【作者】 陈洁；

【导师】 杨汉春； 何启盖；

【作者基本信息】 中国农业大学 ， 兽医博士（专业学位）， 2018， 博士

【摘要】 中草药在我国具有悠久的历史,尤其在病毒性疾病防治方面具有独特的优势。一些中草药及其成分不仅能直接影响病毒复制,还能调节机体免疫功能。猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)是危害全球养猪业的重要病原,均可导致机体免疫抑制,引起其他病原微生物的继发或并发感染,从而造成巨大的经济损失。因此,除了依靠疫苗免疫,探讨控制PCV2和PRRSV感染的新策略十分必要。本研究从牛蒡子中提取活性成分牛蒡苷元(ACT),分析其在体内外对PCV2和PRRSV的抗病毒活性,以期为猪圆环病毒病(PCVAD)、猪繁殖与呼吸综合征(PRRS)的防控以及ACT的开发应用提供科学依据。牛蒡子粗粉经脱脂后用含5%盐酸的50%乙醇溶液回流5h,过滤后药渣用50%乙醇溶液重复提取1h,合并滤液,用5mol/L NaOH溶液调节至中性,氯仿萃取两次,减压旋转蒸干后获得ACT粗品,经两次硅胶柱层析纯化获得ACT;三批次提取的ACT平均百分含量为98.2%,平均提取率为1.62%。分析了 ACT对PCV2在PK-15细胞及小鼠体内增殖的影响。结果显示,15.6-62.5μg/mLACT能够明显抑制PCV2在PK-15细胞上的增殖(p<0.01);进一步小鼠试验显示,按每公斤体重0.2mg腹腔注射ACT能够明显抑制PCV2在小鼠肺、脾及腹股沟淋巴结组织内的增殖(p<0.01)。分析了 ACT对人工感染PCV2仔猪体内病毒增殖及组织病理学影响。结果显示,攻毒前按剂量20μg/kg和50μg/kg连续给药3天均能显著抑制攻毒后7、14和21d仔猪外周血病毒载量(p<0.05,p<0.01);攻毒后21d将全部试验仔猪剖杀,发现给药组1(20 μg/kg)能显著抑制PCV2在腹股沟淋巴结(p<0.05)、肠系膜淋巴结(p<0.05)组织中的增殖,给药组2(50μg/kg)还能显著抑制PCV2在扁桃体(p<0.05)和肺(p<0.05)组织中的增殖;病理切片和组织病理学评分结果显示,两个剂量ACT均能减轻PCV2感染造成的上述脏器组织的病理损伤。分析了 ACT对PRRSV体外增殖的影响。结果显示,12.5-100μg/mL的ACT对1OOTCID50及10TCID50PRRSV均显示出显著的直接灭活作用(p<0.01);0.39-100μg/mLACT能显著抑制100TCID50及 10TCID50PRRSV 在 MARC-145 细胞的增殖(p<0.01);而 0.19-100μg/mLACT 预处理细胞并不能影响PRRSV的感染和增殖。从细胞周期调控角度探讨了 ACT抑制PCV2增殖的机理。结果显示,62.5μg/mLACT能抑制PCV2感染导致的PK-15细胞S期延迟(p<0.01);同时抑制CyclinE2、CDK2、p53的转录和表达。表明ACT可能通过抑制CyclinE2、CDK2、p53蛋白的表达影响细胞周期分布,从而抑制PCV2在PK-15细胞的增殖。综上,本研究建立了一种简便、稳定的ACT制备方法,并对ACT在体内外抗PCV2和PRRSV感染的作用和效果进行了研究分析。结果表明,ACT对PCV2在细胞、小鼠及仔猪的增殖均具有良好的抑制效果,在体外对PRRSV也具有一定的直接灭活和抑制作用,并发现ACT可能通过调控细胞周期抑制PCV2在PK-15细胞的增殖。研究结果为进一步挖掘ACT抗病毒机理及临床防控PCVAD和PRRS提供了试验依据。更多还原

【Abstract】 The Chinese herbal medicines,with a long history in our country,have unique advantages in the prevention and treatment of viral diseases.Some medicines can not only share direct effects on virus replication,but also regulate the immune function with low side effects.Porcine circovirus type 2(PCV2)and porcine reproductive and respiratory syndrome virus(PRRSV)are important pathogens that endanger the global pig industry.They can cause immunosuppression in pigs,leading to the secondary infection of other pathogens,and result in huge economic losses.Vaccination alone cannot achieve the desired effect of prevention and control.Therefore,it is necessary to develop novel prevention and control measures.In this study,we extracted and purified the arctigenin(ACT)from the herbal medicine fructus arctii and analyzed its antiviral activity for PCV2 and PRRSV in vitro and vivo,in order to provide scientific evidence for the development and clinical use of ACT.A mixture of degreased powder of fructus arctii and 50%alcohol solution containing 5%hydrochloric acid was refluxed for 5 h.After filtration,the solid residue was re-suspended in 50%alcohol solution,and refluxed for 1 h again.The pH value of the filtrate was adjusted to neutral by 5mol/L NaOH,and then extracted twice with chloroform.Then,the combined organic phase was evaporated using a rotary evaporator under reduced pressure.The crude extract was purified by silica gel column chromatography twice,and finally the ACT was obtained.The average ACT concentration in the three batches was 98.2%,compared with reference substance.And the average ratio of extraction reached 1.62%.The effect of ACT on the proliferation of PCV2 in PK-15 cells and mice was analyzed.The results showed that 15.6-62.5μg/mL of ACT could significantly inhibit the replication of PCV2 in PK-15 cells(p<0.01).In vivo,the ACT could significantly inhibit the replication of PCV2 in the lung,spleen,and inguinal lymph nodes of mice at the dose of 0.2mg/kg by intraperitoneal injection(p<0.01).The effects of ACT on PCV2 replication and histopathology induced by PCV2 infection were analyzed in piglets.Compared to the control group,the viral loads in drug-treated groups(20μg/kg and 50μg/kg)decreased significantly on day 7,14,and 21 post-challenge(p<0.05,p<0.01).The piglets were sacrificed on day 21 post-challenge and related tissues were collected for pathological,virological and immunohistochemical examinations.In group 1(20μg/kg),the proliferation of PCV2 significantly lowered in the inguinal lymph nodes(p<0.05)and mesenteric lymph nodes(p<0.05),and in group 2(50μg/kg),the proliferation of PCV2 significantly decreased in mesenterium lymph nodes(p<0.001),inguinal lymph nodes(p<0.01),tonsilla(p<0.05)and lungs(p<0.05).The results of histopathological section and scores showed that both doses of the ACT could reduce the pathological damage of the aforementioned organs caused by PCV2 infection.The effects of ACT on the proliferation of PRRSV were analyzed.The results showed that the ACT had direct inactivation on PRRSV in 100TCID50 and 10TCID50,at dosage of 12.5-100μg/mL(p<0.01),and significantly inhibited the proliferation of PRRSV with 100TCID50 and 1OTCID50 in MARC-145 cells at dosage of 0.39-100μg/mL(p<0.01).However,the proliferation of PRRSV was not inhibited when MARC-145 cells was pretreated with the ACT at the dosage of 0.19-100μg/mL.The mechanism of ACT inhibition on PCV2 proliferation was analyzed from the perspective of cell cycle regulation.The results showed that 62.5 μg/mL ACT could inhibit PK-15 cells S-phase arrest(p<0.01)caused by PCV2 infection.Meanwhile,the increase of CyclinE2,CDK2 and p53 mRNA caused by PCV2 infection were significantly inhibited,and further Western Blot results were consistent with qPCR.It is suggested that the ACT may inhibit the cell cycle distribution by inhibiting the expression of CyclinE2,CDK2 and p53 proteins,thus inhibiting the proliferation of PCV2 in PK-15 cells.Taken as a whole,we established a simple and stable method for ACT extraction.Moreover,the antiviral activity of ACT against PCV2 and PRRSV were analyzed in vitro and vivo.The results showed that the ACT can effectively inhibit the proliferation of PCV2 in vitro,mice and piglets.ACT was also shown to inhibit the growth of PRRSV in vitro.Further research found that ACT could inhibited PCV2 proliferation in PK-15 cells by regulating of cell cycle.Our findings provided novel ideas and experimental data for further understanding the antiviral mechanism of ACT and the clinical control of PCVAD and PRRS.更多还原