【作者】 高雅；

【导师】 周斌；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2021， 硕士

【摘要】 姜黄素（Curcumin）是姜科植物姜黄根茎提取出的脂溶性酚类色素,呈橙色晶体状,分子式为C₂₁H₂₀O₆,相对分子质量368.39,主链为芳香族及不饱和脂肪族基团,有光热敏性。目前国内外研究已证实其具有天然的生物活性,包括抗肿瘤、抗突变、抗氧化、抗菌和抗病毒等。因其可提高动物免疫力,促进动物生长等作用,也有研究其在动物饲料添加剂等方面的应用。猪瘟（Classical swine fever,CSF）是由猪瘟病毒（Classical swine fever virus,CSFV）引起的猪的高度接触性传染病,具有高发病率和致死率,典型特征是高热、出血以及白细胞减少,还可导致怀孕母猪流产,对养猪业危害极大。我国主要以疫苗接种为主,以生物安全措施为辅助防治猪瘟,通过尽可能降低猪瘟流行概率,以减少经济损失。虽然现在已有商品化疫苗应用于养猪业并产生良好的保护作用,但由于猪瘟病毒毒株复杂多变,导致现有的疫苗保护力不足以完全避免猪瘟病毒的爆发,仍需继续研制安全、稳定、高效、低毒的新型疫苗或抗病毒制剂。目前,姜黄素的抗病毒作用及机制已在人类免疫缺陷病毒（HIV）、寨卡病毒（ZIKV）、日本脑炎病毒（JEV）、登革热病毒（DENV）、猪繁殖与呼吸综合征病毒（PRRSV）、传染性胃肠炎病毒（TGEV）等中得到验证。大量研究结果表明,姜黄素可影响多种代谢途径或信号转导途径,发挥抗病毒感染、减少病毒代谢物和子代病毒粒子的释放等多种作用以保护宿主细胞。基于姜黄素丰富的抗病毒特性以及猪群猪瘟感染的复杂特性,我们对姜黄素抗CSFV的特性进行探究。本实验前期证明姜黄素对CSFV的抑制作用,而后检测姜黄素对脂质代谢的影响,继而针对其促进先天性免疫的特性进行验证,进而对于姜黄素抑制CSFV复制的具体作用机制进行阐述。具体研究内容如下:1对姜黄素抑制猪瘟病毒的感染研究使用安全剂量下的姜黄素处理感染CSFV和JEV的PK-15细胞,通过RT-q PCR、Western blot、IFA等多种方式来证明药物对病毒的抑制效果,设置不同加药时间点来确定药物作用的具体阶段。结果显示,姜黄素对CSFV的抑制具有浓度依赖性,共同孵育可抑制病毒的感染性,但对于病毒的吸附和内化过程无明显作用,主要影响病毒复制的后期阶段。另外,通过实验证实姜黄素对JEV的抑制也具有浓度依赖性。由此可见,姜黄素具有良好的抗病毒活性,能抑制CSFV和JEV的复制,为后续的研究奠定基础。2对姜黄素影响脂质代谢调控猪瘟病毒的复制的研究为探究姜黄素在猪瘟病毒复制中的分子机制,首先通过激光共聚焦检测姜黄素处理后感染病毒细胞中的脂肪酸合成酶（Fatty acid synthase,FASN）和脂滴（lipid droplets,LDs）的变化。结果显示,在感染和未感染的细胞中,姜黄素处理均能抑制脂肪酸合成酶（FASN）和脂滴（LDs）的产生和积累。另外,活化转录因子6（activating transcription factor 6,ATF6）是调控脂质代谢的相关因子。一系列实验证明姜黄素抑制CSFV感染引起的ATF6的上调。敲低细胞内源性ATF6后,FASN蛋白表达下降,同时,姜黄素的抗病毒作用减弱,但未敲低ATF6的处理组中,姜黄素仍显示出明显的抗病毒活性,也抑制ATF6和FASN的m RNA水平和蛋白表达水平。由此可见,姜黄素可通过抑制ATF6来调控细胞脂质的积累从而抑制病毒的复制。3对姜黄素增强病毒感染后的先天性免疫应答的研究姜黄素的抗病毒机制并不单一。为继续挖掘姜黄素抗CSFV的潜在机制,探究姜黄素处理后对宿主细胞先天性免疫的影响。通过RT-q PCR检测I型干扰素（Interferon-I,IFN-I）通路相关的干扰素调节因子3（Interferon regulatory factor 3,IRF3）、干扰素调节因子7（Interferon regulatory factor 3,IRF7）、干扰素刺激基因15（Interferon-stimulated gene 15,ISG15）、干扰素α1（IFN-α1）的m RNA水平,发现姜黄素可上调它们的m RNA水平。敲低PK-15细胞抗病毒关键因子ISG15后,RT-q PCR验证敲低效率并检测药物处理后子代病毒含量,发现敲低ISG15后姜黄素仍可发挥抗病毒作用,但效果被削弱。使用商品化试剂盒检测细胞上清中IFN-β的含量,发现姜黄素可促进IFN-β的分泌。据此证明姜黄素会增强病毒感染后的先天性免疫反应。通过RT-q PCR和Western blot检测同样处理方式之下,姜黄素对NF-κB通路的影响。结果显示姜黄素对NF-κB通路上的p65和磷酸化p65无影响。由此可见,姜黄素可以增强宿主细胞的先天性免疫,促进ISG15的表达,同时可以上调I型干扰素（IFN-I）通路相关的因子。更多还原

【Abstract】 Curcumin is a fat-soluble phenolic pigment extracted from the the Curcuma longa.It is in the form of orange crystals.Its molecular formula is C₂₁H₂₀O₆.The relative molecular weight is 368.39.The main chain is unsaturated aliphatic and aromatic groups.At present,domestic and foreign studies have confirmed that it has natural biological activities,including anti-tumor,anti-mutation,anti-oxidation,anti-bacterial and anti-virus.Because it can improve animal immunity and promote animal growth,it has also been studied for its application in animal feed additives.Classical swine fever is a highly contagious infectious disease of pigs caused by classical swine fever virus,with high morbidity and mortality which caused huge economic losses.The typical features of CSF are fever and hemorrhage.As well as the reduction of leukocytes,it can also cause abortion in pregnant sows,which is extremely harmful to the pig industry.At present,the prevention and treatment of CSF in China is still based on vaccination to reduce the economic loss by reducing the outbreaks of CSF.Although commercial vaccines are now used in the pig industry and have a certain protective effect,it is still necessary to continue to develop new vaccines or antiviral agents due to the complex and variable strains of swine fever virus.Notably,curcumin exhibits inhibitory ability against the proliferation of diverse viruses,such as human immunodeficiency virus（HIV）,Zika virus（ZIKV）,Japanese encephalitis virus（JEV）,dengue fever virus（DENV）,porcine reproductive and respiratory syndrome virus（PRRSV）,transmissible gastroenteritis virus（TGEV）and so on.A large number of research results show that curcumin have effects on different signal pathways or metabolic pathways to resist the viral infections,reducing the release of infectious virus particles,and protecting host cells.However,there is no report confirm that curcumin can inhibit CSFV.In the early stage of this experiment,the inhibitory effect of curcumin on CSFV was proved.And we tested the effect of curcumin on lipid metabolism,and then the characteristics of promoting innate immunity were verified.At last,the specific mechanism of curcumin in inhibiting CSFV replication was explained.The specific research content is as follows:1 Study On Curcumin Inhibits Classical Swine Fever Virus InfectionThe PK-15 cells were infected with CSFV and JEV and treated with different concentrations of curcumin.Then we use RT-q PCR,Western blot,IFA and other methods to confirm the inhibitory effect of curcumin on CSFV,and set a series experiments to determine the exact stage to inhibit CSFV of curcumin.The results showed that curcumin inhibited CSFV in a time-and dose-dependent manner,and mainly affected the late stage of virus replication,and had nothing to do with the process of virus adsorption and internalization.Curcumin can also affect the infectivity of CSFV after co-incubation.Curcumin can inhibit JEV in a dose-dependent manner.In short,curcumin has good antiviral activity and can inhibit the replication of CSFV and JEV,supporting the follow-up research.2 Study On Curcumin Regulates Lipid Metabolism During the Replication of Classical Swine Fever VirusIn order to explore the specific mechanism of curcumin in the CSFV replication,firstly,the changes of fatty acid synthase（FASN）and lipid droplets（LDs）in infected virus cells after curcumin treatment were detected by confocal.Regardless of normal cells or virus-infected cells（CSFV,JEV）,curcumin treatment can inhibit the production and accumulation of fatty acid synthase（FASN）and lipid droplets（LDs）.In addition,cells infected with the virus can cause endoplasmic reticulum stress,which in turn activates the unfolded protein response,and upregulates the transcription factor ATF6,which is a related factor regulating lipid metabolism.A series of experiments proved that curcumin inhibited the up-regulation of ATF6 caused by CSFV infection.After knocking down ATF6,FASN expression was decreased.At the same time,the ability of curcumin to anti-CSFV disappeared.However,in the treatment group without knocking down ATF6,curcumin still showed antiviral activity,and it also inhibited the m RNA levels and the expression levels of ATF6 and FASN.3 Study On Curcumin Enhances Innate Immunity Response of PK-15 Cells with CSFV infectionCurcumin can exert a broad-spectrum antiviral effect,which proves that curcumin affects multiple signaling pathways.In order to explore the potential mechanism of curcumin against CSFV,the effect of curcumin treatment on the innate immunity of cells was explored.The m RNA levels of IRF3,IRF7,ISG1,IFN-α1 related to the type I interferon（IFN-I）pathway were detected by RT-q PCR,and it was found that curcumin can up-regulate the m RNA levels.After knocking down ISG15,the key factor of host cell antiviral ability,RT-q PCR verified the knockout efficiency and tested the virus content after drug treatment.It was found that curcumin could still exert antiviral effect after knocking down ISG15,but the effect was weakened.Using a commercial kit to detect the content of IFN-βin the cell supernatant,we found that curcumin can up-regulate IFN-β.This proves that curcumin enhances the innate immune response after viral infection.The effect of curcumin on the NF-κB pathway was detected by RT-q PCR and Western blot with the same treatment.Curcumin has no effect on p65 and phosphorylated p65 in the NF-κB pathway.In short,curcumin can enhance the innate immunity of host cells,promote the expression of ISG15,and at the same time it can up-regulate factors related to the type I interferon（IFN-I）pathway.更多还原