去甲乌药碱通过抑制心脏成纤维细胞激活抗心肌纤维化的作用及机制研究

Cardiac fibroblasts comprise the major number of all the cells in the healthy adult heart, Over the last decade, the pleiotropic roles of fibroblasts in cardiac biology and disease have been studied.Extensively, The differentiation from fibroblast to myofibroblast marked by the overexpression of alpha-smooth muscle actin(α-SMA) is promoted by transforming growth factor (TGF)-β, cytokines, the ECM, and other growth factors, this phenotype change also is thought to be essential for cardiac fibrosis, during the activation of cardiac fibroblast by TGFβ stimulation, TGFβ/Smad pathway is the canonical signaling..Higenamine, known as the main activated water-soluble compound of Chinese herd “Fuzi”, was found by our previous work and other labs as β2 agonist with the function of anti-apoptosis through β2/PI3K/AKT signaling in cardiomyocytes, more recently, we found Higenamine can attenuate cardiac fibrosis in TAC mice, reverse the overexpression of α-SMA which induced by TGFβin cardiac fibroblast, and this function is a new pharmacological target that independent on its well knownβ2 agonist like function..To find out more details of the wide used Chinese herb, we prepare to load normal and 1/10 of normal dosage of Higenamine in myocardial infarction mice, after 1 or 8 weeks, cardiac fibrosis markers and related pathway protein will be test to demonstrate the anti-fibrosis function..In vitro experiment will be done in primary cultured cardiac fibroblast, 1) To make sure the anti-fibrosis function of the compound through a new, independent of its canonicalβ2 target, the reverse of transfer fibroblast to myofibroblast triggered by Higenamine will be test in the condition of with or without inhibitor or knock down of its well knownβ2/PI3K/AKT signaling, 2) plasmids that overexpress of protein involved in TGFβ/Smad with constitutively activated mutant will be transfected in CFs to mimic protein phosphorylation, knock down express of Smad7 will also be induced to define the precise new target of anti-fibrosis of Higenamine..These findings will provide strong evidence of a whole new anti-fibrosis function of Higenamine pathologic heart, and also may have potential to develop new candidate for therapy of heart disease.

心脏成纤维细胞已经成为心脏研究的热点领域；其活化转化为肌成纤维细胞被认为是心肌纤维化的核心病理过程，TGFβ/smad信号通路是其经典分子机制；我们及同仁的前期研究证明中药附子主要活性成分去甲乌药碱（Higenamine）通过激活β2/PI3K/AKT通路抗心肌细胞凋亡；最近，课题组发现其体内减少TAC模型小鼠的心肌纤维化，体外抑制TGFβ1诱导的心脏成纤维细胞激活（α-SMA的表达增加），且此作用不能被β2受体抑制剂阻断，提示其独立于已知药理机制的新靶点；在此基础上，我们拟探讨1）体外采用转染组成型激活突变体激活相关信号蛋白的方法探讨其作用靶点，2）体外原代培养心脏成纤维细胞经药物处理后增殖、分化及纤维化相关基因及蛋白表达的变化，3）体内采用小鼠心梗后心肌纤维化模型探讨去甲乌药碱在体内的抗心肌纤维化功能，以期发现该单体抗心肌纤维化的新作用靶点，结合前期结果开发心血管多靶点的先导化合物。