小檗碱靶向MD-2抑制TLR4/MD-2复合物形成抗内毒素作用的机制研究

Pathogenic Escherichia coli is one of the most common and serious pathogens in intensive pig farms. The high fatality rate of Escherichia coli is directly related to endotoxin. Therefore, anti-endotoxin therapy has become one of the key factors to enhance the therapeutic effect. Research results show that myeloid differential protein (MD-2) plays an important role in the regulation of the signal transduction induced by LPS,MD-2 is one of the potential targets for anti-endotoxin treatment.MD-2/TLR4 complex plays a key role in the signal transduction induced by LPS.If inhibition of MD-2/TLR4 complex formation,can block or inhibit pathogen signaling into cells.Our preliminary studies have proved that Pathogenic Escherichia coli is one of the most common and serious pathogens in intensive pig farms.The high fatality rate of Escherichia coli is directly related to endotoxin.Therefore,anti-endotoxin therapy has become one of the key factors to enhance the therapeutic effect.Research results show that myeloid differential protein (MD-2) plays an important role in the regulation of the signal transduction induced by LPS,MD-2 is one of the potential targets for anti-endotoxin treatment.MD-2/TLR4 complex plays a key role in the signal transduction induced by LPS.If inhibition of MD-2/TLR4 complex formation,can block or inhibit pathogen signaling into cells. Our preliminary studies have proved that berberine could regulate MD-2 expressions in intestinal mucosal microvascular endothelial cell and block endotoxin injury in the treatment of intestinal Escherichia coli disease.This project would based on previous studies proposed the hypothesis that berberine inhibits the formation of MD-2/TLR4 complex against endotoxin damage,MD-2 is the target of berberine.It is suggested that MD-2/TLR4 is the target of berberine.This project would based on previous studies intend to explore the anti-endotoxin mechanism of berberine with MD-2 as the target by Molecular docking technique,bis-Ans fluorescence Spectrometric Analysis Technology and co-immunoprecipitation technique.this study would provid a new theoretical basis for revealing the anti-inflammatory mechanism of berberine,and a new way for the study of drug mechanism for the prevention and treatment of bacterial diseases.

致病性大肠杆菌是养猪场常见的严重仔猪病原菌之一。仔猪大肠杆菌病高致死率与内毒素直接相关，因此阻断内毒素作用是提高治疗效果的关键环节。髓样分化蛋白-2（MD-2）对LPS诱导的信号转导具有重要的调控作用，MD-2已成为抗内毒素的靶点之一。MD-2/TLR4复合物在LPS诱导的信号转导中起着关键性的作用。抑制MD-2/TLR4复合物形成，可以阻断或抑制病原体信号向细胞内传递。本课题组前期研究初步证明小檗碱可以通过抑制肠黏膜微血管内皮细胞MD-2的表达阻断内毒素损伤。本项目在前期研究基础上提出“小檗碱通过抑制MD-2/TLR4复合物的形成而阻断内毒素作用，MD-2是小檗碱的抗炎作用靶点”。本项目拟以MD-2靶标，通过分子对接、bis-ANS荧光光谱分析技术、免疫共沉淀等技术进一步研究小檗碱抗内毒素作用的机制，本研究为揭示小檗碱的抗炎新机制提供理论依据，为防治细菌性疾病的药物机理研究提供新的思路。

致病性大肠杆菌是养猪场常见的严重仔猪病原菌之一。仔猪大肠杆菌病高致死率与内毒素直接相关，因此阻断内毒素作用是提高治疗效果的关键环节。髓样分化蛋白-2（MD-2）对LPS诱导的信号转导具有重要的调控作用，MD-2已成为抗内毒素的靶点之一。MD-2/TLR4复合物在LPS诱导的信号转导中起着关键性的作用。抑制MD-2/TLR4复合物形成，可以阻断或抑制LPS信号转导。本课题组前期研究基础上提出“小檗碱通过抑制MD-2/TLR4复合物的形成而阻断内毒素作用，MD-2是小檗碱的抗炎作用靶点”。本项目以MD-2靶标，从小檗碱与MD-2 蛋白的结合、小檗碱对TLR4/MD-2生成的影响、小檗碱对TLR4/MD-2/NF-κB信号通路、TLR4/p38MAPK 信号通路和TLR4/NF-κB/NLRP3信号通路的调控作用和小檗碱抗内毒素的转录组学四个方面进行研究，进一步探讨小檗碱抗内毒素作用的机制，完成了预期研究目标。实验结果证实小檗碱可以和MD-2疏水口袋特异性结合，并能减少MD-2/TLR4复合物的形成，通过TLR4/MD-2/NF-κB信号通路、TLR4/p38MAPK 信号通路和TLR4/NF-κB/NLRP3信号通路阻断内毒素的作用从而发挥抗炎作用，证实了MD-2是小檗碱的作用靶点，且小檗碱具有多通路多靶点的作用特性。本研究结果为揭示小檗碱的抗炎机制提供了理论依据，填补了小檗碱对TLR4/MD-2/NF-κB信号通路、TLR4/p38MAPK 信号通路和TLR4/NF-κB/NLRP3调控研究，本研究为揭示小檗碱的抗炎新机制提供理论依据，为防治细菌性疾病的药物机理研究提供新的思路，为进一步筛选防治细菌性疾病的新型中药制剂提供研究平台。本项目对于加快中兽药的研发和成果转化步伐，实现健康养殖，保障畜产品安全具有重要的意义。