β3 -AR/RhoA-ROCK1通路介导心肌重构的机制研究

Three subtypes of β-adrenoceptor are involved in the sympathetic nervous system, which plays an important role in the biology activity of cardiovascular system. The newfound β3-adrenoceptor is different from β1 and β2-adrenoceptor in its structure and function. Stimulation of the β(3)-adrenoceptor (β(3)-AR) is thought to be a valuable approach for the treatment of obesity, type 2 diabetes, heart failure, frequent urination, preterm labor, anxiety and depression. In chief, β(3)-AR mediates a negative inotropic effect.Therefore, the β(3)-AR is recognized as an attractive target for drug discovery. After ?(3)-AR cloning in the late 1980s, convincing evidence for ?(3)-AR expression and function in cardiovascular tissues recently initiated a reexamination of their involvement in the pathophysiology of cardiovascular diseases. Their upregulation in diseased cardiovascular tissues and resistance to desensitization suggest they may be attractive therapeutic targets. In cardiac ventricle, their contractile effects are functionally antipathetic to those of ?(1/2)-AR; in normal heart, ?(3)-ARs may mediate a moderate negative inotropic effect, but in heart failure, it may protect against adverse effects of excessive catecholamine stimulation by action on excitation-contraction coupling, electrophysiology, or remodelling. Some research suggest that β(3)-AR can promote myocardial remodeling and deteriorate cardiac function by increasing neurohormone and activating cytokines. However, the exact role of β(3)-AR in myocardial remodeling is not clear yet. Myocardial remodeling is the main pathogenesis of heart failure, whether β3-adrenoceptor deteriorate cardiac function by promoting myocardial remodeling is a question. Rho / Rock signal transduction pathway is closely linked with the myocardial remodeling development.Rho kinase inhibitor fasudil can significantly reduce myocardial fibrosis, inhibit ventricular remodeling and the inhibition of TGF-β2 production is one of the important mechanisms. In vascular tissue, β3-adrenoceptor can cause a effect of vasodilatation through the β(3)-AR/Rho-ROCK1 signal pathway. Maybe, the main signaling pathway that associated with β(3)-AR promoting myocardial remodeling is Rho/Rock1 pathway. Our study is to myocardial cells in vitro and pressure afterload heart failure wide-type and ROCK knockout(ROCK-/-) mice model as the research object, to investigate the effect of β(3)-AR on the myocardial remodeling, and to probe into its mechanism for provide theory to chronic heart failure by using β(3)-AR agonist and β(3)-AR inhibitor,and by blocking the Rho/Rock pathway. Thus, prospective studies in animals and patients at different stages of heart failure should lead to identify the best therapeutic window to use ?(3)-AR antagonists.

交感神经系统主要通过其3个β肾上腺素能受体介导心脏的生物活动。新近发现的β3-AR与β1-AR和β2-AR有着明显不同的作用特点，表面的作用是通过负性变力效应恶化心衰进程。但个别研究显示，β3-AR可能会通过增加循环中神经激素及大量细胞因子的活化加速心肌重构恶化心衰，但具体的信号通路不明确。RhoA是参与心肌重构的起分子开关作用的小分子G蛋白，是引起细胞骨架重组、细胞肥大的重要分子，以激活下游激酶ROCK1通路介导上述效应。在血管组织中，?3-AR/RhoA-Rock1模式可引起血管舒张效应，但在心肌细胞中，这一通路是否参与?3-AR的心脏重构作用呢？本项目以培养心肌细胞和压力负荷型野生型及ROCK-/-型心衰小鼠为研究对象，分别通过激动和阻断β3受体效应，并相应阻断Rho激酶通路，以观察β3-AR/RhoA-ROCK1通路在心肌重构中的作用，为心衰的生物学代偿机制及药物治疗提出新的展望。

交感神经系统调节心脏的生物活动主要是通过β肾上腺素能受体的3个亚型进行的。而在这三个亚型中，β3-AR的作用特点与β1-AR、β2-AR有着明显的不同，因此针对β3-AR在心肌重构中的作用机制进行研究对于全面认识心衰的发生、发展有着十分重要的意义。β3-AR作为一种新近发现的受体亚型，其在心衰进展中的作用机制尚未在学界形成共识。本项目以构建压力负荷型野生型及ROCK-/-型心衰小鼠模型、体外培养心肌细胞和心肌成纤维细胞为研究对象，分别通过激动和阻断β3-AR效应，并相应阻断Rho激酶通路，以观察β3-AR/RhoA-ROCK1通路在心肌重构中的作用。分别利用TUNEL法及AnnexinV-FITC/PI双染法检测心肌细胞凋亡率， WST-1法检测成纤维细胞增殖情况，Western blot和RT-PCR检测心肌细胞肥厚相关因子β-MHC和c-myc、成纤维细胞纤维化中I型和III型胶原、β3-AR及Rho激酶通路相关分子蛋白和mRNA的表达。结果显示：与正常组相比，心肌肥厚组β3-AR、RhoA、Rho激酶的蛋白及mRNA在心肌细胞及成纤维细胞中均显著表达，与心肌肥厚组比较，BRL组RhoA、Rho激酶、β3受体表达升高，SR组RhoA、Rho激酶、β3受体表达显著下降。针对β3-AR在心脏组织中表达量极低这一现实困境，我们创新性地给予携带β3-AR基因片段的慢病毒进行转染。同时，在β3-AR过表达的水平上，进一步探索β3-AR介导心室重构的机制。我们发现：在体外β3-AR过表达后，也可以通过激活MAPK通路和TGF-β/Smad信号通路介导心肌肥厚和心肌成纤维细胞纤维化，促进心肌重构，参与心衰的进展。上述β3-AR在心室重构中的作用机制，为临床治疗心衰及心室重构等疾病提供了新的思路和方法，为进一步的研究提供相关的理论基础。