几种中药甙糖类成分磷酸化修饰物抗病毒性肝线粒体损伤及其信号转导机制研究

Based on the research results of the first two national natural science foundation, these Glycosides and polysaccharides ingredients of several Chinese herbal medicines with fine antiviral activities and hepatoprotective effective including Baicalin, Epimedium glycoside, Paeoniflorin, Bush Sophora Root polysaccharide, Codonopsis pilosulapolysaccharide, Wild chrysanthemunindici polysaccharide were chosen as the research object and phosphorylated modificated. Methods including Real-time PCR, Western blot, laser confocal microscopy, electron microscopic techniques and so on were applied to determinate the influence of these ingredients on the protective functions and its important links of liver injury and mitochondrial damage in liver and liver cell. Also, the regulation of gene and protein expression level of the Nrf2 gene and oxidases and Ⅱphase detoxifying enzymes such as heme oxygenase 1 (HO-1), benzene quinone oxidoreductase (NQO1) and so on, of the Nrf2 signaling pathways downstream were deeply understood. The design was to explore the damage effect of animal virus to liver mitochondria and the protective mechanism of signal transduction regulation of phosphorylated modificated Glycosides and polysaccharides ingredients. The aim of this study was to determine treatment for animal viral diseases, screen and develop good clinical curative effect of veterinary drugs for liver damage caused by animal virus disease, provide a reference for the prevention and control of animal virus disease with Chinese traditional medicine.

基于前两项国家自然科学基金的研究结果，选择黄芩苷、淫羊藿苷、芍药苷、山豆根多糖、党参多糖、野菊花多糖等有较好抗病毒和保肝作用的几种中药甙糖类成分为研究对象，采用磷酸化修饰法修饰并对其工艺进行优化，采用Real-time PCR、Western blot、电镜等技术比较研究这些甙糖类成分磷酸化修饰物对动物源病毒（如鸭肝炎病毒）感染引起的肝组织中和体外培养的肝细胞中线粒体损伤的保护作用及其主要作用环节，并研究Nrf2信号通路中Nrf2基因及下游氧化酶和Ⅱ相解毒酶如亚铁血红素加氧酶1（HO-1）、苯醌氧化还原酶（NQO1）等的调控基因和蛋白表达水平变化，并干预法验证，探讨动物病毒感染对肝线粒体的损伤作用及中药甙糖类成分磷酸化修饰物的保肝作用的信号转导调控机制。旨在为动物病毒性疾病确定治疗方案,筛选和研发对引起明显肝损伤的动物病毒病有良好临床疗效的中兽药，为利用中药防控动物病毒病提供参考

为比较筛选具有较好保肝作用的中药成分，磷酸化修饰了黄芩苷、淫羊藿苷、山豆根多糖、生地多糖和野菊花多糖。采用DHAV攻毒雏鸭的方法，比较发现5种多糖及其磷酸化修饰物都可缓减DHAV-1染毒雏鸭肝损伤，生地多糖作用较差。.为研究DHAV-1感染造成DEHs及雏鸭肝组织线粒体损伤的机制和山豆根多糖、淫羊藿苷、黄芩苷、野菊花多糖磷酸化修饰物的缓解作用。检测了各药物对感染DHAV-1体外肝细胞线粒体膜电势、ROS水平及Ca2+浓度、COX和SDH的酶活和ATP的含量影响；DHAV-1感染雏鸭肝线粒体的超微结构及氧化损伤评价指标SOD、GSH、GPX和ATP含量等的变化。结果DHAV-1可通过氧化应激破坏肝细胞线粒体膜通透性改变线粒体结构，抑制线粒体呼吸链酶活性，降低ATP生成来而致病，药物可维持线粒体氧化呼吸链和三羧酸循环等内部功能的稳定以抵御DHAV-1对线粒体的损伤。DHAV-1感染雏鸭后会破坏肝脏线粒体的形态、引起严重的氧化应激状态和线粒体功能紊乱。各药物基于其抗氧化作用缓解了DHAV-1感染雏鸭造成的线粒体损伤和氧化应激状态，提高了ATP含量，保证了雏鸭肝脏正常功能。各磷酸化修饰药物作用更为理想，以磷酸化修饰的淫羊藿苷、黄芩苷、野菊花多糖作用最佳。.为研究DHAV-1造成DEHs线粒体损伤及黄芩苷、野菊花多糖和淫羊藿苷磷酸化修饰物的缓解作用的机制，检测了Nrf2/ARE信号转导通路相关基因和蛋白的活性。同时，检测了DHAV-1感染雏鸭肝组织中Nrf2的蛋白表达量。结果显示：DHAV-1感染导致DEHs中SOD、GPX-1、Nrf2、NQO1和HO-1的mRNA基因相对表达量降低，SOD、GPX和Nrf2蛋白相对表达量降低，肝组织中Nrf2蛋白表达量降低。黄芩苷、野菊花多糖和淫羊藿苷修饰前后均能调节上述基因和蛋白表达量且磷酸化修饰物效果更佳。说明黄芩苷、野菊花多糖和淫羊藿苷及其磷酸化修饰物可以通过激活DEHs和感染肝组织中Nrf2/ARE信号通路实现抗氧化和线粒体保护作用，野菊花和淫羊藿苷的修饰物效果更佳。.培养博士4名，硕士4名，其中6名研究生毕业；获得江苏省科学技术二等奖1项；发表论文9篇，第一标注8篇，其中SCI收录论文8篇，TOP10共4篇。