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。

心肌纤维化（cardiac fibrosis, CF）是大多数心血管疾病的一个共同的病理变化，如心肌梗塞、心脏肥大等，最终诱发心力衰竭（heart failure, HF）。心脏成纤维细胞（cardiac fibroblasts, CFs）激活转化为肌成纤维细胞（myofibroblasts, myoCFs）被认为是心肌纤维化的关键步骤，其导致的细胞外基质蛋白沉积（extracellular matrix, ECM）被认为是心肌纤维化的基础。附子作为治疗心力衰竭症状的关键药物，其水溶性强心碱去甲乌药碱（Higenamine, HG）被研究的最多。在心血管领域，各研究发现HG可以治疗心律失常、抗凋亡和抗氧化应激，有正性肌力和正性频率等作用。然而，目前尚无HG干预心脏成纤维细胞激活从而抑制心肌纤维化的研究。在此我们体内采用TAC手术以及多柔比星建立心脏纤维化模型和ISO诱导心室重构模型探讨HG对心脏功能、心肌细胞肥大及心肌纤维化的影响，发现HG可以明显抑制小鼠心功能障碍、心脏肥大以及小鼠病理性心室重塑。体外外源性加入TGF-β1 刺激CFs 转化建立FMT细胞模型，探讨HG对心脏成纤维细胞激活的作用，RT-PCR提示HG干预之后可以明显抑制ACTA2，Col I, Col III和Fn mRNA基因的表达。Western blotting和细胞免疫荧光提示表型蛋白α-SMA，通路蛋白 P-Smad 表达增加。用血清诱导FMT，并刺激 MFBs 增殖和迁移，经HG预处理细胞后，发现MFBs的迁移和增殖不能被HG抑制。探讨乙酰化去甲乌药碱对心脏成纤维细胞激活以及ISO诱导的心脏纤维化的影响，结果显示AH在体内外均能抑制成纤维细胞的激活，改善心脏纤维化。此外，我们探讨HG对血管平滑肌细胞增殖、迁移、凋亡以及新生内膜增生的影响，以及HG对AngⅡ诱导心肌细胞肥大的影响。本课题研究结果揭示了HG与经乙酰化改造后的HG在体内外均能抑制心肌间质纤维化，改善心脏功能；同时发现HG 抑制心脏成纤维细胞激活的机制与其已知的在心肌细胞上作用于β2-AR 信号无关，而是通过调节TGF-β/Smad 信号通路抑制心脏成纤维细胞的激活及基质蛋白的沉积，从而抑制心肌纤维化。此外，HG可以抑制AngⅡ诱导的心肌细胞肥大。并抑制血管平滑肌的异常增殖、迁移以及诱导平滑肌细胞凋亡，起到延缓血管重塑的病理进程