黄芪丹参药对通过保护细胞骨架改善高血压肾损害的实验研究

The deficiency of Qi and blood stasis is one of the main pathogenesis of the hypertensive nephropath. The change of cytoskeleton is crucial in the pathology of the change of histomorphology and function of renal arterioles, glomerular and tubular, the renal insufficiency and ESRD. This study was to analyze the genome profile of rat renal arteriolar endothelial cells, podocytes and tubular epithelial cells treated with Ang II and Earle's buffer (lack of tissue ischemia, nutrition), to assess the changes and mechanism of cytoskeleton of the cells in hypertensive condition. The spontaneously hypertensive rats (SHR) are to be administrated with calycosin-tanshinoneⅡA (double-drugs compatibility of Salvia miltiorrhiza and Astragalus membranaceus) according to "Buqi Huoxue" Chinese prescription and calycosin-tanshinoneⅡA (C-T) combined with valsartan, to observe the effect of C-T on cytoskeleton of aorta and intrinsic kidney cells in hypertension and the regulation between them mutually, thus, to conclude reno-protective efficacy of the C-T in uncontrolled hypertensive rats and rats of effective control of blood pressure and to understand drug targets and pharmacodynamic mechanism.We hope to introduce new ideas for hypertensive target organ protection, discuss and make clear the mechanism of "Buqi Huoxue" Chinese prescription and promote the professional acceptation of Salvia miltiorrhiza and Astragalus membranaceus in foreign scholars.

气虚血瘀是高血压肾损害的主要病机之一，细胞骨架的改变，在高血压肾损害病理过程中至关重要，与肾小动脉、肾小球、肾小管结构和功能的改变、肾功能不全、ESRD等密切相关。本研究拟建立分析大鼠肾小动脉内皮细胞、足细胞和小管上皮细胞分别在Ang II和Earle's平衡盐（组织缺血、营养缺乏）作用下的基因表达谱的改变，推断各细胞细胞骨架在高血压状态下的改变和机制；以补气活血类中药黄芪丹参有效单体（毛蕊异黄酮-丹参酮ⅡA）药对、药对联合缬沙坦分别对自发性高血压大鼠（SHR）进行干预，评估大鼠高血压程度和大血管（主动脉）损害、肾脏损害的各自进展和互相影响，并观察细胞骨架在其中的作用；探讨药对分别在未控制高血压和在血压得到有效控制的情况下的肾脏保护作用，及其作用靶点和药效机制，为高血压靶器官损害的治疗提供新的思路，对明确补气活血类中药的作用机制和国外学者对黄芪丹参药对的认可有重要的意义。

高血压肾损害的过程中，气虚血瘀贯穿始终，证属本虚标实。随着高血压的进展，高灌注肾单位逐渐减少，缺血性损害逐步占据主导地位。本项目应用补气活血药对“黄芪-丹参”用于高血压肾病的体内、体外（体外以黄芪-丹参有效单体毛蕊异黄酮-丹参酮ⅡA配伍）研究，证实黄芪-丹参药对的主要靶细胞为肾小管上皮细胞和肾小球内皮细胞，可显著降低大鼠尿β2-MG、NAG、mALB和血清AngⅡ水平，改善肾血流、降低血流阻力指数（RI），改善肾脏组织形态，减轻系膜区增生、足突融合和肾间质小灶纤维化，提高肾脏eNOS的合成，降低TGF-β1、GTGF分布。黄芪-丹参药对与缬沙坦共同用药的情况下，并未表现出协同作用；但在缬沙坦有效降压的情况下，表现出更好的肾脏保护作用。基因芯片、转录组测序提示黄芪-丹参药对在Ang II作用下的基因表达谱的改变富集中于ATP依赖DNA解旋酶的活动，选择性mRNA剪接，有丝分裂细胞周期的G2/M期转变，调节糖质新生，胰岛素受体信号通路的调控等；黄芪-丹参药对在缺血作用下的基因表达谱的改变富集于核苷酸代谢，辅助因子和维生素的代谢，脂质代谢，糖的生物合成和代谢，能量代谢等。实时荧光定量PCR和Western Blotting结果提示黄芪-丹参药对的作用靶点集中于Rho/ROCK通路和AMPK通路，可显著降低Vinculin、Rock、FAK、RhoA、PI3K，升高AMPK。其中，黄芪单独应用对RhoA和PI3K表现出较强的调节，丹参单独应用对AMPK表现出较强的调节，黄芪-丹参药对和黄芪-丹参-缬沙坦联用对Vinculin和FAK表现出较强的调节作用。本研究首次明确了黄芪-丹参药对改善高血压肾损害的靶细胞和分子靶点，基本明确了黄芪-丹参药对的作用机制。