Knockdown of Alpha-1 Antitrypsin with antisense oligonucleotide does not exacerbate smoke induced lung injury.

Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein's primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Effects of simvastatin on iNOS and caspase­3 levels and oxidative stress following smoke inhalation injury.

The overexpression of inducible nitric oxide synthase (iNOS) induces cell apoptosis through various signal transduction pathways and aggravates lung injury. Caspase­3 is an important protein in the apoptotic pathway and its activation can exacerbate apoptosis. Simvastatin, a hydroxymethyl glutaryl­A reductase inhibitor, protects against smoke inhalation injury by inhibiting the synthesis and release of inflammatory factors and decreasing cell apoptosis. Following the establishment of an animal model of smoke inhalation injury, lung tissue and serum were collected at different time points and the protein and mRNA expression of iNOS and caspase­3 in lung tissue by immunochemistry, western blot and reverse transcription­quantitative polymerase chain reaction, the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in lung tissue and serum were analyzed using thiobarbituric acid method and the WST­1 method. The results were statistically analyzed. The lung tissues of the rats in the saline group and the low­, middle­ and high­dose groups exhibited clear edema and hemorrhage, and had significantly higher pathological scores at the various time points compared with the rats in the control group (P<0.05). Furthermore, lung tissue and serum samples obtained from these four groups had significantly higher mRNA and protein expression levels of iNOS and caspase­3 (P<0.05), significantly lower SOD activity and higher MDA content (P<0.05). Compared with the saline group, the low­, middle­ and high­dose groups had significantly lower pathological scores (P<0.05), significantly lower mRNA and protein expression levels of iNOS, caspase­3 and MDA content in lung tissues (P<0.05) and significantly higher SOD activity in lung tissues and serum. The middle­ and high­dose groups had significantly lower pathological scores (P<0.05), significantly decreased iNOS and caspase­3 mRNA and protein expression in lung tissues, significantly higher SOD activity in lung tissues and serum and a significantly lower MDA content (P<0.05) compared with the low­dose group. With the exception of SOD activity in lung tissues at 24 and 72 h and MDA content in serum at 48 h, no significant differences were observed between the middle­ and high­dose groups. The present study demonstrated that there was an association between the therapeutic effect and dosage of simvastatin within a definitive range. In rats with smoke inhalation injury, simvastatin inhibited iNOS and caspase­3 expression in lung tissues and mitigated oxidative stress, thereby exerting a protective effect. In addition, the effect and dose were associated within a definitive range.

Bone marrow mesenchymal stem cells protect lungs from smoke inhalation injury by differentiating into alveolar epithelial cells via Notch signaling.

To examine the protective effect of transplanting bone marrow mesenchymal stem cells (BMSCs) in treating lung injuryinduced by smoke exposure and to investigate the underlying mechanisms of this protection. SD rats were randomlydivided into four groups: normal group, normal +BMSCGFP group, smoke group, and smoke +BMSCGFP group. Todetect lung injury, we measured arterial blood gas, the wet-to-dry weight ratio, and levels of interleukin-1b, tumor necrosisfactor-a, interleukin-10, and interleukin-13 in bronchoalveolar lavage fluid and lung tissues. We also conductedhistopathology examinations. The protein markers of alveolar epithelial cells were measured to determine the BMSCdifferentiation. The protein levels of Notch1, Jagged-1, and Hes-1 also were detected. In the present study, BMSCtransplantation significantly decreased the wet-dry weight ratio of the lung, reduced the production of inflammatorymediators, and alleviated lung injury simply through differentiating into alveolar type II cells and alveolar type I cells. Western blot analysis confirmed that the protein expression of Notch-1, Jagged-1, and Hes-1 increased significantly aftersystemic BMSC transplantation. No significant difference was observed between the normal group and the nor-mal +BMSCGFP group. Our findings indicate that systemic transplantation of BMSCs alleviated lung injury induced bysmoke exposure, which may be associated with BMSCs' ability to differentiate into alveolar-type cells via the Notchsignaling pathway.

Identification of differentially expressed proteins in the injured lung from zinc chloride smoke inhalation based on proteomics analysis.

BACKGROUND: Lung injury due to zinc chloride smoke inhalation is very common in military personnel and leads to a high incidence of pulmonary complications and mortality. The aim of this study was to uncover the underlying mechanisms of lung injury due to zinc chloride smoke inhalation using a rat model. METHODS: Histopathology analysis of rat lungs after zinc chloride smoke inhalation was performed by using haematoxylin and eosin (H&E) and Mallory staining. A lung injury rat model of zinc chloride smoke inhalation (smoke inhalation for 1, 2, 7 and 14 days) was developed. First, isobaric tags for relative and absolute quantization (iTRAQ) and weighted gene co-expression network analysis (WGCNA) were used to identify important differentially expressed proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to study the biological functions of differentially expressed proteins. Then, analysis of lung injury repair-related differentially expressed proteins in the early (day 1 and day 2) and middle-late stages (day 7 and day 14) of lung injury after smoke inhalation was performed, followed by the protein-protein interaction (PPI) analysis of these differentially expressed proteins. Finally, the injury repair-related proteins PARK7 and FABP5 were validated by immunohistochemistry and western blot analysis. RESULTS: Morphological changes were observed in the lung tissues after zinc chloride smoke inhalation. A total of 27 common differentially expressed proteins were obtained on days 1, 2, 7 and 14 after smoke inhalation. WGCNA showed that the turquoise module (which involved 909 proteins) was most associated with smoke inhalation time. Myl3, Ckm, Adrm1 and Igfbp7 were identified in the early stages of lung injury repair. Gapdh, Acly, Tnni2, Acta1, Actn3, Pygm, Eno3 and Tpi1 (hub proteins in the PPI network) were identified in the middle-late stages of lung injury repair. Eno3 and Tpi1 were both involved in the glycolysis/gluconeogenesis signalling pathway. The expression of PARK7 and FABP5 was validated and was consistent with the proteomics analysis. CONCLUSION: The identified hub proteins and their related signalling pathways may play crucial roles in lung injury repair due to zinc chloride smoke inhalation.

Alterations in airway microbiota in patients with PaO2/FiO2 ratio ≤ 300 after burn and inhalation injury.

BACKGROUND: Injury to the airways after smoke inhalation is a major mortality risk factor in victims of burn injuries, resulting in a 15-45% increase in patient deaths. Damage to the airways by smoke may induce acute respiratory distress syndrome (ARDS), which is partly characterized by hypoxemia in the airways. While ARDS has been associated with bacterial infection, the impact of hypoxemia on airway microbiota is unknown. Our objective was to identify differences in microbiota within the airways of burn patients who develop hypoxemia early after inhalation injury and those that do not using next-generation sequencing of bacterial 16S rRNA genes. RESULTS: DNA was extracted from therapeutic bronchial washings of 48 patients performed within 72 hours of hospitalization for burn and inhalation injury at the North Carolina Jaycee Burn Center. DNA was prepared for sequencing using a novel molecule tagging method and sequenced on the Illumina MiSeq platform. Bacterial species were identified using the MTToolbox pipeline. Patients with hypoxemia, as indicated by a PaO2/FiO2 ratio ≤ 300, had a 30% increase in abundance of Streptococcaceae and Enterobacteriaceae and 84% increase in Staphylococcaceae as compared to patients with a PaO2/FiO2 ratio > 300. Wilcoxon rank-sum test identified significant enrichment in abundance of OTUs identified as Prevotella melaninogenica (p = 0.042), Corynebacterium (p = 0.037) and Mogibacterium (p = 0.048). Linear discriminant effect size analysis (LefSe) confirmed significant enrichment of Prevotella melaninognica among patients with a PaO2/FiO2 ratio ≤ 300 (p<0.05). These results could not be explained by differences in antibiotic treatment. CONCLUSIONS: The airway microbiota following burn and inhalation injury is altered in patients with a PaO2/FiO2 ratio ≤ 300 early after injury. Enrichment of specific taxa in patients with a PaO2/FiO2 ratio ≤ 300 may indicate airway environment and patient changes that favor these microbes. Longitudinal studies are necessary to identify stably colonizing taxa that play roles in hypoxemia and ARDS pathogenesis.

Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke.

Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase alpha subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke.

Mainstream tobacco smoke causes paternal germ-line DNA mutation.

Despite the presence of known mutagens and carcinogens in cigarette smoke, there is currently no evidence to show that smoking, or exposure to cigarette smoke, can result in heritable genetic mutation. We show that male mice exposed to mainstream tobacco smoke (MTS) exhibit a significant increase in germ-line mutation frequency in spermatogonial stem cells. We exposed mature male mice to MTS for 6 or 12 weeks and investigated mutations arising in exposed spermatogonial stem cells at the expanded simple tandem repeat locus Ms6-hm. A generalized score test showed a significant treatment effect (P = 0.0214). Ms6-hm mutation frequency was 1.4 and 1.7 times higher in mice exposed to MTS for 6 and 12 weeks, respectively, compared with sham controls. The data suggest that mutations accumulate in the spermatogonial stem cells with extended exposures. Mutation spectra were identical between exposed and sham individuals, supporting the hypothesis that tandem repeat mutations arise through indirect mechanisms of mutation. Mutations in sperm that are passed on to offspring cause permanent, irreversible changes in genetic composition and can persist in future generations. Our research suggests that the consequences of smoking extend beyond the smoker to their nonsmoking descendents.

Smoke-induced inhalation injury: effects of retinoic acid and antisense oligodeoxynucleotide on stability and differentiated state of the mucociliary epithelium.

Rabbit tracheal explants, exposed to burning pine wood smoke, were cultured in a chemically defined medium with and without retinoic acid (+/- RA). Exposures of 15-20 minute led to RA-independent degeneration of the mucociliary epithelial sheath. In 10 minute exposures tissue integrity was retained, but epithelial morphology changed from normal pseudostratified columnar to the flattened appearance typical of the squamous phenotype. Despite the dramatic shift in morphology, explants exhibited normal RA-dependent mucin gene expression characteristic of the mucociliary phenotype. Furthermore, electron micrographs showed continued presence of both secretory granules and cilia. RA(+) cultures also showed a normal pattern of adherent epithelial cells. In RA(-) cultures, however, there were prominent intercellular spaces indicating an RA dependence for maintaining adhesive contacts following smoke exposure. An 18-mer mucin antisense oligomer that suppressed mucin gene expression also unexpectedly blocked the smoke induced metaplasia in RA(+) cultures, but the sense oligomer had no effect.

A study on genotoxicity of cooking fumes from rapeseed oil.

The present article reports the genotoxic potential of rapeseed oil cooking fume investigated by a battery of short-term tests (Ames test, SCE/V79 in vitro and mice micronucleus in vivo test). The results showed that the cooking fume contained mutagenic activity. In the presence of S9 mix, an increase in the number of the Salmonella TA98 was observed at doses ranging from 1.0 to 5.0 mg/plate, and the SCE frequencies of V79 cell were markedly raised at doses ranging from 0.05 to 0.5 mg.ml-1. The positive result was also obtained in mice micronucleus assay, the mice had inhaled the cooking fume a week earlier. The frequency of mice bone marrow MN-PCE was increased and it showed a remarkable time-dose-response relationship during the 4 weeks exposure. The results suggested that this cooking fume exposure may be a risk factor of lung cancer in Chinese women.