| Bronchial asthma is one of common chronic inflammatory diseases that ischaracterized by airway hyperresponsiveness and airway mucus hypersecretion，with ahigher incidence and costing. In asthmatics, mucus hypersecretion obstructs airway andleads to airflow limitation, which is an independent risk factor for decline of forcedexpiratory volume in one second (FEV1) and a cause of asthmatics death. However, thereis no effective way for controlling mucus hypersecretion. New clinical therapeuticstrategies are needed. Airway goblet cell hyperplasia is an important pathological basis ofairway mucus hypersecretion. It was confirmed that hyperplasia with and highly specificexpression of hCLCA1and decreased apoptosis in airway goblet cells was closely relatedto airway goblet cell hyperplasia and mucus hypersecretion in asthmatics. Further research also showed that mCLCA3expression was closely related to airway goblet cellhyperplasia and mucus hypersecretion in a mouse model of asthma, and the increasingnumber of airway goblet cells can be reduced by increasing apoptosis to keep the originalbalance.Therefore, CLCA and apoptosis may be effective targets for inhibiting mucushypersecretion, and there may be some correlating with each other. To changephysiological characteristics of airway goblet cells by blocking CLCA could affectapoptosis and mucus secretion of goblet cells and reach the aim for treating mucushypersecretion.Objective:To observe expression of hCLCA1and mCLCA3in airway tissues, and apoptosis ofairway goblet cells in asthmatic patients and mice, and analyze the correlation with eachother and high airway mucus secretion. To search a new therapeutic strategy for mucushypersecretion of asthma, we study the effects of monoclonal antibody of mCLCA3onapoptosis of airway goblet cells and airway mucus secretion in a mouse model of asthma..Methods:1. Collecting airway tissue samples from10cases of asthmatics and10cases ofnormal human by bronchoscopy, we used HE staining, PAS staining,immunohistochemistry, TUNEL and RT-PCR methods to detect the number and apoptosisof airway goblet cells, distribution of apoptotic protein and expression of MUC5AC andhCLCA1mRNA in airway tissues.2. Eighty BALB/c mice were sensitized with ovalbumin and then randomly dividedinto sixteen groups (A~P groups) based on the intranasal challenge days. HE staining, PASstaining, immunohistochemistry, TUNEL, RT-PCR and ELISA methods were used todetect the number and apoptosis of airway goblet cells, distribution of apoptotic protein,expression of MUC5AC and hCLCA1mRNA in airway tissues and MUC5AC level inBALF.3. Thirty BALB/c mice were randomly divided into three groups: normal group,asthmatic mice group and mCLCA3monoclonal antibody treatment group. Asthmamodels were established in the last two groups, and then we treated mice of the last group through intranasal instilling mCLCA3monoclonal antibody. We detected the airwaymucus secretion, number and apoptosis of airway goblet cells by HE staining, PASstaining, immunohistochemistry, TUNEL and ELISA methods.Results:1. The proportion of goblet cells in airway epithelial cells and proportion of Bcl-2+goblet cells (0.456±0.072,0.372±0.068, respectively) in the asthmatics group wassignificantly higher than that of the healthy control group (0.230±0.051,0.233±0.044,respectively)(all P <0.05)，but the Bax+proportion (0.152±0.033) and apoptosis radio(0.022±0.009)of goblet cells was significantly lower than that of the healthy controlgroup(0.606±0.081,0.076±0.015, respectively)(all P<0.05). Airway tissues in theasthmatics group expressed hCLCA1mRNA (0.219±0.043), but the healthy control grouphardly expressed. The expression of MUC5AC mRNA in airway tissue in the asthmaticsgroup(0.603±0.150) was higher than that in the healthy control group (0.190±0.095)(P<0.05). In the asthmatics group, expression of hCLCA1mRNA in airway tissues waspositively correlated with the proportion of goblet cells and expression of MUC5ACmRNA in airway tissues (r=0.67, r=0.71, respectively, P<0.05). In these two groups, theapoptosis radio of airway goblet cells was positively correlated with the Bax+proportionof airway goblet cells(r=0.62,P<0.05), and was negatively correlated with the Bcl-2+proportion and the proportion of goblet cells (r=-0.59, r=-0.477,respectively, P<0.05).2. The proportion of goblet cells in airway epithelial cells of mice was graduallyincreased with challenge days, and was maximum in H group (7challenge days), couldmaintain to K group (10challenge days), then was gradually decreased. The proportion ofgoblet cells in airway epithelial cells of mice from H group, K group and P group (15challenge days)(0.910±0.140,0.896±0.091and0.460±0.150, respectively) were morethan that from A group(0challenge day)(0.070±0.035)(all P<0.05); The airway gobletcells of mice from H group expressed high level of mCLCA3protein (P<0.05), but thatfrom A group hardly expressed; The Bax+proportion of airway goblet cells of mice fromH group,K group and P group (0.201±0.055,0.358±0.103and0.443±0.192, respectively)was much higher than that from A group (0.103±0.021)(all P<0.05), but the Bcl-2+ proportion of airway goblet cells of mice from the three groups (0.252±0.044,0.204±0.090and0.096±0.041, respectively) was significantly lower than that from Agroup(0.103±0.021)(all P<0.05). The apoptosis radio in airway goblet cells of mice fromH group, K group and P group (0.107±0.033,0.175±0.048and0.251±0.103) were morethan that from A group (0.024±0.009)(all P<0.05); In H group, K group and P group,the apoptosis radio of airway goblet cells was positively correlated with the Bax+proportion of airway goblet cells (r=0.73,P＜0.05), and was negatively correlated with theBcl-2+proportion of airway goblet cells and the MUC5AC content of BALF (r=-0.51, r=-0.69, respectively. P＜0.05). In these three groups, expression of MUC5AC mRNA inairway tissues was positively correlated with mCLCA3mRNA (r=0.81, P＜0.05).3. The goblet cell proportion of airway epithelial cells in mice from the monoclonalantibody treatment group (0.665±0.067) was significantly lower than that from theasthmatic mice group (0.896±0.091)(P<0.05), but the apoptosis radio of airway gobletcells(0.310±0.059) was significantly higher than that from the asthmatic mice group(0.177±0.050)(P<0.05). From the monoclonal antibody treatment group the Bax+proportion (0.411±0.095) of goblet cells was significantly higher than that that from theasthmatic mice group（0.252±0.062）(P<0.05), but the Bcl-2+proportion(0.145±0.044)of goblet cells was significantly lower than that that from the asthmatic mice group（0.220，±0.032）(P<0.05).MUC5AC content in BALF from the monoclonal antibodytreatment group (5.9±1.9ng/mL) was significantly lower than that from the asthmatic micegroup (14.7±4.8ng/mL)(P <0.05).Conclusion:1. The hCLCA1mRNA was specifically expressed in the asthmatic airways epithelialtissues, and apoptosis of airway goblet cells was reduced, which is closely related toairway goblet cell hyperplasia and airway mucus hypersecretion. The low Bax and highBcl-2protein expression of airway goblet cells may result in reduced apoptosis.2. The mCLCA3was specifically expressed in the airway goblet cells of asthmaticmice, which is closely related to airway goblet cell hyperplasia and airway mucushypersecretion. With increasing challenge days, the higher Bax protein and lower Bcl-2 protein expression of airway goblet cells could be the cause of reducing the increasednumber of airway goblet cells by apoptosis in asthmatic mice.3. Blocking mCLCA3by specific monoclonal antibody could changeapoptosis-related proteins expression in airway goblet cells to increase apoptosis andinhibit airway mucus hypersecretion in asthmatic mice. |
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