尾加压素II促进糖尿病心肌纤维化的机制研究

Cardiac fibrosis is a key pathological feature of diabetic cardiomyopathy. However, its pathogenesis remains to be clarified. Urotensin II (UII) is a potent vasoconstrictive peptide. Its gene expression is associated with susceptibility to type 2 diabetes. We have demonstrated that UII could stimulate the phenotypic differentiation of vascular fibroblasts and promote the development of vascular fibrosis and cardiac fibrosis in a paracrine / autocrine manner. Therefore, in this project, we will use in vivo models of type 2 diabetes and cardiac microvascular endothelial cells in vitro to study UII effects and signaling mechanisms. Our aim is to investigate the relationship between the expression of endogenous UII and its receptor and the acquisition of endothelial-to-mesenchymal transition (EndMT), including expression of endothelial markers CD31, E-cadherin, and fibroblast markers fibroblast specific protein 1 (FSP1) and α-smooth muscle actin (α-SMA). We also aim to investigate the relationship between the changes in expression of endogenous UII and its receptor and progression of cardiac fibrosis by evaluating levels or activities of collagen, fibronectin, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP1). The focus will be on the underlying signaling mechanisms of the upregulation of UII and its receptor in response to high glucose and advanced glycosylation end products and the molecular mechanisms of EndMT and matrix synthesis induced by UII. We aim to test whether UII may contribute to cardiac fibrosis in diabetic cardiomyopathy by stimulating EndMT in a paracrine/autocrine manner. The results of this study may provide new insights into the pathogenesis of cardiac fibrosis in diabetic cardiomyopathy and reveal novel therapeutic strategies and targets for its prevention and treatment.

糖尿病心肌病的重要病理特征之一是心肌纤维化，其机制仍待阐明。尾加压素II（urotensin II，UII）是一种和2型糖尿病易感性相关的缩血管活性肽。本项目在我们发现UII以旁/自分泌方式刺激成纤维细胞表型转化、促进血管和心肌纤维化基础上，拟于2型糖尿病动物模型和体外培养的心肌微血管内皮细胞上，以内皮细胞标志CD31、上皮型钙粘蛋白下调，成纤维细胞标志FSP1、α-SMA等上调作为内皮细胞向间质成纤维细胞表型转化（EndMT）指标，以胶原、纤连蛋白、MMPs/TIMP1表达与活性变化作为纤维化相关指标，研究内源性UII及受体变化和EndMT、心肌纤维化的关系；研究高糖和糖基化终产物对UII及其受体的上调机制；UII诱导EndMT转化促进基质合成的分子机制，以论证UII作为心肌旁/自分泌因子，通过刺激EndMT促进糖尿病心肌纤维化发病的假说，以揭示该病的新机制，为其防治提供新思路和新靶点。

目的：在离体培养的心肌微血管内皮细胞(CMEs)及2型自发性糖尿病小鼠模型上，探讨了尾加压素II（urotensin II, UII）在内皮间质转化中的作用和机制、UII受体阻断对心肌纤维化的影响，Smad2/3信号在UII刺激内皮间质转化中的作用、以及血管外膜成纤维细胞（AFs）激活中的作用，然后探讨了UII可否介导同型半胱氨酸（HCY）、糖基化终产物（AGEs）对AFs的激活作用、UII阻断对血管钙化的影响。方法：细胞分别采用新生大鼠CMEs、成年大鼠胸主动脉AFs；自发性2型糖尿病模型采用KK/Upj-Ay/J小鼠，UII基因敲除小鼠模型采用TALEN技术制备；以成纤维细胞标志FSP-1、α-SMA上调、内皮细胞标志CD31、VE钙粘蛋白下调为内皮间质转化标志，UII、UII受体UT、FSP1、α-SMA、VE钙粘蛋白、p-smad2/3、CD31、TGF-β、CTGF变化分别采用免疫组化、western blot或实时定量PCR检测。采用放免法/ELISA 检测血浆UII、TGF-β1或细胞分泌MCP-1和IL-6水平。结果：UII刺激CMEs激活Smad2/3信号，上调α-SMA，而下调CD31、VE钙粘蛋白，阻断UII受体或沉默Smad2/3信号能抑制该作用；糖尿病小鼠血糖升高、体重增加，心肌纤维化明显，UII/UT上调，伴FSP-1、α-SMA上调、CD31和VE钙粘蛋白下调，p-Smad2/3、CTGF和TGF-β1上调，伴肾纤维化。UT受体阻断剂urantide能逆转上述变化，减轻心、肾纤维化；UII基因敲除小鼠繁殖力明显减弱，需进一步验证；在AFs上，UII促进Smad2/3激活, 促进胶原合成、细胞迁移, 该作用可被UII受体阻断剂以及沉默Smad2/3基因抑制。最后发现HCY、AGEs可促进AFs上UII/UT表达；UII受体阻断剂能够抑制HCY促细胞增殖和迁移以及AGEs诱导的α-SMA上调，抑制血管钙化。结论：UII能够刺激心肌微血管内皮间质转化，促进糖尿病心肌病心肌纤维化和肾纤维化发展，其中Smad2/3信号可能介导了UII的促内皮间质转化作用；UII还参与了HCY、糖基化终末产物刺激AFs活化的作用，而阻断UII能够逆转内皮间质转化、减轻纤维化和血管钙化发展，提示阻断UII可能是防治糖尿病心肌病、心肌纤维化、血管钙化的新策略之一。