摘要
为探究绿原酸对鸭肠炎病毒(DEV)感染的鸭胚成纤维细胞(DEF)代谢组学的影响,本研究首先通过CCK-8法筛选绿原酸对DEF的最适作用浓度,结果显示绿原酸最佳作用浓度为0.250 mg/mL。将DEF分别设置空白对照组(DEF)、绿原酸对照组(经0.250 mg/mL绿原酸溶液作用的DEF)、DEV感染组(DEV病毒液感染的DEF)和绿原酸干预组(0.25 mg/mL绿原酸溶液作用DEF后接种DEV病毒液),培养至24 h、36 h和48 h时的各组DEF,经CCK-8检测细胞活性后,采用液相色谱-质谱联用技术(LC-MS)测定各组细胞间的差异代谢物,经MetaX软件筛选各组细胞间显著差异代谢物,KEGG分析显著差异代谢物富集的信号通路,并进一步采用荧光定量RT-PCR检测上述各组细胞中DEV NP基因拷贝数,分析DEV的增殖情况。结果显示:与空白对照组相比,绿原酸对照组在3个时间点的细胞活性差异均不显著(P>0.05),而DEV感染组的细胞活性均极显著下降(P<0.01),绿原酸干预组在24 h和36 h时的细胞活性均显著下降(P<0.05),48 h时极显著下降(P<0.01);与DEV感染组相比,绿原酸干预组在3个时间点的差异代谢物分别有28、31和21个,差异代谢物主要是六亚甲基双乙酰胺、S-乳酰谷胱甘肽、L-谷氨酸,L-蛋氨酸、吲哚-3-乙酸、甲酰基-L-蛋酰肽等,富集的信号通路主要有半胱氨酸与蛋氨酸代谢、酪氨酸代谢、苯丙氨酸代谢、色氨酸代谢等代谢通路。荧光定量RT-PCR检测结果显示,与空白对照组相比,DEV感染组DEV NP基因拷贝数在3个时间点均显著上升(P<0.05);与DEV感染组相比,绿原酸干预组DEV NP基因的拷贝数在3个时间点均显著下降(P<0.05)。上述结果表明,绿原酸通过调控DEF细胞半胱氨酸和蛋氨酸等氨基酸代谢通路抑制DEV增殖,该结果为绿原酸在鸭病毒性肠炎等疾病防治中的应用提供了科学依据。
To investigate the effect of chlorogenic acid on the metabolome of duck embryo fibroblast(DEF)infected by duck enteritis virus(DEV),DEF cells were prepared from 11-day-old SPF duck embryos.After the chlorogenic acid concentration was screened by CCK-8 method,four experimental groups were established:a blank control group(DEF cultured for 24 hours,36 hours and 48 hours),chlorogenic acid control group(DEF treated with 0.25mg/mL chlorogenic acid for 1 hour,followed by culture for 24 hours,36 hours and 48 hours);a DEV infection group(DEF infected with DEV for 1 hour,then cultured for 24 hours,36 hours,and 48 hour),and a chlorogenic acid intervention group(DEF treated with 0.25mg/mL chlorogenic acid for 1 hour,followed by infection with DEV for 1 hour and subsequent culture for 24 hours,36 hours,and 48 hours).Cell samples were collected at each time point and analyzed by liquid chromatography-mass spectrometry(LC-MS)to identify differential metabolites,MetaX software was used to screen these metabolites,and KEGG analysis was performed to identify enriched metabolic pathways.Meanwhile,the proliferation of DEV was detected by fluorescence quantitative RT-PCR targeting the DEV NP gene.Results:Compared to the control group,cell viability in the chlorogenic acid control group showed no significant differences at any time point(P>0.05).However,cell viability in the DEV infection group decreased significantly(P<0.01).In the chlorogenic acid intervention group,cell viability decreased significantly at 24 hours and 36 hours(P<0.05)and further decreased at 48 hours(P<0.01).Compared to the DEV infection group,the chlorogenic acid intervention group exhibited 28,31,and 21 differential metabolites at 24 hours,36 hours,and 48 hours,respectively.These metabolites included hexamethylene diacetylamine,S-lactoylglutathione,L-glutamic acid,L-methionine,indole-3-acetic acid,formyl-l-egg acylpeptide etc.The enriched signaling pathways mainly included cysteine and methionine metabolism,tyrosine metabolism,phenylalanine metabolism,tryptophan metabolism.The results of fluorescence quantitative RTPCR showed that compared with the virus infection group,the copy number of the DEV NP gene in the chlorogenic acid intervention group decreased significantly at the three time points(P<0.05).These results indicated that chlorogenic acid inhibited DEV proliferation by altering the metabolism of amino acids such as cysteine and methionine in DEF cells,which provided scientific basis for the prevention and treatment of duck viral enteritis and other viral diseases.
作者
杨芸芸
张黔东
冯轶
文安林
杨颖
文明
刘江
YANG Yun-yun;ZHANG Qian-dong;FENG Yi;WEN An-lin;YANG Ying;WEN Ming;LIU Jiang(College of Animal Science,Guizhou University,Guiyang 550025,China;Huishui County Mengjiang Street Agricultural and Rural Comprehensive Service Center,Huishui 550600,China;Guizhou Center of Animal Disease Prevention and Control,Guiyang 550025,China;Bijie City Agriculture and rural Bureau,Bijie 551000,China)
出处
《中国预防兽医学报》
CSCD
北大核心
2024年第12期1236-1244,共9页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(32360872和315060703)
贵州省百层次创新型人才项目(黔科合平台人才[2016]6009号)
贵州省科技平台项目[黔科合平台人才[2018]5253号]。
关键词
鸭肠炎病毒
绿原酸
鸭胚成纤维细胞
代谢组学分析
代谢通路
duck enteritis virus
chlorogenic acid
duck embryo fibroblast
metabolomic analysis
metabolic pathway
