The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca concentration ([Ca]) in ASM cells. The results showed that EA significantly inhibited the high K (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) μmol/L and (56.22 ± 7.62) μmol/L, respectively. EA decreased the K and ACh-induced elevation of [Ca] from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca influx and decreases of [Ca], leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; Enzyme Inhibitors ; pharmacology ; Ethacrynic Acid ; pharmacology ; Mice ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Respiratory System ; cytology ; drug effects

Chronic obstructive pulmonary disease (COPD), a common preventable and treatable disease, is characterized by airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways. Its main pathological manifestations include airway inflammation, mucus hypersecretion, oxidative stress and apoptotic epithelial cells. Recent research suggests that MAP kinases and Keap1-Nrf2-ARE signaling pathway are involved in the pathological process of inflammation and oxidative stress. This review explores the potential role of the cross talk of these signaling pathways in airway inflammation, mucus hypersecretion, oxidative stress and apoptotic epithelial cells. To clarify the roles of cross talk between MAP kinases and Keap1-Nrf2-ARE signaling pathway, we also focus on the drugs related to that in the treatment of COPD, and it provides ideas for more drug research in the treatment of COPD.
Apoptosis ; Epithelial Cells ; cytology ; Humans ; Inflammation ; Intracellular Signaling Peptides and Proteins ; Kelch-Like ECH-Associated Protein 1 ; Mitogen-Activated Protein Kinases ; NF-E2-Related Factor 2 ; Oxidative Stress ; Pulmonary Disease, Chronic Obstructive ; metabolism ; Respiratory System ; Signal Transduction

OBJECTIVETo investigate the antiinflammatory activities of aqueous extract of Occimum gratissmium (OGE) with emphasis on expression of proinflammatory cytokines in Lipopolysaccharide (LPS)-stimulated epithelial cell BEAS-2B.

METHODSEffects of OGE on cell viability were determined by MTT assay. mRNA expression were analyzed by and reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Activation of kinase cascades was investigated by immunoblot. Intracellular reactive oxygen species (ROS) was analyzed by flow cytometry.

RESULTSOGE (<200 μg/mL) treatment or pretreatment and following LPS exposure slightly affected viability of BEAS-2B cells. Increase of interleukin (IL)-6 and IL-8 and the elevated level of intracellular ROS in LPS-stimulated BEAS-2B cells were diminished by OGE pretreatment in a dose-dependent manner. OGE suppressed inflammatory response-associated mitogen-activated protein kinases (MAPKs) and Akt activation. Additionally, OGE pretreatment increased level of cellular inhibitor of κBα (IκBα) and inhibited nuclear translocation of nuclear factor kappa B (NF-κB).

CONCLUSIONThese findings indicate that significant suppression of IL-6 and IL-8 expressions in LPS-stimulated BEAS-2B cells by OGE may be attributed to inhibiting activation of MAPKs and Akt and consequently suppressing nuclear translocation of NF-κB.

Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Cytosol ; drug effects ; metabolism ; Epithelial Cells ; drug effects ; enzymology ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; I-kappa B Proteins ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Interleukin-8 ; genetics ; metabolism ; Intracellular Space ; drug effects ; metabolism ; Lipopolysaccharides ; pharmacology ; Mitogen-Activated Protein Kinases ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Ocimum ; chemistry ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Protein Transport ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Respiratory System ; cytology ; Water

OBJECTIVETo investigate the effect of spearmint oil on emphysema-like changes and the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta(IL-1beta), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-9) in lipopolysaccharide (LPS) treated rats.

METHODEmphysematous changes model was induced by intratracheal instillation of LPS once a week for up to 8 weeks in rats. Rats were divided into control, dexamethasone (0.3 mg x kg(-1)), and spearmint oil (10, 30,100 mg x kg(-1)) groups. Each group was treated with saline, dexamethasone, and spearmint of oil respectively for 4 weeks. Then total and different white blood cell counts in bronchoalveolar lavage fluid(BALF) were carried out. The pathologic changes of lung tissue such as alveolar structure, airway inflammation, and goblet cell metaplasia were observed by HE and AB-PAS staining. Expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9 were measured.

RESULTBoth spearmint and dexamethasone decreased the destruction of pulmonary alveolus. The total and different white blood cell counts in BALF including neutrophile and lymphocyte of spearmint oil 100 mg x kg(-1) and dexamethasone group were significantly reduced, and the goblet cell metaplasia was also inhibited. Dexamethasone had inhibitory effect on the expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9. Spearmint oil 30, 100 mg x kg(-1) significantly reduced TNF-alpha and IL-1beta respectively. Spearmint oil 10, 30 and 100 mg x kg(-1) had no effect on the expression of TIMP-1, but could decrease the expression of MMP-9 significantly in lung tissues.

CONCLUSIONSpearmint oil has protective effect on rats with emphysematous changes, since it improves alveolar destruction, pulmonary inflammation, and goblet cell metaplasia. The mechanism may include reducing TNF-alpha, IL-1beta content and inhibiting overexpression of matrix metalloproteinase-9 in lung tissues.

Animals ; Azo Compounds ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Goblet Cells ; drug effects ; Interleukin-1beta ; drug effects ; metabolism ; Leukocytes ; drug effects ; metabolism ; Lipopolysaccharides ; Lymphocytes ; drug effects ; metabolism ; Matrix Metalloproteinase 9 ; drug effects ; metabolism ; Mentha spicata ; chemistry ; Metaplasia ; Monocytes ; drug effects ; metabolism ; Neutrophils ; drug effects ; metabolism ; Phytotherapy ; Plant Oils ; therapeutic use ; Pulmonary Emphysema ; chemically induced ; drug therapy ; enzymology ; pathology ; Rats ; Respiratory System ; drug effects ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; metabolism ; Tumor Necrosis Factor-alpha ; drug effects ; metabolism

Adrenergic beta-2 Receptor Agonists ; pharmacology ; Anti-Inflammatory Agents ; pharmacology ; Asthma ; drug therapy ; physiopathology ; Chloroquine ; pharmacology ; Humans ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Quaternary Ammonium Compounds ; pharmacology ; Receptors, Adrenergic, beta-2 ; metabolism ; Receptors, G-Protein-Coupled ; agonists ; metabolism ; Receptors, Interleukin-4 ; antagonists & inhibitors ; metabolism ; Respiratory System ; drug effects ; metabolism ; physiopathology

BACKGROUNDIncreased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle.

METHODSASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE + pcDNA3.1 group and CSE + pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting.

RESULTS(1) The percentage of S + G2M phase, absorbance value at 490 nm wavelength (A(490)) and the expression rate of PCNA protein in CSE group were (31.22 +/- 1.17)%, 0.782 +/- 0.221, (90.2 +/- 7.0)% respectively, which were significantly increased compared with those of control group ((18.36 +/- 1.02)%, 0.521 +/- 0.109, and (54.1 +/- 3.5)%, respectively) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S + G2M phase, A(490) and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P < 0.01). (2) The ratios of A(490) of cyclin D1 mRNA in CSE group was 0.288 +/- 0.034, which was significantly increased compared with that of control group (0.158 +/- 0.006) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P < 0.01). (3) The ratios of A(490) of cyclin D1 protein expression in CSE group was 0.375 +/- 0.008, which was significantly increased compared with that of control group (0.268 +/- 0.004) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P < 0.01).

CONCLUSIONCSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.

Animals ; Asthma ; metabolism ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; genetics ; metabolism ; Disease Models, Animal ; Female ; Flow Cytometry ; Immunohistochemistry ; Microscopy, Phase-Contrast ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Plant Extracts ; toxicity ; Rats ; Respiratory System ; cytology ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Smoking ; adverse effects ; Tobacco ; chemistry

OBJECTIVETo investigate the changes of neutrophils in airway inflammation in children with severe asthma.

METHODChildren with mild to moderate asthma (n=23), severe asthma (n=16) and healthy control subjects (n=16) underwent lung function tests and sputum induction. The sputum specimens were assayed for cellular differential count, the supernatant and peripheral blood were assayed for the concentrations of IL-8 by "sandwich" enzyme linked immunosorbent assay (ELISA). Sputum supernatant, IL-8 and mifepristone were assessed for their abilities to prolong the in vitro survival of blood-derived neutrophils.

RESULTThe percentage of sputum neutrophils was significantly higher in severe asthmatics [59.54 (41.99-74.65)%] than mild-moderate asthmatics [30.03 (15.94-47.71)%] and healthy control subjects [29.72(16.53-45.74)%] (P < 0. 01); the level of IL-8 in sputum was significantly higher in severe asthmatics [2907.78 (331.67 - 3457.93) ng/L] than mild-moderate asthmatics [287.58 (130.75-656.84) ng/L] and healthy control subjects [179.2 (58.55-346.59) ng/L] (P < 0.01); the percentages of neutrophilic apoptosis respectively cultured with LPS [(10.57 +/- 1.97)%], severe asthmatics supernatant [(11.82 +/- 2.96)%], IL-8 [(10.47 +/- 1.93)%], dexamethasone [(9.93 +/- 1.95)%], severe asthma supernatant + mifepristone [(12.15 +/- 2.86)%] in vitro were lower than that cultured with PBS [(17.98 +/- 2.27)%], healthy control supernatant [(17.37 +/- 2.50)%], mild-moderate asthmatics supernatant [(16.35 +/- 3.26)%], mifepristone [(17.89 +/- 2.38)%], and dexamethasone + mifepristone [(17.06 +/- 2.59)%] (P < 0.01).

CONCLUSIONSuppression of neutrophilic apoptosis seems to play a potential role in airway neutrophilic inflammation in severe asthmatics, and the level of IL-8 in sputum was significantly higher in patients with severe asthmatics.

Adolescent ; Apoptosis ; Asthma ; metabolism ; pathology ; physiopathology ; Case-Control Studies ; Child ; Female ; Humans ; Inflammation ; Interleukin-8 ; metabolism ; Leukocyte Count ; Male ; Neutrophils ; cytology ; Respiratory System ; metabolism ; pathology ; Sputum ; metabolism

PURPOSE: Obesity is a major risk factor for asthma and it influences airway smooth muscle function and responsiveness. Adiponectin is inversely associated with obesity and its action is mediated through at least 2 cell membrane receptors (AdipoR1 and AdipoR2). Leptin is positively associated with obesity. We investigated whether human airway smooth muscle (ASM) cells express adiponectin receptors and whether adiponectin and leptin regulate human ASM cell proliferation and vascular endothelial growth factor (VEGF) release. MATERIALS AND METHODS: Human ASM cells were growth-arrested in serum-deprived medium for 48 hours and then stimulated with PDGF, adiponectin and leptin. After 48 hours of stimulation, proliferation was determined using a cell proliferation ELISA kit. Human AdipoR1 and -R2 mRNA expressions were determined by RT-PCR using human-specific AdipoR1 and -R2 primers. Concentrations of VEGF, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha in cell culture supernatant were determined by ELISA. RESULTS: Both AdipoR1 and AdipoR2 mRNA were expressed in the cultured human ASM cells. However, adiponectin did not suppress PDGF-enhanced ASM cell proliferation, nor did leptin promote ASM cell proliferation. Leptin promoted VEGF release by human ASM cells, while adiponectin did not influence VEGF release. Neither leptin nor adiponectin influenced MCP-1 secretion from human ASM cells. Adiponectin and MIP-1alpha were not secreted by human ASM cells. CONCLUSION: Human ASM cells expressed adiponectin receptors. However, adiponectin did not regulate human ASM cell proliferation or VEGF release, while leptin stimulated VEGF release by human ASM cells.
Adiponectin/metabolism/*pharmacology/physiology ; Cell Proliferation/*drug effects ; Cells, Cultured ; Chemokine CCL2/metabolism ; Chemokine CCL3/metabolism ; Humans ; Leptin/metabolism/*pharmacology/physiology ; Myocytes, Smooth Muscle/cytology/drug effects/*metabolism ; Obesity/metabolism ; Platelet-Derived Growth Factor/metabolism ; Receptors, Adiponectin/*metabolism ; Respiratory System/cytology/metabolism ; Vascular Endothelial Growth Factor A/metabolism

Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Mucin 5AC ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Respiratory System ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation ; drug effects

AIMTo investigate the effect of antisense oligonucleotides (ASON) of ryanodine receptor on proliferation and [Ca2+]i concentration of airway smooth muscle cells (ASMCs).

METHODSASMCs were cultivated with collagen enzyme digestion method. Different concentrations of ASON were added to the cultures with Lipofectamine 2000 to observe the ASMCs proliferation using MTS/PES method. The changes of ASMCs [Ca2+]i were also observed by flow cytometry. The expression of mRNA of subtypes of RyR was assayed by RT-PCR.

RESULTSRyR ASON restrained the proliferation of ASMCs, decreased the expression of RyR and reduced the concentration of [Ca2+]i.

CONCLUSIONRyR ASON could inhibit the proliferation of ASMCs by influencing the concentration of [Ca2+]i after excited.

Animals ; Calcium ; metabolism ; Calcium Channels ; Cell Division ; Cell Proliferation ; drug effects ; Cells, Cultured ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; Rats ; Respiratory System ; Ryanodine Receptor Calcium Release Channel ; genetics ; pharmacology