Target molecule for anti-inflammatory therapy and drug development

红参皂苷粗品具有抗炎作用。几种提取物也显示出抗炎作用。然而，这些抗炎作用的确切机制尚未阐明。本研究观察了黄芪皂苷Ro对Thy-1治疗的实验性肾小球肾炎的影响，并寻找影响黄芪皂苷Ro的效应分子。将10只8周龄的Wistar雄性大鼠以0.25mg/kg体重的剂量注射抗大鼠CD 90（Thy-1）/小鼠CD 90.1（Thy-1.1）（PharMingen，USA），并分为两组：非治疗对照组（n=5）和黄芪皂苷-Ro治疗组（30 mg/kg I. p./天）。将大鼠饲养在代谢笼中，连续6天收集24小时尿液样品。黄芪皂苷-Ro治疗后尿蛋白排泄量减少约15%。将肝或肾组织的细胞溶质部分应用于BIACORE系统（Biacore，日本）。在固定化人参皂苷-Ro的CM-5传感器芯片中观察到特异性结合。采用亲和层析法对三七皂苷-Ro的特异性靶分子进行鉴定。人参皂苷-Ro与EAH Sepharose 4 B凝胶结合。8周龄的Wistar大鼠以1 ml/kg的剂量用CCl 4处理，24小时后处死大鼠。用含有0.25 M蔗糖的50 mM Tris/HCl缓冲液（pH 7.6）（含蛋白酶抑制剂混合物（1 ml））对肝脏进行匀浆，并通过几个步骤进行离心，以制备核酸级分、微粒体级分和上清液级分。将样品上样至亲和凝胶柱，并收集含有黄芪糖苷-Ro的缓冲液。用SDS-PAGE法对冻干的黄芪进行分析。凝胶电泳鉴定出几条特异性条带和许多非特异性条带。以上结果提示，黄芪皂苷-Ro具有抗肾小球肾炎作用，其作用机制可能与其反应分子有关

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