| Background Bronchial asthma (asthma) is the common chronic respiratory disease in the worldwide scale. It has become a serious threat to human health in the countries of the world and has an increasing trend. According to statistics, there are around300million asthma patients in the world, the global prevalence of1%～18%, about10～30million asthma patients in China. Asthma is a kind of complex disease caused by genetic and environmental factors. Although the basic research on asthma has made great progress, the pathogenesis and development aspects are not yet completely clear, the treatment of exacerbation of asthma and severe asthma not very satisfied. Therefore, to study the pathogenesis of airway inflammation in asthma and to find a new target for treatment and prevention have become respiratory professional research priorities and hotspots.Asthma is an allergic disease characterized by chronic airway inflammation, increased mucus secretion, airway remodeling and airway hyperresponsiveness and lead to bronchial obstruction and airway hyperresponsiveness. With the more thorough study on asthma, the hypothesis on pathogenesis of asthma is increasing, the most widespread concern is the "hygiene hypothesis".Its core of the theory is Thl/Th2imbalance in asthma.The hypothesis consider that the pathogenesis of asthma is Thl/Th2balance disorder, Th1dominant in the healthy while Th2 dominant in the asthmatic because of Th2excessive differentiation. Imbalance of Thl/Th2is an important foundation of asthma search, and Th2advantage is a key mechanism in development of allergic asthma. In addition to inflammatory cells and inflammatory mediators, airway structural cells such as human bronchial epithelial cells are involved in airway inflammation. Firstly, human bronchial epithelial cells (HBE) as the first line of defense against invaders play an important role in innate immune response. Secondly, some study showed that HBE bridge innate and adaptive immune response. With the in-depth study of inflammatory diseases and the development of the inflammatory pathways, inflammasomes become a hotspot in inflammatory diseases, in which NLRP3inflammasome was the most studied. NLRP3(nucleotide-binding oligomerization domain-leucine-rich repeats containing pyrin domain3) inflammasome is composed of NLRP3protein, ASC (apoptosis-associated speck-like protein containing a CARD) and Caspase-1, NLRP3protein joint pro-Caspase-1(45KDa) by ASC and assembles into NLRP3inflammasome, then pro-Caspase-1can be activated automatically, the activated Caspase-1(10kDa and20KDa) promotes cytokines (IL-1β, IL-18, IL-33, etc.) of the IL-1family maturation and secretion, thus participate in the inflammatory response.In this study, C57BL/6mice and human bronchial epithelial cells (HBE) as an object of the study, observed expression Caspase-1and cytokines IL-1family cytokine secretion in a mouse model of asthma and16HBE inflammatory microenvironment, and effect of the IL-1family cytokine in which Caspase-1blocked by Ac-YVAD-cmk.It may be helpful to improve the mechanism and treatment of the asthma airway inflammation.Part I Establishment of the asthmatic mouse modelObjectiveTo establish a C57BL/6mouse asthmatic model by OVA sensitization and rechallenged.MethodSPF female C57BL/6mice (6-8weeks)18～20g, were purchased from Laboratory Animal Services Center of Southern Medical University, were randomly divided into three groups, group A (control group), group B (asthma group), group C (Ac-YVAD-cmk intervention group), Mice were sensitized on days0,7,14by intraperitoneal injection of OVA-sensitized liquid200μl in group B and C, while only saline in group A. At21-27days mice were challenged with aerosol inhalation of2%OVA40ml for30min, once each day in group B and C, but mice were intraperitoneal injected with Ac-YVAD-cmk(5μg/g) in group C30min before challenge; while mice were challenged with only saline in group A.24h after the last challenge, the mice in each group were nebulized methacholine, Airway responsiveness (Penh value)was determined by BUXCO noninvasive lung function detector. On days28the mice were anesthetized,then sacrificed and were subjected to bronchoalveolar lavage, The bronchoalveolar lavage were stained with Wright and total cell number and the number of eosinophils in bronchoalveolar lavage fluid were counted.The lung tissue was fixed with4%paraformaldehyde, dehydrated with alcohol, embedded in paraffin, conventional HE stained,observed tracheal and perivascular inflammatory cell infiltration with optical microscopy.Result1. Mice behavior observation after challenge:Mice in group B, C displayed various types of allergic response such as shortness of breath, piloerection, scratching the nose, irritability, and then cyanopathy, gatism, while group A showed no such symptoms.2. Airway responsiveness:when mice aerosol inhaled methacholine concentration of3.125g/L,2.5g/L,25g/L and50g/L, The Penh value in group B was significantly higher than that in group A and C (P<0.05)3. The total number of cells and percentage of eosinophil and lymphocyte in BALF:(1)The total number of the cells (28.43±f3.45)×104/ml in group B was significantly higher than in group A (9.01±2.33)×104/ml and group C (22.89±4.61)×104/ml (F=46.682, P=0.000)(2)The percentage of eosinophil (22.35±4.91)%in group B, significantly higher than that in group A (1.07±0.20)%and group C (13.58±2.13)%.(3)The percentage of lymphocyte (32.32±6.12)%in group B, significantly higher than that in group A (10.76±2.02)%and group C (18.50±3.18)%(F=34.688, P=0.000)。4. Histopathology Analysis:The bronchioles and alveolar structure was clear and no leukocyte infiltration in group A. Massive leukocyte infiltration and mucus secretion was present in the lung from group B. The leukocyte infiltration and goblet cell hyperplasia was present in group C, but significantly reduced compared with the group B.ConclusionsC57BL/6mice were injected with OVA and aluminum intraperitoneally then rechallenge with1%OVA can establish the asthmatic mouse model, the symptoms and AHR were consistent with the pathophysiological changes of airway inflammation in asthma. Part Ⅱ Caspase-1expression and IL-1β, IL-33secretion in the lung tissue of asthmatic miceObjectiveTo observe the expression of Caspase-1and cytokine IL-1β, IL-33maturation and secretion in the lung tissue from the asthmatic mice and to explain the possible mechanism.MethodsThe lung tissue were divided into three parts of each groups.The first part was extracted total RNA by Trizol reagent, reverse transcribed into cDNA as a model for PCR amplification. The Caspase-1mRNA expression was detected by Real-time quantitative PCR; the second part containing protease inhibitors (PMSF) RIPA lysis buffer was grinded for3-5min, centrifuged, the protein of supernate was determined by BCA method, calculate the protein concentration, the Caspase-1protein was examined by Western blot assay, Image J software analysis; The third part was grinded for2min, centrifuged, the protein of supernate was determined by BCA method, the IL-1β and IL-33production was detected by ELISA (enzyme-linked immunosorbent assay) in the supernatant..Results1. The comparison of Caspase-1mRNA expression:Caspase-1mRNA expression directly compared to each housekeeping gene GAPDH expression to standardize the amount of initial mRNA.2-△△Ct value in group A(2.12±1.33) and group C(5.99±4.20) were statistically higher than group B (15.97±8.67),(F=10.653, P=0.004).2. The comparison of Caspase-1protein expression:Image J software analysis the Caspase-1/(3-actin value in the groupB (0.56±0.03) were statistically higher than group A(0.47±0.03) and group C (0.28±0.03)(F=73.916, P=0.000)3. The comparison of IL-1β, and IL-33secretion:IL-1β and IL-33secretion (pg/mg) in lung tissue homogenate supernatant of group B [IL-1β (76.49±9.43) and IL-18(73.08±6.67)]were higher than group A [(37.51±9.13) pg/mg, P<0.05;(32.79±5.92) pg/mg, P<0.05) and group C [(64.74±8.65) pg/mg, P<0.05; (46.94±2.38) pg/mg, P<0.05).ConclusionCaspase-1activation is invloved in murine asthma and mediates the maturation and secretion of IL-1p and IL-33. Part Ⅲ The study of Caspase-1expression of HBE induced by LPSObjectiveTo observe the caspase-1expression and IL-1β secretion in HBE induced by LPS and IL-1β secretion.Method16HBE were cultured in RPMI-1640in vitro, the cells were randomly divided into three groups:Negative control group, LPS group and LPS+Ac-YVAD-cmk (LPS+cmk) group. the cell proliferation ability was examined by MTT:The expression of Caspase-1mRNA in the cells was determined by RT-PCR; the Caspase-1protein was examined by Western blot assay. the IL-1β production in the supernatant was detected by ELISA.Result1. The comparison of OD value in the three groups:OD value of the negative control group(0.57±0.10), OD value of the LPS group (0.54±0.06), OD value of LPS+cmk group (0.51±0.08), there is no significant difference (P=0.611,=0.520).2. The comparison of Caspase-1mRNA expression:2-△△Ct value in the control group (1.33±0.33), and LPS+cmk group (1.50±0.24) were lower than LPS group (3.11±0.57).(F=17.564, P=0.003). 3. The comparison of Caspase-1protein expression:grayscale scan results (Z value) in LPS group (0.85±0.11) were significantly higer than LPS+cmk group (0.48±0.06)and control group (0.24±0.03)(F=55.158, P=0.000).4. The comparison of IL-1β level in the supernatant:Compared with LPS group [(52.34±2.47) pg/ml], control group [(15.73±6.83) pg/ml] and LPS+cmk group [(38.15±5.97) pg/ml] were lower (F=46.208, P=0.000).ConclusionCaspase-1activation plays a critical role in human bronchial epithelial cells inflammation and promotes the downstram IL-1family cytokines(IL-1beta) generation. |
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