基于“脾主肌肉”研究苓桂术甘汤对大鼠慢性心力衰竭模型LCC-RyR的锚定调控机制

Chronic cardiac failure is a global public health problem. At present, process of chronic cardiac failure could be intervened by medications functioning on reversing ventricular remodeling and antagonizing neurohormone, but not effectively. Besides, the patients’ compliance and tolerance are affected by the side-effects of the medications. Therefore, the scholars at home and abroad commit themselves to explore new approaches. The latest researches shown that calcium induced calcium release (CICR) injury caused by degeneration anchoring of conjugates formed by L-type Ca2+ channel (LCC) on transverse tubules (TTs) of cardiac muscle cell and ryanodine receptor (RyR) on sarcoplasmic reticulum is one of the molecular mechanisms of chronic heart disease. Thus, anchoring of the conjugates might be a new target of treatment for cardiac failure. It is recorded that the spleen governs the muscles in Plain Questions - Treatise on Flaccidity Disease, and spleen disfunction fails to nourish the muscles and bones to act well in Plain Questions - Taiyin Yangming Theory. If the spleen governs the transformation and transportation well, the muscles are thick and powerful; if not, the muscles are week and debilitating. If the spleen dysfunction affects the heart, the debility of cardiac muscles could cause heart failure. Líng Guì Zhú Gān Tāng (Poria, Cinnamon Twig, Atractylodes Macrocephala and Licorice Decoction), recorded in Synopsis of Golden Chamber, has definite therapeutic effect for cardiac failure with the function of invigorating spleen and promoting urination, warming yang and dissolving rheum (fluid retention). Based on literature reviews and results of previous researches, we propose the hypothesis that Líng Guì Zhú Gān Tāng could treat cardiac failure by regulating the anchorage of LCC-RyR. The project will explore the acting mechanism on molecular level and enrich the scientific connotation of “the spleen governing the muscles” .

慢性心力衰竭是一个全球性公共卫生问题。目前采用逆转心室重塑、拮抗神经内分泌等药物治疗能一定程度干预慢性心衰进程，但疗效却不理想，且因药物副作用，影响患者依从性及耐受性。因此国内外学者致力于探索慢性心衰防治的新方法。最新研究表明：心肌细胞横管上的L型钙通道（LCC）和肌质网上的RyR受体形成的耦联体锚定退化，导致心肌细胞钙致钙释放过程损伤，是慢性心衰的分子机制之一。该耦联体的锚定可能为心衰治疗的新靶点。《素问•痿论》：“脾主身之肌肉。”《素问•太阴阳明论》：“脾病……筋骨肌肉皆无气以生，故不用焉。”脾气健运，肌肉丰盈有活力；脾病，肌肉痿缩不用。责之于心，则现心肌收缩无力，呈心衰诸症。苓桂术甘汤健脾利水、温阳蠲饮，对心衰的治疗疗效确切。我们通过文献复习及前期研究基础，提出苓桂术甘汤通过调控LCC-RyR锚定治疗慢性心衰的研究工作假说，拟从分子水平揭示其抗心衰的机制及丰富“脾主肌肉”的科学内涵。

背景：慢性心力衰竭是一个全球性公共卫生问题，由于目前的经典治疗方案疗效不甚理想，国内外学者致力于探索慢性心衰防治的新方法。中医认为：脾气健运，肌肉丰盈而有活力。脾病，肌肉痿缩不用。责之于心，则现心肌收缩无力，呈心衰诸症。《金匮要略》苓桂术甘汤健脾利水、温阳蠲饮，对心衰的治疗疗效确切。本研究提出“脾主心肌”及温脾蠲饮的苓桂术甘汤通过调控心肌兴奋收缩耦联的分子基础LCC-RyR之间的锚定治疗心力衰竭的假说，展开实验研究。方法：通过在4周内给予8次阿霉素腹腔注射(累积剂量为16 mg/kg)，建立了HF大鼠模型。随后，5、10、15 mL/kg/d给药，每天1次，连续4周。采用体重、心重指数(HWI)、心重/胫骨长比(HW/TL)、血清BNP水平检测LGZG对HF的影响；超声心动图测定心功能；H&E染色观察心肌形态；透射电镜及激光共聚集观察心肌超微结构和TT-SR连接。Western blotting测定JP2；RT-PCR检测CACNA1S、RyR2 mRNA水平和miR-24水平。结果：DOX处理后4周，大鼠发生心肌损伤，BNP水平明显高于对照组（169.6 ± 29.6 pg/mL vs 80.1 ± 9.8 pg/mL, P < 0.001），lGZG组尤其是高剂量组，可显著降低BNP水平(93.8±17.9 pg/mL, P < 0.001)。DOX组大鼠出现左室射血分数较对照组明显下降(58.5±8.7% vs . 88.7±4.0%;P < 0.001)。地高辛和LGZG改善心功能障碍(79.6±6.1%，69.2±2.5%)，保留左室射血分数(77.9±5.1,80.5±4.9,P < 0.01)。LGZG组降低HWI和HW/TL比值，改善LVEDD、LVESD和FS并可减轻心肌形态学异常和线粒体损伤。高剂量的LGZG显著下调了miR-24的表达，上调了JP-2的表达，拮抗了dox诱导的t小管TT-SR微结构重构，因此可改善心衰时LCC-RyR的锚定状态，进而改善了周期性Ca2+瞬变和细胞收缩，这可能是LGZG对HF的改善作用的机制。结论：LGZG对dox诱导的大鼠HF有一定的改善作用，其部分机制是通过改善TT-SR的显微结构重塑，调控LCC-RyR的锚定状态实现。