药理激活p38/MAPK体外抗传染性喉气管炎病毒效应的检测  

Investigating the antiviral function of pharmacological activation of p38/MAPK against ILTV in vitro

作　　者：崔露 刘柘一 张宇[1] 韩宗玺[1] 刘胜旺[1] 李海 CUI Lu;LIU Zhe-yi;ZHANG Yu;HAN Zong-xi;LIU Sheng-wang;LI Hai(State Key Laboratory for Animal Disease Control and Prevention/Avian Respiratory Diseases Division,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室禽呼吸道传染病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第7期723-730,746,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金(JQ2021C006);国家自然科学基金(32072853);现代农业产业技术体系(国家蛋鸡体系岗位科学家)项目(CARS-40-K18)。

摘　　要：鸡传染性喉气管炎病毒(ILTV)是一种严重危害我国和世界养禽业的重要病原。本研究以ILTV-LJS09株感染的鸡LMH细胞系作为研究模型,对该病毒的全基因组转录谱分析,采用基因富集分析(GSEA)分析细胞中的信号通路。结果发现MAPK通路是ILTV感染LMH细胞后最显著激活的通路。进一步采用western blot鉴定,结果显示ILTV感染可以显著激活宿主细胞p38/MAPK信号通路。本研究同时采用脱氢紫堇碱(DHC)和佛波酯(PMA)作为p38/MAPK激活剂,采用特异性荧光定量PCR检测病毒基因组拷贝数,通过测定病毒的TCID_(50)检测病毒滴度,利用荧光定量PCR检测细胞中代谢相关基因的转录水平,使用高内涵成像技术结合Annexin V-FITC/碘化丙啶双染法检测细胞凋亡,从而探究药理激活p38/MAPK对ILTV感染的影响。病毒特异性荧光定量PCR检测结果显示,DHC和PMA均显著降低ILTV基因组的复制(P<0.05)。TCID_(50)测定结果显示,DHC和PMA还显著抑制ILTV感染性病毒粒子的增殖(P<0.05)。荧光定量PCR检测结果显示,无论DHC还是PMA对感染细胞代谢基因的总体转录水平均无明显影响。高内涵成像技术检测结果显示,DHC和PMA显著加剧ILTV感染细胞的凋亡(P<0.05)。综上所述,本研究结果显示DHC和PMA均能够在不影响感染细胞代谢基因表达的情况下,通过加剧ILTV感染细胞的凋亡有效抑制ILTV的感染。本研究在ILTV体外感染模型中证明药理激活p38/MAPK信号通路具有高效的抗病毒作用,为治疗ILTV的感染提供了新的思路,对其他疱疹病毒感染的防治研究也具有借鉴意义。Infectious laryngotracheitis virus(ILTV)constitutes a major threat to the poultry industry worldwide,particularly in China.Using the chicken liver hepatocellular carcinoma(LMH)cell line infected with ILTV-LJS09 as the experimental model,this study conducted a whole-genome transcriptome analysis and found that the MAPK pathway is the most significantly activated pathway in LMH cells in response to ILTV infection.Addtionally western blot detection identified significant activation of host cell p38/MAPK by ILTV infection.To investigate the impact of pharmacological p38/MAPK activation on ILTV infection,two activators,Dehy-drocorydaline(DHC)and Phorbol 12-myristate 13-acetate(PMA),were employed in LMH cells.Fluorescence quantitative PCR was employed to detect the copy number of the virus genome,the 50%tissue culture infectious dose was used to determine viral titers,Fluorescence quantitative PCR was used to detect the transcription level of metabolism-related genes,and high-content imaging technology combined with Annexin V-FITC/propidium iodide double staining was used to detect cell apoptosis,thereby exploring the effect of pharm-acological activation of p38/MAPK on ILTV infection.The results of virus-specific fluorescence quantitative PCR detection showed that both DHC and PMA significantly reduced the replication of the ILTV genome(P<0.05).The detection of 50%tissue culture infectious dose showed that DHC and PMA also significantly reduced the proliferation of infectious ILTV virus particles(P<0.05).The results of fluorescence quantitative PCR detection showed that neither DHC nor PMA had a significant impact on the overall expression of metabolic genes in infected cells.The analysis of high-content imaging showed that DHC and PMA significantly intensified the apoptosis of ILTV-infected cells(P<0.05).In summary,the results of this study show that both DHC and PMA can effectively inhibit ILTV infection by intensifying the apoptosis of ILTV-infected cells without affecting the expression of metabolic genes in infec

关 键 词：传染性喉气管炎病毒 P38丝裂原活化蛋白激酶 转录谱 凋亡 

分 类 号：S852.65[农业科学—基础兽医学]