Evaluation of the anti-inflammatory properties of the active constituents in Ginkgo biloba for the treatment of pulmonary diseases.

Ginkgo biloba has long been used in ancient China for the treatment of cough, asthma, and other lung diseases. However, the active constituents in G. biloba for pulmonary disease treatment remain unclear. The objective of this study was to evaluate the anti-inflammatory active constituents in G. biloba and clarify their associated molecular mechanisms. The biological effects of different G. biloba extracts were evaluated in an ovalbumin-induced allergic mouse model. Anti-inflammatory compounds were present in the ethyl acetate phase of the extract, which were analysed by HPLC-MS. Biflavones were identified as the main compounds, which were further evaluated by docking calculations. Leukocyte elastase showed a high fit score with ginkgetin, one of the identified biflavones. The lowest binding free energy was -6.69 kcal mol-1. The effects of biflavones were investigated in vivo and in vitro. Ginkgetin markedly suppressed the abnormal expression of the Akt and p38 pathways in human neutrophil elastase (HNE)-stimulated A549 cells. Biflavones also decreased MUC5AC mRNA expression in HNE-stimulated A549 cells and the allergic mouse model. Inflammatory cells (neutrophils) and cytokines (IL-8) also decreased in mice treated with biflavones. The results suggest that G. biloba biflavones could inhibit the activity of leukocyte elastase. This in turn implicates G. biloba as a functional food for the treatment of airway inflammation.

Aloin reduces inflammatory gene iNOS via inhibition activity and p-STAT-1 and NF-κB.

Aloin is the major anthraquinone glycoside obtained from the Aloe species and exhibits anti-inflammatory and anti-oxidative activities. Here, we aimed to determine the effects of aloin on heme oxygenase-1 (HO-1) induction and on the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX) 2 in lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs). To the end, aloin was tested whether aloin reduces iNOS protein expression and inflammatory markers (interleukin (IL)-1ß and tumor necrosis factor (TNF)-α) in LPS-treated mice lung tissue. The results indicated that aloin affected HO-1 induction and reduced LPS-activated NF-κB-luciferase activity showed to preferential inhibition of iNOS/NO and COX-2/PGE2 that was partly related to inhibition of STAT-1 phosphorylation. In particular, aloin induced translocation of Nrf2 from cytosol into the nucleus by an increased Nrf2-ARE binding activity, and reduced IL-1ß production in LPS-activated HUVECs. The reduced expression of iNOS/NO by aloin was reversed by siHO-1RNA-transfection. In LPS-treated mice, aloin significantly reduced iNOS protein in lung tissues, and TNF-α levels in the BALF. We concluded that aloin may be beneficial for treatment of lung injury.

Angiotensinogen M235T Gene Polymorphism is a Genetic Determinant of Cerebrovascular and Cardiopulmonary Morbidity in Adolescents with Sickle Cell Disease.

BACKGROUND: Cerebrovascular stroke is a common critical complication of sickle cell disease (SCD). Angiotensinogen (AGT) M235T gene polymorphism is associated with risk of ischemic stroke and cardiovascular disease. AIM: We investigated the potential association between angiotensinogen M235T gene polymorphism and susceptibility to cerebrovascular and cardiopulmonary complications in adolescents with SCD. METHODS: Forty-six patients with SCD in steady state were studied stressing on history of stroke, hydroxyurea/chelation therapy, hematological profile, and echocardiographic findings. Polymerase chain reaction-based restriction fragment length polymorphism analysis was used to detect AGT M235T gene polymorphism. Fifty sex- and age-matched healthy controls were enrolled for assessment of M235T gene polymorphism pattern. RESULTS: The distribution of AGT M235T gene polymorphism was similar between SCD patients and healthy controls. The frequency of T allele of AGT M235T gene polymorphism (TT and MT genotypes) was significantly higher among patients with history of manifest stroke (P < .001). Patients with TT and MT genotypes had higher incidence of cardiopulmonary complications (P = .041) as well as higher percentage of HbS (P < .001) and lower hemoglobin level (P = .008) compared with those with MM genotype. Serum ferritin, liver iron concentration, and cardiac T2* were not related to T alleles or genotypes. Logistic regression analysis revealed that M235T genotype was a significant independent factor related to the occurrence of stroke among patients with SCD (Odds Ratio 14.05, 95% confidence interval 3.82-28.91; P = .001). CONCLUSION: AGT M235T gene polymorphism may represent a genetic modifier to vascular morbidities in Egyptian patients with SCD.

Heterogeneity of lung disease associated with NK2 homeobox 1 mutations.

We retrospectively studied the clinical presentation, treatment modalities and outcome in 16 patients with heterozygous NKX2-1 mutation associated with chronic lung disease. Twelve different NKX2-1 mutations, including 4 novel mutations, were identified in the 16 patients. Nine patients presented with brain-lung-thyroid syndrome, 3 had neurological and lung symptoms and 4 had only pulmonary symptoms. Ten patients had neonatal respiratory distress, and 6 of them developed infiltrative lung disease (ILD). The other patients were diagnosed with ILD in childhood (n = 3) or in adulthood (n = 3). The median age at diagnosis was 36 months (IQ 3.5-95). Patient testing included HRCT (n = 13), BALF analysis (n = 6), lung biopsies (n = 3) and lung function tests (n = 6). Six patients required supplemental oxygen support with a median duration of 18 months (IQ 2.5-29). All symptomatic ILD patients (n = 12) benefited from a treatment consisting of steroids, azithromycin (n = 9), and/or hydroxychloroquine (n = 4). The median follow-up was 36 months (IQ 24-71.5). One patient died of respiratory failure at 18 months and another is waiting for lung transplantation. In summary, the initial diagnosis was based on clinical presentation and radiological features, but the presentation was heterogeneous. Definitive diagnosis required genetic analysis, which should be performed, even in absence of neurological or thyroid symptoms.

Pulmonary Alveolar Microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is a genetic lung disorder that is characterized by the accumulation of calcium phosphate deposits in the alveolar spaces of the lung. Mutations in the type II sodium phosphate cotransporter, NPT2b, have been reported in patients with PAM. PAM progresses gradually, often producing incremental dyspnea on exertion, desaturation in young adulthood, and respiratory insufficiency by late middle age. Treatment remains supportive, including supplemental oxygen therapy. For patients with end-stage disease, lung transplantation is available as a last resort. The recent development of a laboratory animal model has revealed several promising treatment approaches for future trials.

In Vivo Therapeutic Success of MicroRNA-155 Antagomir in a Mouse Model of Lupus Alveolar Hemorrhage.

OBJECTIVE: Diffuse alveolar hemorrhage (DAH) is a rare but life-threatening complication of systemic lupus erythematosus (SLE). Pristane-treated B6 mice develop severe DAH within 2 weeks of treatment. MicroRNA-155 (miR-155) is a pleiotropic microRNA that plays a crucial role in the regulation of immune responses. Recent studies have revealed a pathogenic role of miR-155 in various autoimmune disorders. The purpose of this study was to examine the role of miR-155 in the development of DAH in pristane-induced lupus using miR-155-knockout (miR-155(-/-)) mice and miR-155 antagomir to silence miR-155. METHODS: DAH was induced by an intraperitoneal injection of 0.5 ml of pristane. MicroRNA-155 antagomir was administered intravenously to silence miR-155 expression. Lung tissues were collected for RNA extraction and were embedded in paraffin for sectioning. Gene expression profiling data were analyzed using Ingenuity Pathway Analysis. Real-time quantitative polymerase chain reaction analysis was used for single-gene validation. Luciferase reporter assay and argonaute 2 immunoprecipitation were performed for target validation. RESULTS: MicroRNA-155 expression was significantly increased during the development of DAH. Disease progression was reduced in miR-155(-/-) mice as well as by in vivo silencing of miR-155 using a miR-155 antagomir. MicroRNA-155 silencing dampened pristane-induced ectopic activation of multiple inflammatory pathways and reduced the expression of proinflammatory cytokines. Several negative regulators of NF-κB signaling were inhibited by pristane and were reactivated in miR-155(-/-) mice. In particular, the antiinflammatory factor peroxisome proliferator-activated receptor α was identified as a direct target of miR-155. CONCLUSION: MicroRNA-155 promotes pristane-induced lung inflammation. It contributes to ectopic activation of NF-κB signaling pathways by targeting multiple negative regulators. MicroRNA-155 antagomir may be a promising therapeutic strategy for treating acute lung inflammation in lupus.

Expert opinion in biological therapy: update on developments in lung gene transfer.

INTRODUCTION: Gene therapy may be suitable for a large number of acquired and inherited lung diseases, and research efforts in the field are vast. Although gene transfer to the lung has proven more challenging than initially anticipated, significant progress has been made over the last 10 years. AREAS COVERED: Here, we will first review viral and non-viral gene transfer agents that have been assessed for lung gene therapy and discuss key barriers to pulmonary gene transfer. We will then review progress in gene therapy for various lung diseases. EXPERT OPINION: In our view, one of the most significant developments in recent years is the generation of lentiviral vectors which efficiently transduce lung tissue. Focused and coordinated efforts assessing lentivirus safety and scaling up lentivirus production will be required to move this vector into clinical lung gene therapy studies. Although market authorization for a lung gene therapy product is not yet available, we are optimistic that this key milestone can be achieved in the next few years.

Multiplex Ligation-dependent Probe Amplification improves the detection rate of NKX2.1 mutations in patients affected by brain-lung-thyroid syndrome.

BACKGROUND: NKX2.1 mutations have been identified in patients displaying complete or partial brain-lung-thyroid syndrome, which can include benign hereditary chorea (BHC), hypothyroidism and/or lung disease. AIMS AND METHODS: We evaluated the recently developed Multiplex Ligation-dependent Probe Amplification (MLPA) method to assess the relative copy number of genes. The goal was to determine if MLPA could improve, in addition to direct sequencing, the detection rate of NKX2.1 mutations in a phenotype-selected cohort of 24 patients affected by neurological, thyroid and/or pulmonary disorders. RESULTS: Direct sequencing revealed two heterozygous mutations. Using MLPA, we identified two further heterozygous NKX2.1 gene deletions. MLPA increased the detection rate by 50%. All patients with gene deletions identified were affected by BHC and congenital hypothyroidism. CONCLUSION: MLPA should be considered as a complementary tool in patients with partial or total brain-lung-thyroid syndrome when direct sequencing failed to identify NKX2.1 mutations. All patients with an NKX2.1 mutation had BHC and congenital hypothyroidism, emphasizing the high prevalence of these signs associated with defective NKX2.1 alleles.

Gene expression profiling of flaxseed in mouse lung tissues-modulation of toxicologically relevant genes.

BACKGROUND: Flaxseed (FS), a nutritional supplement consisting mainly of omega-3 fatty acids and lignan phenolics has potent anti-inflammatory, anti-fibrotic and antioxidant properties. The usefulness of flaxseed as an alternative and complimentary treatment option has been known since ancient times. We have shown that dietary FS supplementation ameliorates oxidative stress and inflammation in experimental models of acute and chronic lung injury in mice resulting from diverse toxicants. The development of lung tissue damage in response to direct or indirect oxidant stress is a complex process, associated with changes in expression levels of a number of genes. We therefore postulated that flaxseed might modulate gene expression of vital signaling pathways, thus interfering with the development of tissue injury. METHODS: We evaluated gene expression in lungs of flaxseed-fed (10%FS) mice under unchallenged, control conditions. We reasoned that array technology would provide a powerful tool for studying the mechanisms behind this response and aid the evaluation of dietary flaxseed intervention with a focus on toxicologically relevant molecular gene targets. Gene expression levels in lung tissues were analyzed using a large-scale array whereby 28,800 genes were evaluated. RESULTS: 3,713 genes (12.8%) were significantly (p < 0.05) differentially expressed, of which 2,088 had a >1.5-fold change. Genes affected by FS include those in protective pathways such as Phase I and Phase II. CONCLUSIONS: The array studies have provided information on how FS modulates gene expression in lung and how they might be related to protective mechanisms. In addition, our study has confirmed that flaxseed is a nutritional supplement with potentially useful therapeutic applications in complementary and alternative (CAM) medicine especially in relation to treatment of lung disease.

Effect of diallyl sulfide on in vitro and in vivo Nrf2-mediated pulmonic antioxidant enzyme expression via activation ERK/p38 signaling pathway.

Increasing oxidative stress is intimately involved in the pathogenesis of lung failure. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a key element in redox homeostasis. Nrf2 regulates antioxidant-associated genes that are often the target of phytochemicals in chemoprevention. This study evaluated the effect of diallyl sulfide (DAS), which is present in garlic, on the expression of antioxidant enzymes in the rat lung and the Nrf2 modulation in MRC-5 lung cells. DAS increased the activities of glutathione S-transferase, glutathione reductase, and catalase as well as the GSH/GSSG ratio compared with the lung of untreated control rats (p < 0.05). The pulmonic superoxide dismutase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase 1, and catalase mRNA levels were also significantly increased (p < 0.05) after DAS treatment. Following DAS treatment, DAS level was measured in the plasma after 7 days of oral administration, and the C(max) value was 15 ± 4.2 µM. The total amount of pulmonic Nrf2 and the nuclear translocation of Nrf2 were elevated in DAS-treated rats, clarifying the effect of DAS on the modulation of antioxidant enzymes. Furthermore, DAS could induce nuclear translocation of Nrf2 via ERK/p38 signaling pathway in lung MRC-5 cells. This study demonstrates that DAS administration can significantly induce the activity of antioxidant enzymes in rat lungs and suggests a possible use for DAS as a dietary preventive agent against oxidative stress-induced lung injury.

Proteostasis: a new therapeutic paradigm for pulmonary disease.

Among lung pathologies, α1AT, chronic obstructive pulmonary disease (COPD), emphysema, and asthma are diseases triggered by local environmental stress in the airway that we refer to herein collectively as airway stress diseases (ASDs). A deficiency of α-1-antitrypsin (α1AT) is an inherited genetic disorder that is a consequence of the misfolding of α1AT during protein synthesis in liver hepatocytes, reducing secretion to the plasma and delivery to the lung. Deficiency of α1AT in the lung triggers a similar pathological phenotype to other ASDs. Moreover, the loss of α1AT in the lung is a well-known environmental risk factor for COPD/emphysema. To date there are no effective therapeutic approaches to address ASDs, which reflects a general lack of understanding of their cellular basis. Herein, we propose that ASDs are disorders of proteostasis. That is, they are initiated and propagated by a common theme-a challenge to protein folding capacity maintained by the proteostasis network (PN) (see Balch et al., Science 2008;319:916-919). The PN is a network of chaperones and degradative components that generates and manages protein folding pathways responsible for normal human physiology. In ASD, we suggest that the PN system fails to respond to the increased burden of unfolded proteins due to genetic and environmental stresses, thus triggering pulmonary pathophysiology. We introduce the enabling concept of proteostasis regulators (PRs), small molecules that regulate signaling pathways that control the composition and activity of PN components, as a new and general approach for therapeutic management of ASDs.

[Effect of Shenqi Fuzheng injection on fractalkine expression in lung tissue of rats with lipopolysaccharide-induced acute lung injury].

OBJECTIVE: To observe dynamically the Fractalkine (FKN) expression in lung tissue of rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the effect of Shenqi Fuzheng Injection (SFI) on it. METHODS: Rat model of ALI was established by intravenous injection of 4 mg/kg of LPS. Forty-two Wistar rats were randomly divided into 7 groups, the normal group, the model group and the SFI group, the latter two were separated respectively into three time phases (the 1 h, 2 h and 4 h after modeling ) groups, 6 rats in each group. Pathological changes and wet/dry weight ratio (W/D) of lung were observed, serum TNF-alpha and FKN mRNA expression in the lung tissue were examined by ELISA and RT-PCR respectively. RESULTS: Severe pathological changes of lung presented in the model groups of all three time phases with a higher W/D ratio, as well as increased serum TNF-alpha level and FKN mRNA expression in lung tissue. The peak of abnormality of serum TNF-alpha level and FKN mRNA expression was shown in the 2 h time phase group. All the above-mentioned abnormal changes were alleviated after treatment in the SFI group (P<0.05). In addition, the level of FKN mRNA expression was found to be positively correlated to the serum TNF-alpha concentration. CONCLUSION: SFI treatment in early stage could relieve the pathological changes and edema in lung tissue, decrease serum TNF-alpha and down-regulate FKN mRNA expression, playing a protective role in LPS-induced ALI rats.

[A cytogenetic study of the Chinese hamster lung cell line V-79 infected with Mycoplasma arginini and decontaminated using ciprofloxacin]. / Tsitogeneticheskoe issledovanie linii kletok legkogo kitaiskogo khomiachka V-79, infitsirovannoi mikoplazmoi Mycoplasma arginini i dekontaminirovannoi s pomoshch'iu tsiprofloksatsina.

Karyotypic variability has been studied in a line of the Chinese hamster cells artificially contaminated with Mycoplasma arginini. The contaminated cultures differed from mycoplasma-free cells in cell distribution for chromosome number. The frequency of cells with modal chromosome number 21 decreased, while that of cells with 20 chromosomes increased. Decontamination of cell culture with ciprofloxacin (10 mg/ml) and a subsequent cultivation of cell in the antibiotic-free medium did not restore the original cell distribution for chromosome number. In-53--131 days after infection, the increased frequency of chromosomal aberrations was registered. Eradication of Mycoplasma by ciprofloxacin and a long-term cultivation in antibiotic-free medium restored the frequency of chromosomal aberrations to the control level corresponding to mycoplasma-free cultures. In contaminated cultures no cyto- or genotoxic effect of ciprofloxacin was observed. These data, together with the previous ones, enable the authors to recommend ciprofloxacin to make cell cultures free from mycoplasmas with minimal risk to change the original properties of cell lines.