映山红花总黄酮抗缺血性心肌损伤作用的UT受体-RhoA-Rho激酶信号通路阻断机制

UT receptor, the specific receptor for urotensin II, is involved in the pathological processes of a number of cardiovascular diseases. As same as RhoA/Rho-kinase (ROCK) signal pathway, UT receptor might become a novel target in cardiovascular research. But so far, the role of the blockade of UT receptor in myocardial ischemic injury has not been fully ascertained. Total flavones of rhododendra flower (TFR) is shown to have significant protective effect against myocardial ischemic injury. However, the protective mechanism remains poorly understood. Based on our previous studies, by using UT receptor antagonist and the technique of siRNAs, the role of the blockade of UT receptor on myocardial ischemic injury as well as its relationship with RhoA/ROCK signal pathway will be studied in the project. Our study is going to focus on the study of protective mechanism of TFR against ischemic myocardial injury. By using the radioligand competition assay, vesselexperiment, the measurements of vascular function and contraction of myocardial cells and multiple models of ischenic myocardial injury, together with UT receptor antagonist, inhibitor of ROCK and knockdown of UT receptor or ROCK by siRNA technique, we try to elucidate that TFR exerts protective effect against ischemic myocardial injury and amelioration in ischemia-induced ventricular remodeling through the mechanism of blockade of UT receptor as well as RhoA/ROCK signal pathway. The research results are expected to promote the understanding in the pathological mechanism of myocardial ischemic injury, and provide an enlightenment for the study of traditional Chinese medicine against myocardial ischemic injury.

UT受体，即urotensin Ⅱ受体，参与了多种心血管疾病的病理过程，与RhoA/Rho激酶(ROCK)信号通路一样，可成为心血管研究中新的靶点，但迄今未完全确定UT受体阻断在心肌缺血性损伤中的作用。映山红花总黄酮(TFR)有抗缺血性心肌损伤作用，但机制不明确。本项目拟在前期工作基础上，通过UT受体拮抗剂及siRNA技术，研究确定UT受体阻断对心肌缺血性损伤的保护作用及其与RhoA/ROCK信号通路的关系；重点研究TFR抗缺血性心肌损伤作用机制，在放射配基竞争结合试验、血管实验、心肌细胞收缩性测定及多种缺血性心肌损伤模型上，结合UT受体阻断药、ROCK抑制剂以及下调ROCK或UT受体的siRNA技术，研究阐明TFR通过UT受体及RhoA/ROCK信号通路阻断机制抗缺血性心肌损伤，并改善缺血性性心室重构。本研究预期成果可促进对缺血性心肌损伤机制的认识，为中药抗缺血性心肌损伤的研究提供启示。

人urotensin Ⅱ（hUII）及其受体（UTR）组成的urotensin系统对机体的生理功能有着重要的调节作用，参与了心肌缺血性损伤等心血管疾病的病理过程。hUⅡ是迄今发现的收缩效应最强的血管肽，可通过UTR收缩冠状动脉，减少冠脉血流量，造成心功能的抑制和心肌损伤。因此，UTR与RhoA-Rho激酶（RhoA-ROCK）信号通路一样，可成为心血管系统研究中新的靶点，但迄今未完全确定UTR阻断在心肌缺血性损伤中的确切作用。映山红花总黄酮（TFR）为映山红花的有效部位，有明确的抗缺血性心肌损伤作用，但机制不明确。本项目通过UTR拮抗剂SB-710411及siRNA技术，研究确定了UTR阻断可舒张大鼠冠状动脉及体循环系统的血管，并对心肌缺血性损伤产生保护作用。重点研究了TFR抗缺血性心肌损伤作用机制。利用125I-hUII放射配基竞争结合试验、血管功能测定实验、细胞培养、心肌细胞收缩功能测定及多种缺血性心肌损伤模型，结合UTR阻断药、ROCK抑制剂及下调ROCK或UTR的siRNA技术，阐明了TFR可阻断UTR，抑制缺氧/再给氧损伤和hUII诱导的大鼠心肌细胞收缩频率和收缩幅度的降低。与UTR抑制剂SB-710411一样，TFR在UTR-siRNA在体转染大鼠上的抗心肌缺血再灌损伤作用有明显的减弱，表明TFR抗大鼠心肌缺血再灌损伤作用与其阻断UTR有关；TFR可通过抑制RhoA-ROCK信号通路来舒张大鼠冠状动脉等血管和发挥抗心肌缺血、缺氧性损伤作用。UTR-siRNA转染导致的UTR表达下调明显地减弱TFR对缺血性损伤激活的大鼠心肌RhoA-ROCK通路及其后续效应分子p-MLC蛋白表达的抑制作用，表明TFR抑制大鼠心肌RhoA-ROCK通路是继发于UTR阻断。因此，TFR抗大鼠心肌缺血性损伤作用机制为阻断UTR，继而导致了RhoA-ROCK通路的抑制。TFR通过UTR-RhoA-ROCK信号通路的阻断还可改善大鼠慢性缺血性损伤引起的心室重构。此外，在项目计划研究内容完成的基础上，利用H2S合酶CSE基因敲除小鼠和膜片钳技术，分别探讨了TFR抗心肌缺血性损伤作用的CSE/H2S介导的RhoA-ROCK通路抑制机制和促进大鼠心肌细胞内向整流钾通道和SKCa钾通道开放的机制。本项目研究成果可促进对缺血性心肌损伤机制的认识，为中药抗缺血性心肌损伤的研究提供启示。

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