1、第 57 卷第 1 期河 南 农 业 大 学 学 报Vol 57No 12023 年2 月Journal of Henan Agricultural UniversityFeb2023收稿日期:20220111基金项目:广东省现代农业产业技术体系南方现代草牧业产业项目(2019K127)作者简介:焦豪杰(1995)，男，河南焦作人，硕士研究生，主要从事动物营养与免疫的研究。通信作者:刘铀(1966)，男，湖南湘潭人，教授，博士。引用:焦豪杰，陈绍红，刘铀 异绿原酸 A 体外抗小反刍兽疫病毒活性及其作用机制研究J 河南农业大学学报，2023，57(1):125135 DOI:10 16445/j
2、 cnki 10002340 20220329 001异绿原酸 A 体外抗小反刍兽疫病毒活性及其作用机制研究焦豪杰1，陈绍红2，刘铀2(1 广东海洋大学滨海农业学院，广东 湛江 524088;2 广东海洋大学食品科技学院，广东 湛江 524088)摘要:【目的】研究异绿原酸 A(isochlorogenic acid A，IAA)体外抗小反刍兽疫病毒(peste des petits ruminant virus，PPV)的效果及其作用机制。【方法】采用 MTT 法测定 IAA 对非洲绿猴肾细胞(verda reno，Vero)的最大无毒质量浓度、半数细胞毒性质量浓度(50%cytotoxic
3、 mass concentration，CC50);空斑形成试验测定 IAA 的半数有效质量浓度(median effect mass concentration，EC50)并计算治疗指数(therapeutic index，TI);MTT 法观察 IAA 对 PPV感染的 Vero 细胞活性的影响;采用空斑形成试验、实时荧光定量 PC(quantitative real-time PC，qTPC)、免疫印迹试验和间接免疫荧光试验观察 IAA 对 PPV 增殖和分布的影响;采用活性氧(reactive oxygen species，OS)荧光探针、硫代巴比妥酸(thiobarbituric a
4、cid，TBA)比色法和羟胺比色法分别检测 IAA 对 PPV 感染细胞内OS、丙二醛(malondialdehyde，MDA)含量和超氧化物歧化酶(superoxide dismutase，SOD)活性的影响;采用免疫印迹试验检测 IAA 对 PPV 感染细胞中磷脂酰肌醇 3激酶(phosphatidylinositol 3kinase，PI3K)、蛋白激酶 B(protein kinase B，AKT)、磷酸化 AKT(pAKT)、B 细胞淋巴瘤2 蛋白(B cell lymphoma2 protein，Bcl2)和 Bcl2 相关 X 蛋白(Bcl2 associated X prote
5、in，Bax)表达水平的影响。【结果】IAA 对 Vero 细胞的最大无毒质量浓度、CC50、EC50分别为 7 81、125 60、21 02 mgL1，TI 值为 5 98;IAA 处理能显著抑制 PPV 诱导的细胞病变及病毒在 Vero 细胞中的增殖;细胞活力显著高于病毒感染细胞;病毒噬斑形成数量、病毒 F 和 N 基因表达水平显著降低;宿主细胞胞浆内病毒分布显著减少。IAA 能显著提高 PPV 感染细胞 SOD 活性，清除病毒感染细胞内的OS 和 MDA。经 IAA 处理的 PPV 感染细胞中 PI3K、AKT 表达水平极显著提高，pAKT 表达水平显著降低，Bax/Bcl2 比值显著
6、降低。【结论】IAA 通过调节宿主细胞 PI3K/AKT 通路及其下游细胞凋亡相关蛋白 Bax 和Bcl2 的表达水平来缓解 PPV 感染引起的氧化应激反应，从而发挥其抗 PPV 活性。关键词:异绿原酸 A;小反刍兽疫病毒;抗病毒活性;作用机制;信号通路中图分类号:S859 1文献标志码:A文章编号:10002340(2023)01012511Study on antiviral activity of isochlorogenic acid A against peste despetit ruminant virus in vitro and its action mechanismJIA
7、O Haojie1，CHEN Shaohong2，LIU You2(1 College of Coastal Agricultural Science，Guangdong Ocean University，Zhanjiang 524088，China;2 College of Food Science and Technology，Guangdong Ocean University，Zhanjiang 524088，China)Abstract:【Objective】The present work was conducted to investigate the antiviral act
8、ivity of isochlo-rogenic acid A(IAA)against peste des petits ruminant virus(PPV)in vitro and its mechanism【Methods】The maximum non-toxic mass concentration and the 50%cytotoxic mass concentration126河南农业大学学报第 57 卷(CC50)of IAA on African green monkey kidney cells(verda reno，Vero)were determined by MTT
9、method The median effective mass concentration(EC50)of IAA was determined by plaque formationtest and the therapeutic index(TI)was calculated The effect of IAA on the cell viability of thePPV-infected Vero cells was determined by MTT assay，and the effects of IAA on PPV proliferationand distribution
10、in the host cells were observed by plaque formation experiment，quantitative real-timePC(qTPC)，western-blot(WB)and indirect immunofluorescen assay respectively The effectsof IAA on the intracellular reactive oxygen species(OS)content，malondialdehyde content and activi-ty of superoxide dismutase(SOD)o
11、f the virus-infected cells were respectively evaluated by fluorescentprobe，thiobarbituric acid and hydroxylamine colorimetry method，and the effects of IAA on the expres-sion levels of PI3K，AKT，pAKT，Bcl2 and Bax in the PPV-infected cells were detected by West-ern-blot【esult】The results showed that th
12、e maximum non-toxic mass concentration，the CC50on Ve-ro cells，the EC50on PPV and the TI were 7 81，125 60，21 02 mgL1and 5 98，respectively Thecytopathic effect induced by PPV infection was significantly inhibited by IAA treatment and the via-bility of the virus infected cells was significantly higher
13、than that of PPV-infected cells The numberof plaque formation units and the expression levels of viral structural protein F and N were significantlydecreased The virions in cytoplasm of vero cells were also decreased significantly Moreover，the activityof SOD of the PPV-infected cells was significant
14、ly increased by IAA，and the content of OS andMDA was accordingly decreased The expression levels of PI3K and AKT were significantly up-regula-ted，whereas the experssion of pAKT was significantly down-regulated and the Bax/Bcl2 ratio wasalso significantly decreased in the PPV-infected cells due to IA
15、A treatment【Conclusion】IAA exertsits antiviral activity against PPV by regulating the expression of PI3K/AKT pathway and downstreammolecules Bax and Bcl2，which was closely related to apoptosis of the virus infected cells，and allevi-ating oxidative stress caused by virus infectionKey words:isochlorog
16、enic acid A;peste des petis ruminants virus;antiviral activity;action mecha-nism;signal pathway小反刍兽疫(peste des petits ruminants，PP)是由小反刍兽疫病毒(peste des petits ruminants vi-rus，PPV)引起的急性、高度接触性传染病，主要感染山羊、绵羊及美国白尾鹿等小反刍动物。临床诊断上 PP 主要表现为高热、坏死性口炎、眼鼻出现黏性或脓性分泌物、咳嗽、呼吸困难及剧烈腹泻，发病率和病死率高达 100%和 90%，给世界养羊业带来了严重的经济损失1。弱毒疫苗在控制 PP的暴发流行中发挥了重要作用，但是也存在一些隐患。一是 PPV 对淋巴组织具有高度亲嗜性，会抑制干扰素和炎症因子合成并诱导淋巴细胞凋亡，从而引发免疫动物的短暂免疫抑制2;二是弱毒疫苗热稳定性较差，当缺少冷链运输或在配苗后不能及时接种时其免疫效果就会不佳3;三是一些偏远地区 PP 疫苗覆盖率不高，同时野生动物迁徙又会带来病毒传播和扩散的潜在风险。因此，PP防控形势依然严峻，研