The Role Of Chymase And Renin-Angiotensin System In The Pathogenesis Of Asthma In Mice

ObjectivesBionchial asthma is a kind of chronic inflammatory disease of respiratory tract in which eosinophils, mast cells, T lymphocytes and other inflammatory cells paritcipate. It's also charactered by reversible airflow obstrction, airway hyperresponsiveness, and airway inflammation and remodeling. While the study of pathogenesis and treatment of asthma have got much progress, but asthma prevalence, morbidity and mortality are still on the rise. Therefore, it's always been an important issue to search for methods of the effective prevention and treatmen from various angles in different ways.Renin-angiotensin system(RAS) is an important neuroendocrine system regulating human physiological functions. AngiotensinⅡ(AngⅡ), the vitol facter in RAS, regulates the modulation of salt and water homeostasis, vessels contraction, tissue perfusion, and cell proliferation in both circulation and organizations by connecting with the angiotensinⅡtype 1 recepter(AT1R). In addition to that, AngⅡalso plays a pro-inflammatory part in organisms in the pathological state by modulating the expression of mediators of inflammation, such as chemokines, cytokines and adhesion molecules and promoting accumulation of inflammatory cells, so that it is involved in inflammatory-related diseases.Therefore, RAS in addition to the regulation of cardiovascular functions, its effect in the outcome and prognosis in pulmonary diseases has gradually attracted people's attention.Chymase is a chymotrypsin-like protease stored in high amounts in the secretory granules of mast cells and effects inflammation. To understand the regulatory mechanism of ACE that determines the production of AngⅡprovides new ideas and new methods in the pathogenesis of lung diseases. Recent studies have found that angiotensin I (AngⅠ) can not only be catalyzed into AngⅡby ACE in the classical pathway, but also by chymase in another way. In the pathogenesis of asthma, chronic inflammation of respiratory tract plays the main part, in which a large amount of mast cells take part. Chymase inhibitors in addition to inhibiton of lesion mast cells aggregation, can effectively suppress heart function deterioration after myocardial infarction to reduce mortality, cut down arteriovenous fistula and vascular stenosis after artificial vascular graft as well. A research has confirmed the conection between chymase and respiratory disease, but its correlation with asthma has not been reported. We speculate that chymase may play a role in the process of asthma, except for regulating the function and status of airway epithelial cells, smooth muscle cells, glands in asthma to alleviate asthma inflammatory changes. To our knowledge, RAS is activated in asthmatic disease process, but the results only focus of angiotensin-converting enzyme (ACE) and AngⅡ, which makes it difficult to explain some phenomena in the study on mechanism of asthma. Therefore, the experiment further discussed the role of RAS in the pathogenesis of asthma in the angle of chymase.Therefore, in the experiment, we intended to set the BALB/c mice asthma model, by applying a chymase inhibitor (Suc-Val-Pro-L-PheP(OPh)2), an angiotensin-converting enzyme inhibitor (captopril) and an AngiotensinⅡtype 1 receptor blocker (candesartan) to inhibite the activation of RAS in different ways, respectively, reduce antagonism of AngⅡproduction or its combination with AT1R. We observed the inflammatory changes and whether the RAS system is activated in the lung tissue with asthma in mice.This experiment set further discussion on the role of chymase as a new member of the RAS in the pathogenesis of asthma and whether chymase inhibitors can become a new target for the treatment of asthma in the future.Methods1. Mice asthma model80 BALB/c female mice, weight 18-22g, were assigned to 8 different groups(n=10 per group):①control group (A),②sham group (B),③low dose captopril group (C),④high dose captopril group (D),⑤low dose candesartan group (E),⑥high dose candesartan group (F),⑦low dose Suc-Val-Pro-L-PheP(OPh)2 group (G),⑧high dose Suc-Val-Pro-L-PheP(OPh)2 group (H). Except the control group, the other 7 groups were sensitized to ovalbumin(OVA) by intraperitoneal injection of OVA-aluminum hydroxide(ovalbumin (OVA) 100ug per mouse, aluminum hydroxide 2.25mg per mouse)on Days 0,7, and 14 days. On Day 21, the mice were challenged with inhalation of 5%OVA 30min per day for seven days. As for the observation, restlessness, shortness of breath, abdominal muscle twitching in the mice were regarded as the positive reaction. Except the control group and the sham group, the other 6 groups were treated with intragastric administration of appropriate doses of drugs from the day before inhalation for 7 days in total. After inhalation, all groups of mice were executed and the serum and lung tissue were left by experimental design.2. HE(hematoxylin-eosin) staining and transmission electron microscope were used to show the changes of pulmonary structure and the effects on inflammatory infiltration and structural damages in asthma lung tissue after inhibition of RAS activation and chymase activity.3. Immunohistochemistry staining was used to show the concentration and the expression of ACE, ACE2, AT1R, angiotensin II type 2 receptor(AT2R) and chymase after using the chymase inhibitor, the ACE inhibitor and the AT1R blocker in the asthma lung tissue.4. The expressions of RAS-related proteins and NF-κB were tested and the effects of the chymase inhibitor, the ACE inhibitor and the AT1R blocker on the proteins were evaluated by western-blot analysis.5. The expression changes of AGT, AT1R, AT2R and ACE were determined by RT-PCR and the related gene expressions and the trends after RAS inhibition were analysed.6. We collected mice serum of each group, tested the concentration of Ang I and Ang II by RIA, evaluated the content changes and effects after inhibiting ACE and chymase respectively in serum of asthma model.Results1. The morphological changes of pulmonary tissueWith the HE staining, we found that in the control group tissue slices, the epithelial layer of the small blood vessels and the small bronchial were smooth, with no significant inflammatory cells infiltration around, and the alveolar structure was normal, without submucosal edema or alveolar epithelial cells damaged. In the sham group, there were inflammatory changes of different levels around the bronchial and vessels, including eosinophils around submucosa membrance and vessel-around area, inflammatory cells infiltration such as macrophages and lymphocytes, airway epithelial damaged, submucosal oedema, bronchial wall thickening, airway smooth muscle hyperplasia and peripheral pulmonary tissue excessive inflation. In the captopril-treated groups, the candesartan-treated groups and the (Suc-Val-Pro- L-PheP(OPh)2)-treated groups, the inflammatory cells infiltration around the bronchial and vessels was alleviated obviously, the condition of epithelial cells defulvium was also improved, spasm, thickening of airway smooth muscle and peripheral alveolar tissue emphysematous changes were significantly reduced as well.From the transmission electron microscope results, we saw that in the control group, typeⅠand typeⅡalveolar epithelial cells were normal, with a small amount of alveolar macrophages, in which there were a small amount of relatively small lysosomes, and lipid droplets in the fibroblasts in the alveolar wall were visible. While in the sham group, Alveolar wall was thinner and peripheral pulmonary was over inflation. The number of alveolar macrophages was increased, with a distribution of macrophages in both alveolar wall and alveolar cavity, and the lysosomes in them around the trachea got a large increase. TypeⅡalveolar wall cells reduced in number and became smaller in size, osmiophilic multilamerllar body decreased, and mitochondria was visible in cytoplasm, and surface microvilli reduced. Lipid droplets in the typeⅠalveolar wall cells decreased. In the low-dose-captopril-treated group, part of alveolar wall was thin, without integrity, and in the alveolar cavity, eosinophils, macrophages and neutrophils increased with mitochondrial swelling. TypeⅠalveolar cells were in better condition, typeⅡalveolar cells decreased in the thinner alveolar wall. In the high-dose-captopril-treated group, both typeⅠandⅡalveolar cells were better, and eosinophils in the alveolar cavity were less. In the low-dose-candesartan-treated group, inflammatory cells obviously increased with capillaries almost obstructed and local alveolar cavity was in the condition of hyperemia. Neutrophils increased in alveolar septum with more macrophages and eosinophils seen. In the high-dose-candesartan-treated group, the wall of both typeⅠandⅡAlveolar cells was thicker, the cytoplasm of typeⅠcell was slightly dense, but most typeⅡcells had normal structure, with some individual cell cytoplasm mildly dense, lamellar bodies decreased, and macrophage cells, Eosinophils, neutrophils and lymphocytes reduced in number in alveolar cavity. In both the low-and high-dose-(Suc-Val-Pro-L-PheP(OPh)2)-treated groups, alveolar cell wall structure tended to be normal as the dose increased, and the inflammatory cells were obviously less than the sham group.2. Immunohistochemistry was used to detect the RAS related protein expression and the effects to the expression after inhibiting RAS activation in different pathways.Immunohistochemistry studies of AT1R showed that AT1R was partly positively expressed in the control group, mainly distributed in capillaries' endothelial membrane surface and cytoplasm, and there was no obvious inflammatory cell infiltration in the local tissue; While in the sham group, the AT1R positive expression was more than the controlled group, in the alveolar epithelial cells the positive expression was obvious, and inflammatory cell infiltration around bronchial walls is more, and alveolar epithelial cells defulvium was seen in the alveoler cavity. In the Suc-Val-Pro-L-PheP (OPh) 2 group, the captopril group and Candesartan group, the results showed that as the drugs' dose increased, the AT1R positively expressed less than the sham group, the inflammation alleviated, and bronchial wall thickness was significantly smaller than the model group.ACE immunohistochemistry staining results led to the conclusion that ACE expressed a little in the alveolar epithelial cells and the vessels' endothelial cells in the control group, and the expression became more in the sham group. As the dose increased in the captopril group, ACE significantly reduced positive expression than the model group. In the candesartan group and Suc-Val-Pro-L-PheP(OPh) 2 group, ACE positive expression was more or less the same as in the sham group.Chymase immunohistochemistry staining showed no significant positive expression in the control group and chymase positive expression in the alveolar and bronchioles epithelial cells along with the alveolar space narrowed and infiltration of inflammatory cells increased. As the dose increased in captopril and candesartan groups, positive expressions of chymase gradually diminished, and local tissue infiltration of inflammatory cells was significantly reduced. The trend became more obvious in chymase-inhibitor-treated-groups, and in the high-dose-(Suc-Val-Pro-L-PheP(OPh)2)-treated groups, chymase expression was significantly inhibited.Immunohistochemistry staining of AT2R and ACE2 showed no positive expression in the pulmonary tissue or no difference among groups.3. Wester blot analysis of RAS related protein in pulmonary tissue.Western blot analysis showed that the expression of AT1R, ACE and chymase was significantly increased in the sham group than in the control group, and as the drug dose increased in captopril group, the three protein expression decreased, while AT2R and ACE2 expression showed no trends among the groups, as the dose increased in the candesartan group, expression of AT1R and chymase was significantly reduced compared to the sham group, and expression of AT2R. ACE and ACE2 had no significant trends in different groups. as the dose increased in the Suc-Val-Pro-L-PheP(OPh)2 group, expression of AT1R and chymase was considerably lower than the sham group, while expression of AT2R, ACE and ACE2 was of no significant trends in groups. P65 and P50, subunits of NF-κB, were expressed more in sham group than in the control group, respectively, however, after the application of three kinds of drugs, their expression in lung tissue was significantly reduced compared to the sham group.4. The mRNA of RAS related genes expression was detected through RT-PCRIn the sham group, mRNA expression of ACE was found to be significantly increased compared to the control group, and after applying the ACE inhibitor, its expression was significantly reduced compared to the sham group, while AT1R blockers and chymase inhibitor had no on obvious impact. In the sham group, AT1R mRNA expression was increased significantly than control groups, and after applying the ACE inhibitor and AT1R blocker. its expression was significantly reduced, while chymase inhibitor had no on obvious impact. AT2R mRNA expression was significantly reduced in the sham group than in the control group, and after inhibition of ACE, AT1R and chymase, the AT2R mRNA expressed significantly more. AGT mRNA expression had no significant change trend among the groups.5. The concentration of AngⅡand AngⅠin serum among groupsThe results of concentration AngⅡand AngⅠin serum showed that the content of AngⅡwas significantly higher in the sham group than in the control group, AngⅡcontent in captopril groups and Suc-Val-Pro-L-Phe (OPh)2 groups was obviously lower than in the sham group. In the candesartan groups, serum content of AngⅡhad no significant trends compared to the sham group. The AngⅠcontent was of no significant difference in serum among groups.ConclusionThe experiment can lead to the following conclusions:1. Morphology study of lung tissue showed that in the sham group, there was a large number of inflammatory cells infiltration and alveolar wall structural damaged, it proved that mice asthma model could be successfully copied through OVA-intraperitoneal-injection sensitized and OVA aerosol inhalation.2. In the pathogenesis of asthma in mice. RAS is activated, the relevant ingredients AngⅡ, ACE, AT1R and chymase are involved in lung tissue of asthmatic inflammation processes, and increase the local inflammatory response. AT2R may have the role of confrontation of AT1R in asthma to protect lung tissue and reduce the inflammatory response.3. RAS activation is inhibited to reduce the production of AngⅡin asthma,, so that the inflammation of lung tissue is reduced, particularly the mast cells decrease which results in cut down of chymase expression.4. When asthma, the increased AngⅡbinds with AT1R, activates NF-κB in the cytoplasm, promotes its transfers to the nuclear, helps to transcript into large amounts of proinflammatory factors to release, and increases local inflammation of lung tissue. Inhibition of AT1R, ACE and chymase could reduce pulmonary inflammatory changes.5. Chymase takes part in RAS activation in asthma, and as a member of RAS, it is a new proinflammatory factore by the way to catalyze the AngⅡproduction. On one hand, chymase inhibitor inhibite the catalytic activity of chymase to reduce AngⅡgeneration and reducethe activation of NF-κB, on the other hand, it reduces asthmatic inflammation of lung tissue stabilizing the mast cells and inhibiting the degranulation,This study replicated the mice asthma model successfull, applied chymase inhibitors to inhibit RAS activation, ease the inflammation of lung tissue in mice asthma model and explained the role of chymase and RAS in the pathogenesis of asthma. It provides an important theoretical basis for clinical research and treatment of diseases and the development of effective drugs for asthma so that the chymase inhibitor is expected to become a new class of drugs for treatment of allergic diseases.