| 一、BackgroudBronchial asthma is the common chronic inflammatory disease of the airways characterized by various cells and celelular components. It could lead to airway hyperreactivity (AHR) and the contraction of airway smooth muscle which result in extensive and reversible airflow obstruction and bronchospasm. Present study regards airway remodeling as a key factor leading to airflow limitation. Airway remodeling, characterized by hyperplasia and hypertrophy in smooth muscle mass, thickening of subepithelium, increasing collagenandproteoglycan deposition, fibroblast proliferation and differentiation to myofibroblasts, airway edema and angiogenesis. The airway remodeling, as a result of the severe persistent asthma, leads to less responsive to treatment and irreversible airflow limilation in certain people. There are lots factors regulating the contraction of airway smooth muscle(ASM), including stimulation of allergen, infection of bacteria or virus, activation of Rennin Angiotensin System(RAS). It is a urgent question how to decrease the airway contraction when airway remodeling has happened in asthma.In the past study, it has been approved that RAS play an important role on proliferation, fibrosis and inflammation in cardiovascular system, kidney, liver. RAS and their receptors also exist in lung. In precent study, AngⅡ receptor type1(AT-1R) high expression and the polymorphism of gene ACE were assiociated with asthma attack. Even the level of Ang Ⅱ was incresed in some study. Whatever, RAS is concerned about the contraction of airway smooth muscle, and the discovery of its new components, angiotensin-converting enzyme2(ACE2)-angiotensin-(1-7)-Mas axis provides a new orientation for treatment of asthma. ACE2and ACE hold opposite function, though belong to the same region. ACE is peptidyl dipeptidase of two catalytic domains, but ACE2is carboxypeptidase of a catalytic domain. They have different substrates:ACE2disintigrates C’of Ang Ⅰ and Ang Ⅱ to Ang-(1-9) and Ang-(1-7), and then Ang-(1-9) is disintigrated to Ang-(1-7) by ACE. ACE2is the key enzyme because its more powerful efficiency. Mas is the key receptor of Ang-(1-7). In other side, the axis of ACE2-Ang-(1-7)-Mas plays the opposite effect on proliferation, fibrosis and inflammation versus Ang Ⅱ in many organs. Especially the study in vascular smooth muscle holded the opinion the Ang Ⅱ could induced the contraction of VSM by activation of RhoA. Ang Ⅱ also affect the cytoskeleton and contraction in HSC.The studies indicates that Ang Ⅱ-induced contraction of human airway smooth muscle cells (HASMCs) played a vital role in pathophysiology of asthma. However, which signal pathway is involved in Ang Ⅱ-induced contraction of HASMCs is unkown. How Ang-(1-7) acts and whether Ang-(1-7) could inhibite the Ang Ⅱ-induced contraction of HASMCs need to be confirmed.Rho-kinase is the small GTPase (guanosine triphosphatase). called ROCK2, which is a concernment element of RhoA/ROCK signal pathway associating with the maintenance of the contractive phenotype of smooth muscle cell contraction. According to recent research, Ang Ⅱ acts via AT-1R in vascular smooth muscle cells by Rho/ROCK signal pathway. The pathway also plays a crucial role on AHR in asthma. Rho/ROCK signal pathway plays roles on cell migration, proliferation, inflammation, cytoskeleton and contraction. Cytoskeleton made up of actin is the cruial formation to maintain cell shape, mediate the biological function of eukaryotic cell. It also paticipates the process of cell contraction, migration and so on. And RhoGTP family in Rho/ROCK signal pathway, as a member of Ras superfamily, is a kind protein binding GTP. It could affect downstream ROCK2to play biological aspect and be molecular switch in cell signal transition. There are three vital subfamily:Rho、Rac, Cdc42in RhoGTP superfamily. the small G-protein have the activity GTPase and plays roles on cell migration, proliferation, inflammation, cytoskeleton and contraction.In a word, the activation of is associated with asthma closely. In this study, We hypothesized that Ang Ⅱ induce HASMCs contraction and Ang-(1-7) antagonizes the effect of Ang Ⅱ by RhoA/ROCK signal pathway. We hope to offer an new direction by searching for RAS for treatment of asthma.二、Objectives1. In order to study the effects of Ang Ⅱ and Ang-(1-7) on the contraction of primary cultured HASMCs.2. To confirm the ways of AngⅡand Ang-(1-7) acting on HASMCs by their receptors inhibitors, irbesartan and A779and the role of Rho/ROCK by Y-27632, inhibitor of ROCK2.3. To evaluate cytoskeleton by staining F-actin of HASMCs with fluorophore-conjugated phalloidin and DAPI after treating different stimulus4. To verify that the Rho/ROCK signal pathway participate in regulation of Ang Ⅱ and Ang-(1-7) on the contraction of HASMCs by detecting the key protein.三、Methods1、HASMCs were isolated from the lobar or main bronchus obtained from lung resection from donors, approved by the Division of Thoracic Surgery. All experimental manipulations and protocols in this study were approved by the Human Ethics Committee of Nanfang Hospital, Southern Medical Universi ty(NFEC-201109-K1).2. HASMCs were primary cultured by explant method. HASMCs were initially characterized by their typical morphology and positive immunostaining for a-smooth muscle actin (a-SMA).3. Primary cultured HASMCs were group into6:blank control, AngⅡ+Ang(1-7)、AngⅡ+Ang-(1-7)+A779、AngⅡ+IRB、AngⅡ+Y-27632.4. Typel rat-tail collagen suspension containing cells was prepared according to the manufacturer’s instructions. The area of gel surface of test group were to reflect the degree of contraction macrographically; The surface area of the collagen gels was measured using image J analysis software. The surface%was expressed by the formula:(gel surface area of text group/original gel surface area)x100%.5. To visualize cytoskeleton of HASMCs, F-actin stress fibers were stained with TRITC-conjugated phalloidin. After mounting, images were obtained using an inverted fluorescence microscope to reflect cell contraction.6. HASMCs were group into6:blank control, AngⅡ+Ang(1-7), AngⅡ+IRB, Ang Ⅱ+Y-27632. The key gene ARHGEF、RhoAGTP、ROCK2mRNA expression were measured by real-time PCR.7. Western blot was used to test the expression of moesin, substrate of ROCK2in defferent time pionts.8. Western blot was to measured the phosphorylation level of moesin, substrate of ROCK2in defferent groups.9. All data are represented as means±s, Multiple comparisons were analyzed using one-way analysis of variance (ANOVA) with the Statistical Package for the Social Sciences (SPSS)13.0after homogeneity test of variance. When it is heterogeneity of variance, correcting F-Test was used, and Dunnett T3was to do multiple comparisons. Differences were considered to be statistically significant when p<0.05.四、Result1. Under an inverted light microscope, single cell was a polygon or spindle shape. When confluent, they displayed the typical "clique" or "hill and valley"’ appearance. In pilot studies, Cytoplasm of HASMCs were stained to green by immunofluorescence for a-smooth muscle actin (α-SMA) and had a degree of purity of≥98%.2. Collagen gel assay was utilized to confirm cell contraction. The surface area of defferent groups(%):82.56±1.85,42.24±4.38,75.62±1.40,67.95±1.81,76.16±4.46,78.23±2.33(the initial area was100%). Obviously, The surface area was inversely proportional to the degree of cell contraction. So, Ang Ⅱ could induce the contraction of HASMCs significantly (p<0.001), Ang-(1-7) could inhibit the contraction,(p <0.05); Both of their receptor inhibitors could block their effects. Y-27632, the inhibitor of Rho/ROCK signal pathway produced a stronger inhibitory effect than Ang-(1-7) and irbesartan on thecontraction of HASMCs(p<0.05).3. Primary cultured HASMCs developed a fusiform shape, with a regular arrangement of actin stress fibers, the cytoskeletal reorganization triggered by Ang Ⅱ (10-/M,60min) in HASMCs typically stained with fluorophore-conjugated phalloidin, indicated a significant increase in the number and density of stress fibers and the cells looked tabular and had high tension compared with the control. The Ang Ⅱ-induced increase in the action stress fiber formation was partially reversed by treatment with Ang-(1-7), Y-27632, or irbesartan.Espeacially after Y-27632treatment, the number of stress fibers decreased significantly. The cells looked filamentous and has lower tension.4. Real-time PCR:Ang Ⅱ could activated the Rho/ROCK signal pathway, the expression of gene ARHGEF1, RhoAGTP, ROCK2mRNA increased significantly (p <0.05), and Ang-(1-7), IRB and Y-27632could block its effect partially(p<0.05).5. Western Blot:At the concentration of10-7M of Ang Ⅱ, the level of moesin phosphorylation increased significantly after5min simulated by Ang Ⅱ. this increase reaches to its maximum in15min and then the level decreased as the time went.6. Western Blot:Greyscale ratio of P-moesin/moesin were:0.72±0.03,1.48±0.03,0.67±0.03,0.91±0.03,0.61±0.01,0.83±0.02. the expression of phosphorylated moesin increased after Ang Ⅱ treatment. Ang-(1-7), IRB and Y-27632could block its effect partially(p<0.05). Y-27632treatment produced a stronger inhibitory effect than irbesartan on,(P<0.05).五. ConclusionOur data indicate that Ang Ⅱ induces the contraction of HASMCs by AT-1R, which could be inhibited partially by Ang-(1-7) through down regulation of the Rohm/ROCK signal pathway via Mas. |
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