蜂胶黄酮抗猪细小病毒成分的分离及作用机理研究

Porcine parvovirus (PPV) infection produces great threat to first-timer sows and piglets, so it is urgent need to seek safe and effective drugs for prevention and control. Our previous studies have found that propolis flavone (PF) had good effect on resistance to PPV, but its mechanism is unclear. First, separation and identification of active ingredients in PF was carried out with chromatography in this project, and then anti-PPV activity in vivo and in vitro of propolis flavone ingredients (PFI) was compared and selected by investigating the virus titer and its host or host cell survival rate against PPV. Then the PK 15 cells were treated with the selected PFI, and then immunology and molecular biology technologies such as Western blot, fluorescent quantitative PCR, flow cytometry, electrophoretic mobility shift assay, immunofluorescence were utilized to detect cells apoptosis morphology, signaling pathways regulating factor expression and transcription. And in combination with PPV duplicate key protein NS1 gene expressing and the amount of ROS measuring in cells were going to reveal the regulating molecular mechanisms of PFI to PK-15 cells apoptosis infected by PPV. Two aspects of the host cell apoptosis and virus virulence gene were carried out in this research to clarify the molecular mechanisms of PF in regulating cell resistance to PPV infection and provide theoretical support for screening anti-PPV drug using cell signaling molecules as drug targets.

猪细小病毒（PPV）感染对初产母猪和仔猪产生很大的威胁，急需寻找安全、有效的药物进行防治。我们前期研究发现蜂胶黄酮（PF）具有很好的抗PPV作用，但其机制尚不清楚。本项目首先采用色谱技术分离并鉴定PF中活性成分，然后通过考察病毒滴度和宿主及其细胞存活率进行体内外抗PPV作用比较和筛选；再以筛选出的蜂胶黄酮成分（PFI）处理培养的PK-15细胞后，采用蛋白印迹、荧光定量PCR、流式细胞术、凝胶电泳迁移率、免疫荧光等免疫学和分子生物学技术检测PPV感染细胞凋亡形态学、凋亡信号通路中关键因子表达和转录时相；同时结合PPV复制关键的NS1基因蛋白在细胞中表达以及细胞内ROS含量的测定来揭示PFI对PPV感染致PK-15细胞凋亡的调控作用。研究将从宿主细胞凋亡和病毒致病基因两个方面来阐明PF调控细胞抵抗PPV感染的分子机制，并为以细胞信号分子作为药物靶标进行抗PPV药物筛选提供理论支持。

猪细小病毒（PPV）主要引起母猪繁殖障碍，目前兽医临床上多用疫苗免疫母猪用于预防PPV感染，申请人前期研究发现蜂胶黄酮（PF）具有良好的抗PPV作用，本项目首先提取和分离PF中化学成分，然后体外试验通过给PK-15细胞攻毒PPV后，CCK-8测定细胞活性、RT-PCR检测PPV滴度比较并筛选出效果较好成分。体内实验以豚鼠PPV攻毒为模型，采用分离制备的各成分进行治疗，通过观察死亡率和治愈率等指标，Real-time PCR检测全血及性腺、脾脏等组织中PPV含量以及脏器组织病理变化进一步筛选抗PPV成分。最后使用筛选出的阿魏酸（FA）进一步观察其对PPV引起的细胞凋亡形态学、细胞Bid相关的凋亡基因p53、p38、JNK-MAPKs、Bax、线粒体膜电位、线粒体介导的caspase依赖性凋亡信号通路等的影响，同时结合PPVNS1基因蛋白在细胞中表达以及细胞内ROS含量的测定研究阿魏酸对PPV感染致PK-15细胞凋亡的调控作用；实验结果显示：通过对分离制得的白杨素、高良姜素、槲皮素、阿魏酸、芹菜素等各单体成分进行体外抗PPV试验，发现阿魏酸（FA）处理组病毒含量较低，细胞的存活率较高，体内试验发现FA可显著改善猪细小病毒引起的器官组织变化，作用机理研究发现FA可以逆转PPV激活Bid及其相关信号通路诱导的PK-15细胞凋亡，表明阿魏酸的体外抗病毒活性可能与通过阻断促凋亡因子JNK、Bid等，以及通过抑制与Bid相关的信号传导途径的激活来抑制猪细小病毒的复制；FA处理可降低NF-KB、MyD88和IL-6的表达，从而抑制PPV感染引起的炎症反应；FA还可下调PPV感染的PK-15细胞中NS1结构蛋白的表达；此外，FA下调NS1和TLR4信号、防止活性氧过量产生并且抑制NF-KB 炎性小体和PPV感染的PK-15细胞的凋亡。本项目资助发表SCI论文2篇，under review1篇；受理发明专利2项。项目资助经费21万元，支出19.6061万元，各项支出与预算相符，剩余经费1.3939万元，将用于本项目相关的研究支出。