Role of transforming growth factor-β in the airway inflammation and remodeling in asthma

Transforming growth factor-β (TGF-β) was reported to be increased in asthma in some studies. Accumulation of TGF-β in airway promotes smooth muscle cell mitogenesis and hyperplasia, and induces fibroblast and myofibroblast and smooth muscle proliferation as well as increase in protein synthesis in connective tissue (such as collagen deposition on the reticular basement membrane). The autocrine induction of collagen expression by smooth muscle may contribute to the thickening of the reticular basement membrane, irreversible fibrosis and remodeling seen in the airways in some asthmatics. TGF-β is considered to be a major fibrogenic cytokine. It can increase smooth muscle mass and lead to severe bronchial obstruction in an asthma attack.     

Dynamic observation of asthma model induced by allergen in mice from acute inflammation to chronic remodeling

AIM: To dynamically observe and compare the relative changes of the indexes from the process of acute inflammation to chronic remodeling in asthmatic mice induced by ovalbumin (OVA).METHODS: Female BALB/c mice (n=60) were randomly divided into normal control group and asthma group. The mice in asthma group were sensitized and challenged by OVA, while the mice in normal group received equal volume of normal saline (NS). The challenge was performed for 3 consecutive days from the 21th day to observe the response of acute inflammation, and then the mice in different groups were challenged once per week for 5 weeks. Detailed comparisons of the dynamic changes of cell infiltration, cytokine expression and airway remodeling were conducted.RESULTS: Compared with NS group, the mice in OVA group showed a predominantly eosinophilic infiltration into the airway lumen, increased production of Th2-type cytokines, secretion of epithelial mucus and deposition of subepithelial collagen. In OVA challenge groups, the levels of inflammatory cells and inflammatory factors were remarkably higher in 24 d group, whereas the most obvious changes of goblet cell hyperplasia and airway remodeling were observed in 52 d group.CONCLUSION: Acute asthma model is sufficiently induced by 3 consecutive days of OVA challenge protocol, which is accompanied with high levels of inflammatory cells and inflammatory factors. The OVA challenge protocol once per week for 5 weeks could induce a chronic asthma model with obvious airway remodeling.     

Role of calcineurin in airway remodeling in guinea pig model of asthma

AIM:To investigate the role of calcineurin (CaN) in airway remodeling in guinea pig model of asthma.METHODS:Male guinea pigs were randomly divided into three groups: control, asthma group and CsA group. The following parameters were measured: 1. The protein content, cell count and differential count of BALF; 2. The amount of [³H]-TdR incorporation into central airway smooth muscle; 3. The mean thickness of airway wall and airway smooth muscle of small airwaysl; 4.CaN activity of trachea and lung tissue.RESULTS:1. The protein content, cell count and eosinophil of BALF in CsA group were 46%, 51% and 60% lower than those in asthma group, respectively (P＜0.01); 2. [³H]-TdR incorporation in CsA group was 22% lower than that in asthma group (P＜0.05);3. The mean thickness of airway wall and airway smooth muscle were 34% and 37% less in CsA group than those in asthma group, respectively (P＜0.01); 4. CaN activity of lung tissue and trachea were 52% and 44% lower in CsA group than those in asthma group, respectively (P＜0.01).CONCLUSION:CsA reduced airway remodeling in guinea pig model of asthma, indicating the role of CaN in the airway remodeling.     

The relationship between Proto-oncogenes expression and airway inflammatory cell infiltration in asthma

AIM:To investigate the relationship between inflammatory cell infiltration and proto-oncogenes expression in asthma. METHODS:Guinea pigs were used as asthma models challenged by ovoglobulin. Dot-blot, Northern-blot and immunochemical techniques were used to detect the expression of c-fos, c-myc, c-jun and c-sis. Inflammatory cell infiltration was showed by pathologic study.RESULTS:c-fos and c-myc mRNA could not be detected or expressed at very low level in control group. Those were greatly increased after the animals are challenged by ovoglobulin. Immunochemical study showed that Fos, Myc, Jun and Sis expressed at low level in control group, and those were increased after the challenge. There was little inflammatory cell infiltration in control group. Lymphocyte, neutrophil and eosinophil were detected immediately after the challenge, a great number of inflammation cells could be seen after 12-24 h of the challenge. Majority of neutrophil and eosinophil were under mucosa or in epithelium in airway. CONCLUSION:Oncogenes expression had strong relationship with airway inflammation.     

Effect of airway remodeling on airway responsiveness in asthmatic guinea pigs

AIM: To establish a guinea pig asthma model and to evaluate the effect of airway remodeling on airway responsiveness. METHODS: The guinea pig asthma model was established by ovalbumin (OVA) sensitization and challenge repeatedly. Bronchial provocation tests were conducted through intravenous injection of acetylcholine. The airway morphologic parameters were measured by computer image analysis system. White blood cells and the differential count in bronchoalveolar lavage fluid (BALF) were examined. RESULTS: The resistance of airway was increased significantly after 4 weeks of OVA exposure, but the increase disappeared upon prolonged exposure. After 8 weeks of OVA exposure, fiber tissue in large airway was increased, and the thickness of smooth muscle layer of small airway was enlarged, as compared with that in control animals. CONCLUSION: Airway responsiveness has changed after prolonged OVA exposure in guinea pigs. This change is related to airway remodeling.     

Role of PARP and caspase-3 in the airway epithelial injury induced by peroxynitrite

AIM:To study the mechanism responsible for ONOO^--induced the airway epithelial injury. METHODS:Effects of 3-aminobenzamide(3-AB), a poly-(ADP-ribose) polymerase(PARP) inhibitor, and Ac-DEVD-CHO, a caspase-3 inhibitor, on LDH release and apoptosis of cultured rat tracheal epithelial (RTE) cells induced by ONOO^- were examined. The cleavage of PARP was analysed by Western blot. RESULTS:3-AB inhibited the release of LDH induced by ONOO^- partially, and had no effect on the apoptosis of RTE cells. Caspase-3 inhibitor Ac-DEVD-CHO obviously prevented the apoptosis of RTE cells induced by ONOO^- in a dose-dependent manner. The cleavage of PARP was observed in the process of apoptosis of RTE cells induced by ONOO^-. CONCLUSIONS:PARP activation represents one of the pathways of ONOO^--mediated epithelial injury, and the excessive activation of PARP contributes to the necrosis in RTE cells induced by ONOO^-. Cleavage of PARP by activated caspase-3 plays a crucial role in the apoptosis of RTE cells induced by ONOO^-.     

Role of pentraxin 3 in inflammation and autoimmunity

As a soluble pattern-recognition receptor, pentraxin 3 (PTX3) evolves in various physiological and pathological effects on inflammation, autoimmunity, apoptosis, vessel remodeling, female fertility and vascular endothelial injury. PTX3 is also a sensitive biological marker for detecting severity and activity of various autoimmune diseases. In most cases, PTX3 limits the progress of autoimmunity and plays a protective role in the process of autoimmune diseases.     

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling

AIM: To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS: The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured. The protein expression of β-catenin, glycogen synthase kinase-3β (GSK-3β), c-Myc and cyclin D1 in the ASMC was determined by Western blot. After depressing the interaction between β-catenin and p300/CBP, the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry. Meanwhile, the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS: The protein levels of β-catenin, c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P<0.05). After depressing the interaction between β-catenin and p300/CBP, the cell activity of ASMC was decreased in asthma group compared with control group (P<0.05), and the change of the cell cycle distribution in asthma group was also more obvious (P<0.05). After inhibiting P38 MAPK activity, the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P<0.05).CONCLUSION: Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1, reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.     

Effects of CpG-oligodeoxynucleotides and dexamethasone on airway remodeling and expression of MMP-9 and TIMP-1 in murine model of chronic asthma

AIM: To observe the changes of airway inflammation and remodeling in a murine model of chronic asthma with CpG- oligodeoxynucleotides(CpG ODN) and dexamethasone (DXM) treatments.METHODS: BALB/c mice were sensitized and repeatedly challenged with ovalbumin.Pathological slides were prepared from left lung and stained with hematoxylin-eosin.WAmus (smooth muscle area),Wamuc (mucous area) and WAi (inner wall area) of the airway were measured and standardized by Pbm (basement membrane perimeter). The areas of collagen Ⅰand Ⅲ in the lung tissue were determined by using a Sirius red-polarizing microscopy morphometry method.Expressions of matrix metalloprotease-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were detected by immunohistochemistry.RESULTS: WAmus/Pbm,WAmuc/Pbm and WAi/Pbm decreased significantly in CpG ODN and DXM treated group when compared with asthma group (P<0.05).No statistical significance between CpG ODN and DXM treated group was observed (P>0.05).Collagen deposition in asthma group increased more than that in CpG ODN and DXM treated group (P<0.05).The expressions of MMP-9 and TIMP-1 were much higher in asthma group than those in CpG ODN and DXM treated group (P<0.05).It had no statistical significance between CpG ODN and DXM treated group (P>0.05).CONCLUSION: Airway remodeling occurrs in the chronic asthma.Early intervention with steroid or CpG might partially inhibit its process via lowering expressions of MMP-9 and TIMP-1 in chronic asthma.     

Histamine receptor antagonist prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig

AIM:To investigate the effect of histamine receptor antagonist on airway remodeling and acid-base imbalance in asthma of guinea pig. METHODS:Guinea pigs were divided into 5 groups: the normal control group, the asthma model group, the continued asthma model group, histamine group and histamine receptor antagonist group. For each group, the content of histamine, Na+, Cl-, PaO2, PaCO2, pH, AB, SB in serum, and thickness of airway mucosa and smooth muscle cell layer were measured and compared with each other. RESULTS:(1) According to the content of histamine in serum and thickness of airway mucosa and smooth muscle, the order was: the histamine group>continued asthma model group>the asthma model group>the normal control group (P<0.01), and the histamine receptor antagonist groupthe continued asthma model group (P<0.01), but for PaCO2, the order was conversed. Airway remodeling, increase in histamine in serum, respiratory acidosis and metabolic acidosis in asthmatic guinea pig were observed. Exogenous histamine accentuated the change, however, histamine receptor antagonist attenuated it. CONCLUSION:Histamine may take part in the airway remodeling of asthma. Histamine receptor antagonist can prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig.     

Role of ERK on proliferation of airway smooth muscle cells in chronic asthmatic rats

AIM: To investigate the roles of extracellular signal-regulated kinase(ERK) signaling pathway on regulating proliferation of airway smooth muscle by observing the expression of ERK in airway smooth muscle(ASM) in chronic asthmatic rats.METHODS: Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and proliferating cell nuclear antigen(PCNA) in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, p ERK1/2 and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively.RESULTS: The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.CONCLUSION: ERK signal pathway might be an important pathway on regulating cell proliferation of ASM resulting in asthmatic airway remodeling.     

SO2 derivatives stimulate inflammation of airway epithelial cells through NLRP3 inflammasome

AIM:To investigate the effect of sulfur dioxide (SO₂) derivatives (sodium sulfite and sodium bisulfate) on NLRP3 inflammasome in airway epithelial cells. METHODS:SO₂ derivatives at different concentrations were applied to bronchial epithelial 16HBE cells for 12 h. The production of reactive oxygen species (ROS) was detected by flow cytometry. The protein levels of NLRP3 and caspase-1 p20 were analyzed by Western blot. The level of interleukin-1β(IL-1β) in the cell culture supernatant was measured by ELISA. The cell viability was measued by MTT assay, and the concentration of SO₂ derivatives used in the following experiments was 2 mmol/L. When the NLRP3 gene in 16HBE cells was silenced by RNA interference technique or N-acetyl cysteine (NAC) was used to pretreat 16HBE cells, the intracellular ROS was detected by flow cytometry, and the protein levels of NLRP3 and caspase-1 p20 and the secretion of IL-1β were determined by Western blot and ELISA, respectively. RESULTS:Compared with the control group, the level of intracellular ROS, the protein levels of NLRP3 and caspase-1 p20, and the secretion of IL-1β in cell supernatant were increased significantly in 2 mmol/L and 4 mmol/L SO₂ derivative groups (P<0.05). Compared with the 2 mmol/L group, the protein levels of NLRP3 and caspase-1 p20 were significantly inhibited in NLRP3 siRNA group (P<0.05). The concentration of IL-1β in the cell culture supernatant was significantly decreased (P<0.05). No significant difference of ROS level was observed. Significantly decreased protein levels of NLRP3 and caspase-1 p20, and the concentration of IL-1β in NAC group were found (P<0.05). CONCLUSION:SO₂ derivatives directly promote the production of IL-1β through NLRP3 inflammasome in bronchial epithelial cells.     

Effect of azithromycin on airway inflammation and airway mucus hypersecretion in rats with chronic obstructive pulmonary disease

AIM: To observe the effect of azithromycin on the rats with chronic obstructive pulmonary disease (COPD), and to explore the underlying mechanism about the airway inflammation and mucus hypersecretion. METHODS: Male SD rats were randomly divided into normal control group, COPD model group, azithromycin treatment group. The COPD model was established by the method of cigarette smoking combined with intratracheal injection of LPS. Pathological changes of the bronchi and lung tissues of the rats were observed with HE staining. Pulmonary ventilation function in the rats was detected with pulmonary function instrument. The levels of IL-8, IL-17 and TNF-α in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The expression of MUC5ac and TLR4 at mRNA and protein levels in bronchi and lung tissues was determined by real-time PCR and Western blot.RESULTS: HE staining showed that the changes of bronchi and lung tissues in model group were consistent with typical pathological manifestations of COPD. Compared with model group, these changes were alleviated in treatment group. The pulmonary functions in model group were significantly decreased compared with control group. The levels of IL-8, IL-17 and TNF-α in the BALF in model group were significantly increased compared with control group (P <0.05). The expression of MUC5ac and TLR4 at mRNA and protein levels in model group was significantly higher than that in control group (P <0.05). Compared with model group, the degree of the descent in pulmonary function in treatment group was significantly lessened. Compared with model group, the levels of IL-8, IL-17 and TNF-α in treatment group were significantly inhibited (P <0.05). Furthermore, the expression of MUC5ac and TLR4 at mRNA and protein levels in treatment group was significantly lower than that in model group (P <0.05). CONCLUSION: Azithromycin decreases the levels of IL-8, IL-17 and TNF-α in the BALF of COPD model rats, inhibits the protein expression of MUC5ac and TLR4 in the lung tissues, thus playing a preventive and therapeutic role to reduce airway inflammation and airway mucus hypersecretion.     

Inhibitory effect of 1,25-(OH)2D3 on proliferation of airway smooth muscle cells from passively-sensitized patients

AIM: To investigate the effects of 1,25-dihydroxyvitamin D₃ on the proliferation of passively-sensitized human airway smooth muscle cells (HASMCs), and to explore its potential role in asthmatic airway remodeling.METHODS: HASMCs were passively sensitized with 10% serum from asthmatic patients.1,25-(OH)₂D₃ was used as the interventor.The effect of 1,25-(OH)₂D₃ on the cell proliferation and its optimal concentration were determined by MTT colorimetric assay.The cell cycle analysis was performed by flow cytometry.The expression of proliferating cell nuclear antigen (PCNA) was measured by the method of immunocytochemical staining.RESULTS: 1,25-(OH)₂D₃ at the concentrations of 10^-9-10^-7 mol/L markedly inhibited the cell proliferation and the maximum effect was observed at the concentration of 10^-7 mol/L.This concentration of 1,25-(OH)₂D₃ markedly suppressed the PCNA-positive rate and hampered the G₁/S transition in HASMCs passively-sensitized by asthmatic serum.CONCLUSION: 1,25-(OH)₂D₃ has direct inhibitory effects on the proliferation of passively-sensitized HASMCs in vitro, which may be concerned with the beneficial role of 1,25-(OH)₂D₃ on the prevention and therapy of asthmatic airway remodeling.