防治RA病黄连汤中和内毒素成分确定及其阻断LPS诱导炎症的分子机制

Riemerella anatipestifer disease is a serious infectious disease that threats the development of the duck industry greatly, there is no vaccine for prevention of the desease currently. Once the disease occurred on duck farms, all the ducks including clinically manifestative healthy ducks must rely on the antibiotic for survive, but the treatment result is ineffective.Our research team had developed a Chinese herb medicine prescription named Coptidis rhizoma Decoction, which had a protection rate of 98% for clinically healthy ducks when the RA disease spreads among the ducks,the prescription and production technology of it had been confered a National Invention Patent.On the basis of prelimiary study, the proposal intends to isolate and identify the endotoxin-neutralization specific ingredients of Coptidis rhizoma Decoction by using chemical separation, mass spectrometry, nuclear magnetic resonance, infrared spectroscopy and kinetic turbidimetric Limulus test methods in vitro, investigate impacts on molecular structure of endotoxin. By monitoring the variation of endotoxin in the RA challenged duck with the drug, endotoxin-neutralization effects of the drug is researched in vivo. ELISA method is used to monitor duck endotoxin binding protein (LBP) content, explore the inhibitory effect of the drugs on the secretion of LBP. Real-time quantitative fluorescent PCR technology is used to study the impact of drugs on the quantities of LBP mRNA. The results will clarify material foundation of Coptidis rhizoma Decoction, understand the molecular mechanism of blocking endotoxin-induced inflammation. In the future, by modification of the chemical structure of the drug, a more desirable,new endotoxin-neutralization drug is developed.

鸭疫里默氏菌病是一种严重威胁养鸭业发展的传染病，目前尚无疫苗预防，鸭发病后必须用抗生素治疗,但抗生素治疗效果不佳。本项目负责人研制出一种防治该病的中药复方制剂--黄连汤，对该 病流行时全群鸭的保护率达98%，其配方和制作技术已获国家发明专利授权。本项目拟在此研究的基础上，采用化学、质谱、核磁共振、红外光谱等技术和动态浊度鲎试验方法，分离和确定黄连汤中和内毒素作用的具体有效成分，并研究药物成分对内毒素分子结构的影响；监测用该菌攻毒后再给药鸭体内内毒素含量的变化规律，研究药物体内中和内毒素的效果。应用ELISA法监测鸭体内内毒素结合蛋白(LBP）含量,探究药物对鸭LBP分泌的抑制作用。运用实时定量荧光PCR技术，研究药物对鸭LBP mRNA转录量的影响。研究结果可阐明黄连汤中和内毒素的物质基础，理解其阻断内毒素诱导炎症的分子机制。将来还可通过修饰药物的化学结构，研制更理想的中和内毒素新药。

本项目开展了鸭疫里氏杆菌（RA）内毒素（endotoxin,LPS）的提取、纯化及黄连汤和不同极性溶剂黄连汤萃取物对内毒素清除作用的研究。.以2型RA为实验对象，采用热酚水法结合乙醇纯化技术提取、纯化LPS，通过苯酚硫酸法、考马斯亮蓝法、琼脂糖凝胶电泳等技术，对所提取的内毒素中多糖、蛋白质、核酸检测，并采用显色基质法测定其生物活性。结果表明，所获得LPS与RA菌体湿重比为1.36%，含糖1.48%、蛋白质2.08%，还有部分核酸片段，其生物活性为75.6 EU/mg。相对我国内毒素标准品原料( E.coli 0111)，所提取的LPS含糖和蛋白质较低，说明本实验所提取纯化的LPS纯度较高。.用不同浓度的药液200 μL，在体外分别作用RA（3×109 CFU/mL）、RA超声波破碎液（3×109 CFU/mL）及提取的LPS（10 mg/mL）各11 h，用显色基质鲎试剂法检测各试验组LPS的浓度变化。结果显示，药物作用于RA时，随着药物浓度的增加，LPS浓度首先由1.15EU/mg升高到2.68EU/mg，然后下降到0.43EU/mg，即LPS浓度呈现先上升、后下降现象；药物对RA超声波破碎液和LPS作用时，随着药物浓度的增加，RA超声波破碎组LPS浓度从2.49EU/mg降为0.21EU/mg，LPS组从2.89EU/mg下降到0.26EU/mg，呈现依次下降的趋势。这是因为：药物直接作用于RA时，先破坏菌体结构，导致RA外膜的LPS大量释放，低浓度的药物不足以清除大量的LPS，所以LPS浓度略有升高。随着药物浓度的增加，大量药物可消除多余的LPS，因此表现为LPS浓度先升高、后下降这一现象；药物对RA超声波破碎液组及LPS组处理时，不存在细菌破裂释放内毒素这一环节。所以，随着药物浓度的增加，LPS浓度呈现下降趋势。实验表明：黄连汤对LPS具有明显的消除作用。.将黄连汤水提醇沉部位依次用正己烷、乙酸乙酯、正丁醇萃取，减压浓缩至干得正己烷萃取物、乙酸乙酯萃取物和正丁醇萃取物三个不同极性部位，稀释成1、0.5、0.25 mg/mL溶液。取各萃取物各稀释浓度药液200 μL与10 μL LPS在恒温振荡培养箱中，37℃、200 r/min，孵育11 h后，用显色基质鲎试剂法进行检测，结果表明，黄连汤乙酸乙酯部位具有良好的体外抗内毒素活性。