茵陈蒿提取物体外抗传染性支气管炎病毒的作用研究  

Study on the effects of Artemisia capillaris extracts against Infectious bronchitis virus in vitro

作　　者：李凌 贾艺泉 方守国[1] 刘卫容 章松柏 LI Ling;JIA Yiquan;FANG Shouguo;LIU Weirong;ZHANG Songbai(College of Agriculture,Yangtze University,Jingzhou 434025,China;Health Science Center,Yangtze University,Jingzhou 434023,China;Ministry of Agriculture and Rural Affairs Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River(Ministry-Province Co-Construction),Jingzhou 434025,China)

机构地区：[1]长江大学农学院,湖北荆州434025 [2]长江大学医学部,湖北荆州434023 [3]农业农村部长江中游作物绿色高效生产重点实验室(部省共建),湖北荆州434025

出　　处：《黑龙江畜牧兽医》2024年第20期95-102,130,131,共10页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31572490);荆州市科技计划项目(2022HC34);长江大学大学生创新创业训练计划项目(Yz2022301)。

摘　　要：为了探讨茵陈蒿提取物体外抗传染性支气管炎病毒(Infectious bronchitis virus,IBV)的作用及机制,试验采用人肺癌细胞(H1299细胞)和非洲绿猴肾细胞(Vero细胞)作为供试细胞,分别在IBV感染前及感染后在细胞培养体系中加入茵陈蒿提取物,探索茵陈蒿是否具有抗IBV作用。再将茵陈蒿提取物以最大无毒质量浓度为起点,倍比稀释成不同质量浓度,观察不同质量浓度茵陈蒿提取物抗IBV作用。试验分为空白对照组、IBV组、病毒感染前加药组、病毒感染后加药组,在每组病毒感染H1299细胞24 h后收集细胞,提取总RNA并反转录合成cDNA,并采用一定体积免疫沉淀组织/细胞裂解液与苯甲基磺酰氟(PMSF)混合液裂解各组细胞提取总蛋白,采用实时荧光定量PCR(qPCR)和Western-blot技术检测IBV感染细胞内凋亡相关基因Bad、Bcl-2、Fas和炎症相关因子NF-κB-p65、磷酸化NF-κB-p65(p-p65)、IL-6、IL-8的mRNA基因和蛋白表达情况,采用间接免疫荧光技术检测p-p65的核移位。结果表明:茵陈蒿提取物体外对IBV的复制具有一定的抑制作用,并呈剂量依赖性,体外最大无毒质量浓度为0.5 g/mL。与空白对照组相比,IBV组凋亡相关基因和炎症相关因子的表达水平均不同程度上调;与IBV组相比,病毒感染前加药组、病毒感染后加药组Fas、IL-6、IL-8(除病毒感染后加药组外)、NF-κB-p65 mRNA相对表达量显著或极显著下降(P<0.05或P<0.01),Bad、Bcl-2、Fas、IL-6、IL-8、NF-κB-p65和p-p65蛋白相对表达量均明显下调;间接免疫荧光结果显示,茵陈蒿提取物处理抑制了p-p65核移位。说明茵陈蒿提取物体外具有抗IBV作用,该作用可能与其下调NF-κB信号通路相关炎症因子的表达及减轻IBV感染引起的宿主细胞凋亡有关。In order to investigate the effects of Artemisia capillaris extracts against Infectious bronchitis virus in vitro and its mechanism,in the experiment,human lung cancer cells(H1299 cells)and African green monkey kidney cells(Vero cells)were used as test cells,and Artemisia capillaris extracts were added to the cell culture system before and after IBV infection,respectively.Artemisia capillaris extracts were diluted into different mass concentrations by multiplicative dilution with the maximum nontoxic mass concentration as the starting point;the anti-IBV effects of Artemisia capillaris extracts at different mass concentrations were observed.The experiment was divided into blank control group,IBV group,pre-viral infection addition group,and post-viral infection addition group.The cells in each group were collected 24 h after virus infected H1299 cells,and total RNA was extracted and reverse transcribed to synthesize cDNA;a certain volume of RIPA mixed with phenylmethylsulfonyl fluoride(PMSF)was used to lyse the cells of each group to extract the total proteins.Real-time fluorescence quantitative PCR(qPCR)and Western-blot techniques were used to detect the mRNA and protein expression of apoptosis-related genes Bad,Bcl-2,Fas,and inflammation-associated factors NF-kB-p65,phosphorylated NF-kB-p65(p-p65),IL-6,and IL-8 in IBV-infected cells.The nuclear translocation of p-p65 was detected by indirect immunofluorescence technology.The results showed that the Artemisia capillaris extracts had certain inhibited effect against the replication of IBV in vitro in a dose-dependent manner;the maximum nontoxic mass concentration in vitro was O.5 g/mL.Compared with the blank control group,the expression levels of apoptosis-related genes and inflammation-related factors were upregulated to different degrees in the IBV group.Compared with the IBV group,the relative expression of Fas,IL-6,IL-8(except for the post-viral infection addition group),and NF-kB-p65 mRNA in the pre-viral infection addition group and the post-viral infection a

关 键 词：茵陈蒿 传染性支气管炎病毒 体外 凋亡 炎症 

分 类 号：S853.7[农业科学—临床兽医学]