Target molecule for anti-inflammatory therapy and drug development

红参粗皂苷部分具有抗炎作用。几种提取的人参皂苷也有抗炎作用。然而，这些抗炎作用的确切机制尚未阐明。在本研究中，我们研究了人参皂苷- ro对Thy-1治疗的实验性肾小球肾炎的影响，并寻找影响人参皂苷- ro的应答分子。8周龄雌性Wistar大鼠10只，以0.25mg/kg体重的剂量注射抗大鼠CD90 (Thy-1)/mouseCD90.1 (Thy-1.1) (PharMingen， USA)，分为两组；未处理对照组（n=5）和人参皂苷- ro处理组（30mg/kg I.p./d）。将大鼠置于代谢笼中，连续6天，24小时采集尿液样本。人参皂苷- ro处理可使尿蛋白排泄量减少约15%。肝脏或肾脏组织的细胞质组分应用于BIACORE系统（BIACORE，日本）。在人参皂苷- ro固定化CM-5传感器芯片上观察到特异性结合。为了鉴定人参皂苷- ro的特异性靶分子，采用亲和层析法。人参皂苷- ro与EAH Sepharose4Bgel结合。8周龄Wistar大鼠以1 ml/kg剂量CCl4处理，24小时后处死大鼠。肝脏用50mM Tris/HCl缓冲液Ph7.6与0.25M蔗糖含蛋白酶抑制剂鸡尾酒（1ml）匀浆，离心数步制备核酸部分、微粒体部分和上清部分。样品上附亲和凝胶柱，用含人参皂苷- ro的缓冲液洗脱。用SDS-PAGE法分析冻干洗脱液。通过凝胶电泳鉴定出若干特异性条带和许多非特异性条带。提示人参皂苷- ro具有抗肾小球肾炎作用，可能存在应答分子

Crude saponins fraction of red ginseng showed anti-inflammatory effects. Several kinds of extracted ginsenosides also showed anti-inflammatory effects. However, the precise mechanism of these anti-inflammatory effects has not been elucidated. In the present study, we investigated the effects of ginsenoside-Ro on the Thy-1 treated experomental glomerulonephritis and searched for responsive molecule affecting ginsenoside-Ro. Ten fremale Wistar rats of 8-week old were injected anti-rat CD90 (Thy-1)/mouseCD90.1 (Thy-1.1) (PharMingen, USA) at a dose of 0.25mg/kg body weight, and divided into two groups; non-treatment control group (n=5) and ginsenoside-Ro treated group (30mg/kg I.p./day). Rats were kept in metabolic cages, and urine samples were collected for 24 hours for 6 consecutive days. Urinary excretion of protein was decreased about 15% by the treatment of ginsenoside-Ro. The cytosol fractions of liver or kidney tissues were applied to the BIACORE system (Biacore, Japan). The specific binding was observed in the ginsenoside-Ro immobilized CM-5 sensor chip. To identify the spesific target molecule for ginsenoside-Ro, affinity chromatography method was employed. Ginsenoside-Ro was bound with EAH Sepharose4Bgel. Wistar rats of 8 weeks old were treated with CCl4 at a dose of one ml/kg, and 24 hours later rats were sacrificed. Liver was homogenized by with 50mM Tris/HCl buffer Ph7.6 with 0.25M sucrose containing protease inhibitor cocktail (1ml) and centrifuged by several steps for preparing nucleic fraction, microsomal fraction and supernatant fraction. Sample was applied to the affinity gel column, and eluate by ginsenoside-Ro containing buffer was collected. Lyophilized eluate was analyzed by SDS-PAGE method. Several spesific and many non-specific bands were identified by the gel electrophoresis. These results suggest that ginsenoside-Ro has anti-glomerulonephritis effects, and its responsive molecule may exist