基于非基因组效应研究滋肾活络方克服醛固酮逃逸对高血压LVH疗效的影响及机制

Aldosterone breakthrough is the key to ACEI’s reversion of reduced efficacy on hypertensive LVH. Overall intervention of combined traditional Chinese and western medicine is a safe and effective strategy concerning aldosterone breakthrough. MR-mediated non-genomic effect is the main pathway of remodeling of organs brought about by aldosterone breakthrough. Previous studies show that Zishen Huoluo Formula can improve the effect of ACEI on hypertensive LVH without affecting the draining-potassium function of Aldosterone. Thus a hypothesis is proposed by this project that Zishen Huoluo Formula, based on the theory of “coordination between the heart and kidney”, may influence non-genomic effect by intervening MR membrane translocation and localization, regulate EGFR/ERK signal path, overcome aldosterone breakthrough and improve the effect of ACEI on hypertensive LVH. By using aldosterone-induced cells model and SHR model, and employing methods like siRNA, PCR, Western-blot and Co-IP, this project tries to observe systematically the effect of Zishen Huoluo Formula on ACEI’s treating hypertensive LVH from the whole and cell level as well as gene and protein level；to probe into the influence of the Formula on membrane translocation ,localization and downstream EGFR/ERK signal pathways to uncover its molecular mechanisms in intervening non-genomic effect and overcoming aldosterone breakthrough. The biological basis will also be elucidated as how it helps to nourish kidney and clear heart-fire and balance heart-yang and kidney-yin. Therefore, a new perspective may be provided for the overall intervention of combined traditional Chinese and western medicine on the treating of hypertension and cardiovascular complications.

醛固酮逃逸是ACEI逆转高血压LVH疗效降低的关键，中西医结合整体干预是克服醛固酮逃逸的安全有效策略。盐皮质激素受体（MR）介导的非基因组效应是醛固酮逃逸致有害器官重塑的主要途径。前期研究发现滋肾活络方能提高ACEI逆转高血压LVH疗效，且不影响醛固酮排钾功能。提出假说：基于“心肾相交”理论的滋肾活络方可能通过干预MR的膜转位和定位，影响非基因组效应，调节EGFR/ERK信号通路，克服醛固酮逃逸，提高ACEI逆转高血压LVH疗效。采用醛固酮诱导细胞模型、SHR，运用siRNA、PCR、Western-blot、Co-IP等方法，从整体与细胞、基因与蛋白水平系统观察滋肾活络方对ACEI逆转LVH疗效的影响；对MR膜转位、定位及下游EGFR/ERK通路影响，揭示其干预非基因组效应，克服醛固酮逃逸的机制。阐释其补肾清心，交通心肾的生物学基础。开拓中西医结合整体干预高血压及心血管并发症的新思路。

醛固酮逃逸是ACEI逆转高血压左室肥厚（LVH）疗效降低的关键，盐皮质激素受体（MR）介导的非基因组效应是醛固酮逃逸致有害器官重塑的主要途径。体内实验以16周龄自发性高血压大鼠（SHR）为观察对象，培哚普利干预12w血清醛固酮回升至接近给药前水平，提示存在醛固酮逃逸现象。滋肾活络方联合培哚普利干预，可抑制醛固酮水平回升，降低SHR血压水平，改善心室功能，抑制胶原合成及心肌纤维化进程；增加MR与Cav-1的相互作用，抑制MR与striatin的相互作用；抑制心肌组织pERK1/2、pEGFR蛋白表达,效果优于单用培哚普利。体外实验建立醛固酮诱导心肌细胞和心肌成纤维细胞（CFs）模型，结果显示醛固酮诱导心肌细胞肥大和成纤维细胞增殖自诱导5min即开始，30min达到一定强度，符合非基因组效应特点。滋肾活络方含药血清干预可抑制醛固酮诱导的心肌细胞肥大和CFs增殖及胶原合成，抑制striatin和MR相互作用，增加Cav-1和MR的共定位，抑制EGFR和ERK磷酸化。siRNA 技术沉默striatin能抑制醛固酮诱导的心肌细胞肥大和CFs增殖，抑制EGFR和ERK的磷酸化及膜MR表达；siRNA联合组具有相同效应，且能抑制EGFR和ERK mRNA的表达。应用Filipin干扰Cav-1结构可抑制醛固酮诱导的心肌细胞肥大和CFs增殖，及膜MR表达；Filipin联合组具有相同效应，且能抑制EGFR、ERK mRNA的表达及EGFR和ERK的磷酸化激活。提示滋肾活络方可通过影响striatin和Cav-1介导的MR膜转位和定位，调控EGFR/ERK信号通路的磷酸化激活，抑制心肌肥厚和纤维化，这可能是其克服醛固酮逃逸，干预其非基因组效应，提高培哚普利逆转高血压LVH疗效的机制之一。