磷酸化分子修饰提高中药甙糖类成分抗病毒活性及其机理研究

The previous research of our team indicated that the antiviral activities of glycosides and polysaccharides ingredients and its phosphorylation is relate to multi-links, especially the regulation in the body. Phosphorylated modification invented by our project team possesses prominent antiviral activity, more convenience and good friend to our enviroment. Glycosides and polysaccharides ingredients of Chinese herbal medicine with fine antiviral activities including Baicalin, Epimedium flavone, Gynostemma saponins, Astragalus polysaccharides, Bush Sophora Root polysaccharide and Rehmannia, Wild chrysanthemun indici polysaccharide were chosen as the research object. Phosphorylated modification was applied to modify these ingredients. Phosphorylated products with best antiviral effect were screened via the vivo and vitro experiments. Methods including Real-time PCR, laser confocal microscopy, flow cytometry, Western blot, and so on were applied to determinate the influence on replication and release of duck hepatitis virus, expression of 2A protease and VP1 protein, activity of internal ribosome entry site, hepatocytes apoptosis, lymphocyte subpopulation proliferation, TH1 and TH2 immune balance, function of dendritic cells and macrophages. A deep understanding of antiviral activities of phosphorylated product was by multi-links of virus proliferation regulation, cell apoptosis and virus antigen presentation in vivo and vitro. The aim of this study was to discover the relationship between modification and antiviral activity of the glycosides and polysaccharides ingredients and its phosphorylated modification, to develop traditional Chinese veterinary antiviral herbal medicine.

项目组前期的研究发现中药甙糖类成分及其硫酸化修饰物抗病毒作用是多环节的，体内的调节尤为重要；项目组发明的磷酸化修饰比硫酸（硒）化修饰抗病毒效果更显著、也更方便、环保。选择黄芩苷、淫羊藿黄酮、绞股蓝皂苷、黄芪多糖、山豆根多糖、生地多糖、野菊花多糖等抗病毒作用较好的中药甙糖类成分，优化其磷酸化修饰工艺，通过体内外试验筛选出抗病毒活性最强的磷酸化修饰物。再用Real-time PCR、激光共聚焦、流式细胞术、Western blot等方法在病毒增殖、细胞凋亡和病毒抗原递呈等途径从体内外两方面深入比较研究甙糖类成分及磷酸化修饰物对鸭肝炎病毒复制、释放和2A蛋白酶与VP1蛋白表达、内部核糖体进入位点活性，肝细胞凋亡及淋巴细胞亚群、Th1和Th2型细胞平衡、树突状和巨噬细胞功能的影响，以初步阐明甙糖类成分磷酸化修饰物抗病毒作用的关键机制，探讨甙糖类成分结构改造和抗病毒活性的相关性，创制抗病毒新中兽药

为研究有效抗病毒药物，据项目组前期发明的中药甙类成分磷酸化修饰法成功磷酸化修饰了黄芩苷(BA)、芍药苷(PF)、绞股蓝皂苷(GP）、淫羊藿苷(ICA)、栀子苷(GE）、山豆根多糖（BSRPS）、野菊花多糖（CIPS）、黄芪多糖（APS）等8种甙糖类成分。磷酸化修饰明显提高了8种成分抗DHAV-1感染鸭胚肝细胞(DEHs)的作用，以BA、ICA、GP、APS效果较好，且均可较好抑制DHAV-1复制和释放。并首次成功阐明了DHAV-1的体外肝细胞复制周期。.比较BA、PF、ICA、CIPS和APS磷酸化修饰物抗DHAV-1感染雏鸭作用发现除CIPS外，磷酸化修饰糖苷类成分抗DVH作用均明显提高，降低血液病毒含量，以磷酸化分子修饰的BA、PF、ICA效果更佳。BA、ICA、PF和APS及其修饰物均能调节染毒雏鸭免疫因子分泌平衡，促进Th1型和降低Th2型细胞因子分泌，从而表现出一定的治疗作用，磷酸化分子修饰明显提高其效果。.DHAV-1诱导DEHs发生明显凋亡，TUNEL法和Annexin V/PI流式法检测发现GP、BA、PF、ICA及其磷酸化修饰物可显著抑制DHAV-1诱导的凋亡，修饰物的作用均明显优于未修饰物。雏鸭DHAV-1染毒8h后肝组织即发生显著凋亡；PF、PFPC、BAPC均显著抑制这种凋亡；RT-PCR分析发现PF和PFPC有效下调 Caspase3和TNF-α基因表达，BAPC还可上调Bcl-2基因表达而缓解凋亡；并表现较好的抗DVH作用。.使用放线菌酮抑制蛋白的翻译后，采用RT-PCR法观察BA、BAPC对DHAV-1基因合成的影响，同时用WB观察BAPC对DHAV-1的3D蛋白稳定性的影响。结果BAPC能够抑制DHAV-1的基因合成，而BA则不能抑制DHAV-1基因合成；BAPC能够降低3D蛋白稳定性。再运用干扰RNA技术确定了Hsp70对DHAV-1基因合成的重要性后，采用WB法研究发现BAPC能够通过降低Hsp70表达以抑制DHAV-1基因合成。BA和BAPC均能显著抑制DHAV-1的蛋白质翻译，同时能够抑制DHAV-1的IRES活性，是通过降低DHAV-1的IRES活性以抑制DHAV-1的蛋白质翻译。.培养博士3名，硕士4名。发表SCI收录论文8篇（2篇TOP10）、中文论文1篇；获得授权专利1项，申请专利1项。