从调控RAS两轴平衡角度探讨鹿角方防治慢性心衰的分子机制与物质基础

Lujiao Fang which function was strengthening heart by reinforcing kidney has been demonstrated in the previous studies that it could not only improve the symptoms of patients and function of heart with chronic heart failure, but also partially reverse cardiac hypertrophy, and could significantly reduce AngⅡ, protein expression of ACE and AT1 and aldosterone content. All the improvements have indicated that partly regulation of balance of ACE / AngⅡ/ AT1 and ACE2 / Ang (1-7) / Mas axes may reverse the cardiac remodeling intervened by Lujiao Fang. Therefore, ACE / AngⅡ / AT1 and ACE2 / Ang (1-7) / Mas axis of chronic heart failure will be the target in the current study using heart failure model induced by aorta coarctation, in which the marks are pharmacodynamics, key indicators of the general two axes of RAS system with Lujiao fang interventions, and perindopril as a control. This study will be explored the intervention of Lujiao Fang on RAS system during chronic heart failure and the relevant mechanism and effect links. Fingerprint Spectrum of multi component analysis combined with HPLC-MS in order to clear molecular mechanism of Lujiao Fang recovery RAS system balance through the multi component analysis selection effect substance basis. This study will clarify new molecular mechanisms and material basis of chronic heart failure treated by Lujiao Fang, revealing scientific connotation of “ heart and kidney intersection, treatment from the kidney to the heart”.

前期我们发现具有补肾强心功能的鹿角方在改善慢性心衰（CHF）患者临床症状、心功能同时，还能部分逆转心肌肥厚，且能显著降低AngⅡ、ACE和AT1的蛋白表达、醛固酮含量，进而提示鹿角方可能通过调控RAS恢复ACE/AngⅡ/AT1和ACE2/Ang(1-7)/Mas轴平衡，从而逆转心肌重构。藉此，本研究以RAS系统ACE/AngⅡ/AT1和ACE2/Ang(1-7)/Mas轴为切入点，以腹主动脉缩窄致心衰模型为研究对象，以鹿角方干预，以培哚普利作对照，主要对包括一般药效学，RAS系统两轴关键环节指标等进行观察，研究鹿角方对CHF时RAS系统的改善作用及其效应环节，明确其恢复RAS系统平衡的分子机制；同时结合色谱质谱技术分析血清、心、肾组织中成分化学指纹谱，通过主成分分析法筛选药效物质，明确其物质基础。本研究将阐明鹿角方治疗CHF新的分子机制及物质基础，揭示其心肾相交、从肾治心的科学内涵。

在治疗慢性心衰（CHF）方面，中医药强调“心肾相交，温肾强心，恢复心肾阴阳平衡”与维持RAS平衡的理念一致。本课题从调控RAS两轴（ACE/AngⅡ/AT1轴、AC2E/Ang(1-7)/Mas轴）平衡角度研究具补肾强心功能的鹿角方防治CHF的分子机制与物质基础，揭示其心肾相交、从肾治心的科学内涵。.研究如下：（1）基于LC-MS/MS，建立大鼠吸收入血成分、心肾组织分布成分分析方法。结果表明，鹿角方中与改善心肾功能相关的成分马钱苷、柚皮苷、橙皮苷、朝霍定A、朝霍定B、朝霍定C、淫羊藿苷、补骨脂素、异补骨脂素、宝霍苷Ⅰ、红景天苷、莫诺苷、特女贞苷均能入血；给药2h后，大部分成分在肾的分布大于心的分布。（2）建立腹主动脉缩窄致CHF大鼠模型，以低、中、高剂量鹿角方干预，从血流动力学、心超、左心室重量指数、心肾HE、Masson染色及电镜超微结构评价心室重构、肾脏损伤，采用Elisa、RT-PCR、Western Blot、免疫组化法分析鹿角方对血浆、心、肾中RAS两轴关键环节指标的影响。结果表明，鹿角方可改善CHF大鼠心室重构，中、高剂量优于低剂量；中、高剂量可显著降低血浆中BNP、AngⅡ、肾素、ALD含量，下调肾脏中肾素含量，心肌中AngⅡ含量及ACE、AT1 mRNA和蛋白的表达；高剂量可显著升高血浆中Ang(1-7)含量，心肌和肾脏中Mas mRNA和蛋白的表达，并下调肾脏中AngⅡ、ALD含量，AT1 mRNA和蛋白的表达。（3）LC-MS/MS法分析不同剂量组鹿角方各成分在CHF大鼠血浆、左心室壁与右心室壁、左肾与右肾的含量。结果表明，鹿角方各成分在血浆及各组织中的含量分别随给药剂量升高而升高，与其改善CHF心室重构的药效作用呈正相关；给药2h后，补骨脂素、异补骨脂素在左心室壁的含量与右心室壁含量相当或大于右心室壁含量，其余分析成分在左心室壁的含量仅为右心室壁含量的38-74%，大部分成分在左肾与右肾的分布存在差异。.鹿角方以归经于肾为主，马钱苷等13种成分为其药效物质基础，口服吸收、组织分布行为较优的补骨脂素、异补骨脂素、马钱苷、莫诺苷、红景天苷为主要药效物质；鹿角方改善CHF心室重构、肾损伤的分子机制与调控ACE/AngⅡ/AT1轴、ACE2/Ang(1-7)/Mas轴关键环节指标维持RAS两轴平衡有关，发挥从肾治心作用与减少肾脏肾素、ALD分泌有关。