| BackgroudBronchial asthma is a chronic airway inflammatory disease, there are variety of cells(eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components involved in the disease. This chronic inflammation is correlated with hyperresponsiveness. The pathogenesis of asthma is extremely complex, and so far has not been elucidated, now generally accepted pathogenesis following:①chronic airway inflammation hypothesis;②Th1/Th2imbalance theory;③The immune tolerance function defect doctrine;④regulatory lymphocytes doctrine, mainly including:CD4+Treg cells, CD8+Treg cells, Th17cells, etc. These cells through the regulation of lymphocyte, play activation or inhibition in airway inflammation. However, in recent years from clinnical observation and basic research show that the existing theory can not fully explain the pathogenesis of asthma, and asthma cure is still a problem.NLRP3(nucleotide-binding oligomerization domain-leucine-rich repeats containing pyrin domain3) inflammasome is a cytoplsmic protein polymers, mainly constitute of the nucleotide-binding oligomerization domain receptor(NLRP3), cysteinylacid proteolytic enzyme-1(caspase-1), apoptosis-associated speck-like protein containing a CARD(ASC), which is caspase-1activated platform, then improves IL-1β,IL-18, IL-33and other proinflammatory cytokines modification and activation. NLRP3inflammasome expresses in macrophages, neutrophils, epithelial cells, smooth muscle cells and other cells, in which NLRP3cytoplasmic pattern recognition receptors can sense the intracellular pathogenic microorganisms and metabolites, starts complex assembly, constitutes the core part of the complex.IL-1β、IL-18、IL-33belong to the IL-1family, wherein IL-1β also known as cytokine, mainly is secreted by the monocyte-macrophage, stimulats monocytes and eosinophils degrannulation. IL-1β can promote the differentiation of Th17cells, maintain Th17related cytokine production and may also promote the recruitment of eosinophils. IL-18can promote T cells, mast cells and basophils secrete IL-4, IL-3and enhancing the Thl immune response. Harada found that IL-18gene diversity is closely related to the degree of deterioration of asthma. IL-33can promote a Th2-mediated response. Thl, Th2or Th17response and the recruitment of eosinophils play an important role in asthma, but the issue remains controversial.In our study, we used ovabumin for building allergic asthma model, NLRP3and its downstream cytokine IL-18and IL-1β in lung tissue was detected, the effect of NLRP3inflammasome inhibitor glibenclamide on airway inflammation was observed, the correlation between IL-18or IL-1β and eosinophil percentage of BALF was analyzed, to explore whether NLRP3/IL-1β, IL-18pathway plays an important role in the pathogenesis of asthma.Part one Establishment and assessment of the asthmatic mouse modelObjectiveTo establish and assess a C57BL/6asthmatic mouse model by OVA sensitization and challengeMethods 6-week-old female C57BL/6mice,18-20g, purchased from the Animal Experimental Center of Southern Medical University, were randomly divided into three groups (n=7):normal control(A)group, NLRP3inflammasome inhibitors glibenclamide group(B) and asthmatic(C) group. Group B and group C in the experiment were sensitized by intraperitoneal injection of0.2ml of OVA and alum mixture on days0,7,14. After that, mice were challenged by exposure to an aerosol with1%OVA for30min. The challenge procedure was repeated once each day from days21to days27. B group used glibenclamide500mg/kg for intraperitoneal injection30min before challenging; A group used saline stead of OVA sensitizing and challenging. The airway responsiveness(AHR) of mice was measured24hours after last allergen challenge, the mice were subjected to bronchoalveolar lavage cytology perform and lung tissue sections were prepare for histopathologic examination to evaluate the airway inflammation.Statistical analysisUsing SPSS16.0statistical software to analyze data. Experimental results are expresses as mean±standard deviation. Significant differences in the mean values among multiple groups were analyzed by one-way ANOVA and Bonferroni method for multiple comparisons if homogeneity of variance. However, during heterogeneity of variance, using Welch method to analyze the differences of the mean values and Dunnett’3for multiple comparisons among multiple groups. P value less than0.05was considered to be statistically significant.Results1. The manifestation fo mice aftern sensitization and challengeAsthmatic group and the glibenclamide group dispayed various types fo allergic responses after sensitization and challenge, such as scratching head and face, dispnea, and activation decreased and so on, while control group showed no such symptoms. 2. AHR measurementWhen inhaling methacholine>12.5mg/ml, the airway hyperresponsiveness in asthmatic group was significantly higher than in control (P<0.05); When inhaling methacholine>50mg/ml, the airway hyperresponsiveness in glibenclamide group was significantly higher than in control group (P<0.05); No significant difference was noted between asthmatic group and glibenclamide group.3. The total cells number, EOS%, LYM%in BALFThe total WBC number, the percentage of eosinophils and lymphocytes of BALF in asthmatic group [(29.88±4.97)×104/ml,(21.67±4.83)%,(31.87±5.31)%] were significantly higher than in glibenclamide group [(17.30±1.19)×104/ml,(12.45±1.12)%,(17.16±0.97)%,,P<0.05]and the control group [(9.74±2.88)×104/ml,(1.15±0.26)%,(10.66±1.83)%, P<0.05]. And glibenclamide group’s was higher than control group’s (P<0.05).4. Histological analysisAlveolar structural integrity and almost no inflammatory cell infiltration in the control group. Alveolar septa fracture, inflammatory infiltration around bronchial wall in asthmatic and glibenclamide groups. And asthmatic group’s is more serious than glibenclamide group’s.ConclusionsAccording to airway hyperresponsiveness results,the total cell number and the EOS%of BALF results, lung pathology results, it indicated that we successfully established an asthmatic mouse model by OVA sensitization and challenge. Glibenclamide can alleviate the inflammation of lung in asthmatic mice. Part two The relationship between NLRP3/IL-1β, IL-18signalling and allergic airway inflammation in C57BL/6miceObjectiveTo investigate the NLRP3/IL-1β and IL-18expression in the lung tissue of asthmatic mice and to test the hypothesis that NLRP3-dependent IL-1β and IL-18signalling plays a pathogenic role in asthmatic mice.MethodsA total of C57BL/6mice were randomly divided into3groups:The control(A) group, NLRP3inflammasome inhibitor glyburine(B) group and asthmatic(C) group(n=7).Sensitizing and challenging mice with ovalbumin for allergic asthma model in B and C groups, B group used glyburine for intraperitoneal treatment(500mg/kg)30minetes before inhaling ovabumin. After modeling, lung function was measured, Bronchoalveolar lavage cytogical study was performed and lung tissue sections were prepared for histopathologic examination to evaluate the airway inflammation. NLRP3expression was identified by Western Blot and RT-PCR method, IL-1β and IL-18productions were detected by ELISA(enzyme-linke imminosorbent assay) in mouse lung homogenate supernatant. The correlation between IL-1β/IL-18and the percengtage of eosinophils was analyzed.Results1. NLRP3protein expression in mouse lung:Compared with control and glibenclamide groups, the expression of NLRP3protein were increased in asthmatic group(P<0.05). There was not significant difference between control group and glibenclamide group.2. NLRP3mRNA expression in mouse lung:Compared with control and glibenclamide groups, the expression of NLRP3protein were increased in asthmatic group(P<0.05). There was not significant difference between control group and glibenclamide group.3. The IL-1β and IL-18level in mouse lung supernatant:The level of IL-1β (74.81±17.45) pg/mg and IL-18(426.94±76.05) pg/mg in mouse lung supernatant were higher in asthmatic group than in the control group[(37.54±5.53)pg/mg, P<0.05;(249.62±61.20)pg/mg, P<0.05].4. The correlation between IL-1β/IL-18and the percentage of eosinophils was significant(r=0.833, P=0.02; r=0.856,p=0.014).ConclusionsNLRP3-dependent IL-1β and IL-18signalling plays a crucial role in the allergic airway disease in asthmatic mouse. |
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