysis suggests that lung cancer can be effectively prevented through daily diet adjustments. This review aims to summarize the studies published in the past 20 years exploring dietary phytochemicals using Google Scholar, PubMed, and Web of Science databases. Dietary phytochemicals mainly include medicinal plants, beverages, fruits, vegetables, spices, etc. Moreover, the perspectives on the challenges and future directions of dietary phytochemicals for lung cancer chemoprevention will be provided. Taken together, treatment based on the consumption of dietary phytochemicals for lung cancer chemoprevention will produce more positive outcomes in the future and offer the possibility of reducing cancer risk in society.

Disruption of the Molecular Regulation of Mitochondrial Metabolism in Airway and Lung Epithelial Cells by Cigarette Smoke: Are Aldehydes the Culprit?

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease for which cigarette smoking is the main risk factor. Acetaldehyde, acrolein, and formaldehyde are short-chain aldehydes known to be formed during pyrolysis and combustion of tobacco and have been linked to respiratory toxicity. Mitochondrial dysfunction is suggested to be mechanistically and causally involved in the pathogenesis of smoking-associated lung diseases such as COPD. Cigarette smoke (CS) has been shown to impair the molecular regulation of mitochondrial metabolism and content in epithelial cells of the airways and lungs. Although it is unknown which specific chemicals present in CS are responsible for this, it has been suggested that aldehydes may be involved. Therefore, it has been proposed by the World Health Organization to regulate aldehydes in commercially-available cigarettes. In this review, we comprehensively describe and discuss the impact of acetaldehyde, acrolein, and formaldehyde on mitochondrial function and content and the molecular pathways controlling this (biogenesis versus mitophagy) in epithelial cells of the airways and lungs. In addition, potential therapeutic applications targeting (aldehyde-induced) mitochondrial dysfunction, as well as regulatory implications, and the necessary required future studies to provide scientific support for this regulation, have been covered in this review.

Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract.

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

Gastric cancer stem cell-derived exosomes promoted tobacco smoke-triggered development of gastric cancer by inducing the expression of circ670.

As one of the most common malignant cancers in the world, gastric cancer is caused by mang factors among which tobacco smoke is an important risk factor. Gastric cancer stem cells (GCSCs) and the derived exosomes play a key role in the occurrence and development of gastric cancer, and exosomal circRNA is considered as a new regulatory factor in the development of gastric cancer. However, it is unclear whether tobacco smoke can affect exosomes and their transport circRNAs to promote the development of gastric cancer. Herein, we provided a new insight into tobacco smoke promoting the progression of gastric cancer. In the present study, we demonstrated that tobacco smoke-induced exosomes promoted the spheroidizing ability, stemness genes expression, and epithelial-mesenchymal transition (EMT) process of GCSCs. We further found that hsa-circRNA-000670 (circ670) was up-regulated in tissues of gastric cancer patients with smoking history, tobacco smoke-induced GCSCs, and their exosomes. Functional assays have shown that circ670 knockdown inhibited the stemness and EMT process of GCSCs, whereas circ670 overexpression appeared to have an opposite effect. Our findings indicated that exosomal circ670 promotes the development of tobacco smoke-induced gastric cancer, which may provide insight into the mechanism of tobacco smoke promoting the progression of gastric cancer.

Piperine Attenuates Cigarette Smoke-Induced Oxidative Stress, Lung Inflammation, and Epithelial-Mesenchymal Transition by Modulating the SIRT1/Nrf2 Axis.

Piperine (PIP) is a major phytoconstituent in black pepper which is responsible for various pharmacological actions such as anti-inflammatory, antioxidant, and antitumor activity. To investigate the effects and mechanisms of PIP on cigarette smoke (CS)-induced lung pathology using both in-vitro and in-vivo models. BEAS-2B and A549 cells were exposed to CS extract (CSE) for 48 h; BALB/c mice were exposed to CS (9 cigarettes/day, 4 days) to induce features of airway disease. PIP at doses of (0.25, 1.25, and 6.25 µM, in vitro; 1 and 10 mg/kg, in vivo, i.n) and DEX (1 µM, in vitro; 1 mg/kg, in vivo, i.n) were used to assess cytotoxicity, oxidative stress, epithelial−mesenchymal transition (EMT), Sirtuin1 (SIRT1), inflammation-related cellular signaling, and lung function. PIP treatment protects cells from CSE-induced lung epithelial cell death. PIP treatment restores the epithelial marker (p < 0.05) and decreases the mesenchymal, inflammatory markers (p < 0.05) in both in vitro and in vivo models. The PIP treatment improves the altered lung function (p < 0.05) in mice induced by CS exposure. Mechanistically, PIP treatment modulates SIRT1 thereby reducing the inflammatory markers such as IL-1ß, IL-6 and TNF-α (p < 0.05) and enhancing the epigenetic marker HDAC2 (p < 0.05) and antioxidant marker Nrf2 (p < 0.05) expressions. Thus, PIP alleviates pulmonary inflammation by modulating the SIRT1-mediated inflammatory cascade, inhibits EMT, and activates Nrf2 signaling.

Cigarette Smoke Extract Induces p38 MAPK-Initiated, Fas-Mediated Eryptosis.

Eryptosis is a physiological mechanism for the clearance of senescent or damaged erythrocytes by phagocytes. Excessive eryptosis is stimulated under several pathologies and associated with endothelial injury and thrombosis. Cigarette smoke (CS) is an established risk factor for vascular diseases and cigarette smokers have high-levels of eryptotic erythrocytes. This study, for the first time, investigates the mechanism by which CS damages red blood cells (RBCs). CS extract (CSE) from commercial cigarettes was prepared and standardized for nicotine content. Cytofluorimetric analysis demonstrated that treatment of human RBCs with CSE caused dose-dependent, phosphatidylserine externalization and cell shrinkage, hallmarks of apoptotic death. CSE did not affect cellular levels of Ca2+, reactive oxygen species (ROS) or glutathione (GSH). Immununoprecipitation and immunoblotting revealed the assembly of the death-inducing signaling complex (DISC) and oligomerization of Fas receptor as well as cleaved caspase-8 and caspase-3 within 6 h from the treatment. At the same time-interval, CSE elicited neutral sphyngomielinase (nSMase) activity-dependent ceramide formation and phosphorylation of p38 MAPK. Through specific inhibitors' nSMase, caspase-8 or p38 MAPK activities, we demonstrated that p38 MAPK activation is required for caspase-8-mediated eryptosis and that ceramide generation is initiator caspase-dependent. Finally, ex vivo analysis detected phosphorylated p38 MAPK (p-p38) and Fas-associated signaling complex in erythrocytes from cigarette smokers. In conclusion, our study demonstrates that CSE exposure induces in erythrocytes an extrinsic apoptotic pathway involving p38 MAPK-initiated DISC formation followed by activation of caspase-8/caspase-3 via ceramide formation.

From Differential DNA Methylation in COPD to Mitochondria: Regulation of AHRR Expression Affects Airway Epithelial Response to Cigarette Smoke.

Cigarette smoking causes hypomethylation of the gene Aryl Hydrocarbon Receptor Repressor (AHRR), which regulates detoxification and oxidative stress-responses. We investigated whether AHRR DNA methylation is related to chronic obstructive pulmonary disease (COPD) and studied its function in airway epithelial cells (AECs). The association with COPD was assessed in blood from never and current smokers with/without COPD, and in AECs from ex-smoking non-COPD controls and GOLD stage II-IV COPD patients cultured with/without cigarette smoke extract (CSE). The effect of CRISPR/Cas9-induced AHRR knockout on proliferation, CSE-induced mitochondrial membrane potential and apoptosis/necrosis in human bronchial epithelial 16HBE cells was studied. In blood, DNA methylation of AHRR at cg05575921 and cg21161138 was lower in smoking COPD subjects than smoking controls. In vitro, AHRR DNA methylation at these CpG-sites was lower in COPD-derived than control-derived AECs only upon CSE exposure. Upon AHRR knockout, we found a lower proliferation rate at baseline, stronger CSE-induced decrease in mitochondrial membrane potential, and higher CSE-induced late apoptosis/necroptosis. Together, our results show lower DNA methylation of AHRR upon smoking in COPD patients compared to non-COPD controls. Our data suggest that higher airway epithelial AHRR expression may lead to impaired cigarette smoke-induced mitochondrial dysfunction and apoptosis/necroptosis, potentially promoting unprogrammed/immunogenic cell death.

Transcription Factor p300 Regulated miR-451b Weakens the Cigarette Smoke Extract-Induced Cellular Stress by Targeting RhoA/ROCK2 Signaling.

Background: A previous study identified miR-451b as a potential biomarker in smoker with or without chronic obstructive pulmonary disease (COPD). However, the function and molecular mechanisms of miR-451b in the pathogenesis of COPD remain elusive. Methods: Macrophages and lung fibroblasts were exposed to 10% cigarette smoke extract (CSE) solution for 24 h. Expression miR-451b and its potential transcription factor p300 were detected. The association between p300 and miR-451b, miR-451b and RhoA was validated by luciferase reporter assay. The release of IL-12 and TNF-αby macrophages was measured by ELISA assay, and Transwell assay was performed to analyze its migration and invasion. Collagen protein of fibroblasts was detected by Western blotting. Results: Results showed that p300 and miR-451b was downregulated, while RhoA was upregulated in CSE-induced macrophages and lung fibroblasts. The stimulation of CSE promoted the degradation of p300 by ubiquitination, and RhoA was confirmed as the target gene of miR-451b. MiR-451b overexpression significantly decreased the release of IL-12 and TNF-α, downregulated the expression of RhoA, ROCK2, and p65, and suppressed cell migration and invasion in CES-induced macrophages. In addition, miR-451b overexpression decreased the expression of RhoA, ROCK2, COL1A1, and COL2A1 in lung fibroblasts. Conclusions: Our data suggest that p300/miR-451b protects against CSE-induced cell stress possibly through downregulating RhoA/ROCK2 pathway.

Anti-inflammatory and anti-oxidant properties of Ipomoea nil (Linn.) Roth significantly alleviates cigarette smoke (CS)-induced acute lung injury via possibly inhibiting the NF-κB pathway.

Acute respiratory distress syndrome (ARDS), a serious manifestation of acute lung injury (ALI), is a debilitating inflammatory lung disease that is caused by multiple risk factors. One of the primary causes that can lead to ALI/ARDS is cigarette smoke (CS) and its primary mode of action is via oxidative stress. Despite extensive research, no appropriate therapy is currently available to treat ALI/ARDS, which means there is a dire need for new potential approaches. In our study we explored the protective effects of 70 % methanolic-aqueous extract of Ipomoea nil (Linn.) Roth, named as In.Mcx against CS-induced ALI mice models and RAW 264.7 macrophages because Ipomoea nil has traditionally been used to treat breathing irregularities. Male Swiss albino mice (20-25 ± 2 g) were subjected to CS for 10 uninterrupted days in order to establish CS-induced ALI murine models. Dexamethasone (1 mg/kg), In.Mcx (100 200, and 300 mg/kg) and normal saline (10 mL/kg) were given to respective animal groups, 1 h before CS-exposure. 24 h after the last CS exposure, the lungs and bronchoalveolar lavage fluid (BALF) of all euthanized mice were harvested. Altered alveolar integrity and elevated lung weight-coefficient, total inflammatory cells, oxidative stress, expression of pro-inflammatory cytokines (IL-1ß and IL-6) and chemokines (KC) were significantly decreased by In.Mcx in CS-exposed mice. In.Mcx also revealed significant lowering IL-1ß, IL-6 and KC expression in CSE (4 %)-activated RAW 264.7 macrophage. Additionally, In.Mcx showed marked enzyme inhibition activity against Acetylcholinesterase, Butyrylcholinesterase and Lipoxygenase. Importantly, In.Mcx dose-dependently and remarkably suppressed the CS-induced oxidative stress via not only reducing the MPO, TOS and MDA content but also improving TAC production in the lungs. Accordingly, HPLC analysis revealed the presence of many important antioxidant components. Finally, In.Mcx showed a marked decrease in the NF-κB expression both in in vivo and in vitro models. Our findings suggest that In.Mcx has positive therapeutic effects against CS-induced ALI via suppressing uncontrolled inflammatory response, oxidative stress, lipoxygenase and NF-κB p65 pathway.