地西他滨对猪圆环病毒2型的体外抑制作用

Inhibitory Effect of Decitabine on Porcine Circovirus Type 2 in vitro

猪圆环病毒2型(PCV2)作为无囊膜单股负链环状DNA病毒,能引起感染猪的免疫抑制,是猪圆环病毒相关疾病的主要病原体,广泛传播于世界各地,给全球养猪业造成了巨大损失。为探究地西他滨(DAC)对PCV2增殖的影响及其作用机制。本研究构建了感染PCV2的PK-15细胞模型,使用CCK-8法检测DAC对PK-15细胞的细胞毒性,进而使用荧光定量PCR、间接免疫荧光和Western blot检测DAC对PCV2增殖水平的影响;通过间接免疫荧光法标记5甲基胞嘧啶表征DAC对细胞基因组DNA全局甲基化的影响;通过RNA干扰技术在PK-15细胞中沉默DAC的作用靶点DNA甲基转移酶1(Dnmt1),进一步探究DAC抑制PCV2增殖的作用机理。结果显示,DAC在20μmol·L^(-1)时才具有轻微的细胞毒性,并在浓度为10μmol·L^(-1)时即表现出明显的抗PCV2活性,可以抑制不同时间的PCV2增殖。DAC处理细胞后能显著降低PK-15细胞基因组DNA的甲基化,并诱导Ⅰ型干扰素(Ⅰ-IFN)和干扰素刺激基因(ISGs)的表达。沉默DAC的作用靶点Dnmt1能显著抑制PCV2的增殖,且同样诱导I-IFN和ISGs的表达。本研究证实DAC能够有效抑制PCV2的增殖,且发现DAC能促进I-IFN和ISGs的表达并抑制DNA的甲基化,推测DAC抗PCV2病毒的功能与I-IFN和ISGs的表达上调有关,提示DAC具备开发成PCV2临床抗病毒药物的潜力,还揭示Dnmt1是激活宿主固有免疫抗病毒通路的潜在靶点,为新的PCV2抗病毒药物研发提供理论依据。

Porcine circovirus type 2(PCV2)is a non-enveloped single-stranded negative-strand circular DNA virus that can cause immunosuppression in infected pigs.It is the main pathogen of porcine circovirus associated diseases and is widely spread around the world.The purpose of this paper is to explore the effect of decitabine(DAC)on the proliferation of PCV2 and its mechanism.In this study,a PK-15 cell model infected with PCV2 was constructed,and the cytotoxicity of DAC to PK-15 cells was detected by CCK-8 method,and then the effect of DAC on the proliferation of PCV2 was detected by fluorescent quantitative PCR,indirect immunofluorescence and Western blot.The effect of DAC on the global methylation of cellular genomic DNA was characterized by labeling 5-methylcytosine by indirect immunofluorescence method;Dnmt1(DNA methytransferase 1),as the target of DAC in PK-15 cells,was silenced by RNA interference technology,to further explore the inhibitory effect of DAC on PCV2 proliferation.The results showed that DAC had slight cytotoxicity at 20μmol·L^(-1),and showed obvious anti-PCV2 activity at a concentration of 10μmol·L^(-1),and could inhibit the proliferation of PCV2 at different times.DAC treatment of cells can significantly reduce the methylation level of genomic DNA in PK-15 cells and induce the expression of typeⅠinterferon(Ⅰ-IFN)and interferon-stimulated genes(ISGs).Silencing Dnmt1,the target of DAC,significantly inhibited the proliferation of PCV2 and induced the expression of I-IFN and ISGs.This study confirmed that DAC can play an anti-PCV2 function by inhibiting DNA methylation and promoting the expression of I-IFN and ISGs.Furthermore,DAC has the potential to be developed into a clinical antiviral drug against PCV2,Dnmt1 is a potential target for activating the host′s innate immune antiviral pathway,providing a theoretical basis for the development of new PCV2 antiviral drugs.