大豆皂苷I及类似物抑制成熟自由肾素以及干预（前）肾素-受体系统的机理研究

Renin is the rate-limiting step of the renin-angiotensin system (RAS) and can induce hypertension and cardiovascular diseases through the over-activated renin-ACE-AngII-AT1R axis.Real-time binding of the inhibitor soyasaponin I (or II)to renin is observed using surface plasmon resonance ,and the association (ka) and dissociation(kd) rate constants are measured and the dissociation constant (KD) is determined through the Langmuir 1:1 kinetic-binding model.Nuclear magnetic resonance is used to acquire the nonselective,selective and double selective spin-lattice relaxation rates of protons, and the molecular motional correlation times and 2D transferred nuclear overhauser enhancement of proton pairs in soyasaponin I (or II) in the absence and presence of renin,which will indicate affinity and active sites of interaction. Molecule docking analysis explores active sites, types (such as hydrogen bond,van der Waals,hydrophobic and ionic interactions), and energy of the interaction. Comparative molecular field analysis and comparative molecular similarity index analysis establish 3D-QSAR models through which the active sites and activities of renin inhibitory saponins can be easily obtained.These results will systematically show how soyasaponin I and its analogues exert their inhibitoy effects on mature/free renin. .Binding of prorenin to the (pro)renin receptor induces a conformational change in the molecule, thus increasing its enzymatic activity from virtually zero to a value comparable to that of active renin in solution without proteolytic removal of the prosegment. Renin bound to the (pro)renin receptor displays a four-fold increase of the catalytic efficiency of angiotensinogen conversion to Ang I compared with free renin.In addition to the Ang-dependent function on the cell surface,the (pro)renin-receptor system also has Ang-independent intracellular effects leading to the expression of profibrotic genes.Values of ka, kd and KD for binding of soyasaponin I (or II) to the renin-receptor and prorenin-receptor complexes are obtained using the real-time surface plasmon resonance,and the inhibitory mode (competitive, noncompetitive,uncompetitive or mixed)and Ki are determined.Values of ka,kd and KD for binding of soyasaponin I (or II)-renin complex to the receptor are also determined to explore the interference of soyasaponin in binding of active renin to its receptor.Real-time fluorescent quantitative PCR assay explores the effects of soyasaponin I (or II) on genes expression in human aortic smooth muscle cells of the (pro)renin receptor,transforming growth factor, plasminogen-activator inhibitor-1 and collagen 1. These results will show whether and how soyasaponin I (or II) interferes with the (pro)renin-receptor system.

肾素是RAS的限速酶，它通过肾素-ACE-AngII-AT1R轴诱导高血压和CVD。表面等离子共振确定结合、解离常数和亲和力，NMR确定作用前后质子弛豫速率、质子间交叉弛豫速率、分子运动相关时间和转移NOE 等的变化，分子对接确定作用位点、作用力类型和结合自由能，CoMFA和CoMSIA分析建立3D-QSAR模型，确定立体、静电、疏水和氢键场三维等势图,揭示大豆皂苷I及类似物抑制成熟自由肾素的分子机理。.受体非酶激活的前肾素和与受体结合的肾素不仅促进AngI产生（酶），还上调促纤维因子表达（激素）。研究确定大豆皂苷I(或II)与和受体结合的（前）肾素作用的动力学和亲和力、抑制（前）肾素-受体降解Ang原的形式和Ki、皂苷-肾素与受体的亲和力，RT-qPCR确定主动脉HSMC受体、TGF-β、PAI-1和1型胶原mRNA表达量变化，揭示大豆皂苷对（前）肾素-受体系统两种信号功能的干预机制。

肾素是肾素-血管紧张素系统中的一级频率限速酶，它通过肾素-血管紧张素转化酶-血管紧张素II-1型受体轴诱导高血压和心血管疾病。八个皂苷有抑制体外肾素活性功能，尤其其中的大豆皂苷I具有体内抗高血压活性。项目通过分子模拟、核磁共振、等温滴定量热及RT-qPCR研究了皂苷对肾素的抑制，结果发现：大豆皂苷I与II在最优构象位置易与肾素结合，Ser230、Tyr231、Ser42与Ser84是产生氢键的关键氨基酸，而Ala229、Arg82、Asp38、Tyr83、Met303、Gly228、Asp226、Ser41是肾素与皂苷类抑制剂形成疏水作用的重要氨基酸，S1'口袋内没有氢键作用，而6个口袋均存在疏水作用；静电与范德华力是结合主要驱动力，非极性溶剂化能也有促进效果，而极性溶剂化能则阻碍相互作用，总结合自由能与皂苷的IC50值具有线性相关性；分子动力学模拟1000 ps后均方根偏差趋向稳定，八个体系平均值均小于0.25 nm，其中大豆皂苷I-肾素均值最小且涨落幅度最小，肾素氨基酸均方根波动RMSF总体偏大，体系主要存在于(Thr39-Trp45)、(Met107-Thr112)、(Leu224-Ser235)与(Ala314-Thr318)四大低RMSF值区域；加入成熟自由肾素前后不同皂苷单个被测质子化学位移、非选择性自旋-晶格弛豫速率未明显变化，而选择性自旋-晶格弛豫速率增强，游离态正值交叉弛豫速率变为结合体负值，分子运动相关时间延长而速率减慢，反相NOE信号在结合后变为同相；八个皂苷降低与受体结合肾素催化反应速率常数，其中大豆皂苷II对酶活影响最大；大豆皂苷I还显著性抑制人血管平滑肌细胞促纤维化因子TGF-β1与PAI-1基因表达。研究不仅揭示了大豆皂苷及类似物对肾素的抑制机制，也为大豆皂苷开发为预防高血压和轻度高血压前期治疗的食源性功能因子提供理论支持。