百里香药茶对人冠状病毒OC43体内外增殖的影响  

Effect of Thyme Herbal Tea on Proliferation of Human Coronavirus OC43 in vitro and in vivo

作　　者：田纪祥 张彤彤 常雨宁 谢佩芳 董书维 赵晓昂 王云[2] 赵春晖 吴宏伟[2] 张阿梅[3] 李海舟[3] 夏雪山 张华敏[1] TIAN Jixiang;ZHANG Tongtong;CHANG Yuning;XIE Peifang;DONG Shuwei;ZHAO Xiaoang;WANG Yun;ZHAO Chunhui;WU Hongwei;ZHANG Amei;LI Haizhou;XIA Xueshan;ZHANG Huamin(Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China;Yunnan Provincial Key Laboratory of Public Health and Biosafety,Kunming Medical University,Kunming 650500,China)

机构地区：[1]中国中医科学院中医基础理论研究所,北京100700 [2]中国中医科学院中药研究所,道地药材与品质保障全国重点实验室,北京100700 [3]昆明理工大学生命科学与技术学院,昆明650500 [4]昆明医科大学云南省公共卫生与生物安全重点实验室,昆明650500

出　　处：《中国实验方剂学杂志》2024年第23期81-89,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中央本级重大增减支项目(2060302);国家“重大新药创制”科技重大专项(2019ZX09201005);中国中医科学院中医药防治流感技术体系资助项目(ZZ13-035-04)。

摘　　要：目的:探究百里香药茶(BLX)对人冠状病毒OC43(HCoV-OC43)的体内外抗病毒活性及作用机制。方法:通过超高效液相色谱-质谱(UPLC-MS)分析BLX化学成分,利用HCoV-OC43细胞模型检测BLX的体外毒性及其对该病毒的抑制作用,采用时间差异给药法检测药物作用阶段,实时荧光定量聚合酶链式反应(Real-time PCR)法检测病毒基因拷贝数,利用HCoV-OC43小鼠模型检测BLX的体内毒性及其对小鼠体质量和生存周期变化的影响,通过免疫组化技术检测脑和肺组织中病毒蛋白表达。结果:从BLX中鉴定出11种化合物;在HCoV-OC43感染的HRT-18细胞中,BLX的半数毒性浓度(CC50)为(13859.56±319)mg·L^(-1),半数抑制浓度(IC50)为(1439.09±200)mg·L^(-1),其选择指数范围为8.26~11.44。与病毒组比较,BLX在1500、1000、500 mg·L^(-1)剂量下均可明显抑制病毒复制(P<0.05,P<0.01),该药物在病毒复制早期即可发挥抗病毒作用,且对病毒吸附过程的干扰大于对病毒入胞过程的干扰(P<0.05)。在HCoV-OC43感染的小鼠模型中,BLX在1200、600 mg·kg^(-1)·d^(-1)剂量下可缓解被感染小鼠的症状、延长生存期并降低死亡率,有效抑制脑组织中(P<0.01)和肺组织(P<0.01)中病毒核衣壳mRNA表达水平;BLX在1200mg·kg^(-1)剂量下可抑制脑组织(P<0.01)和肺组织(P<0.01)中病毒核衣壳蛋白的表达。结论:BLX含有多种具有抗病毒活性的化学成分,可在体内外抑制HCoV-OC43复制,该药物可能通过干扰病毒吸附等方式发挥其抗病毒活性。该研究为冠状病毒感染治疗提供了候选药物,为BLX的进一步开发应用提供了理论参考。Objective:To investigate the effects of thyme herbal tea(BLX)on the proliferation of human coronavirus OC43(HCoV-OC43)in vitro and in vivo.Method:The chemical composition of BLX was analyzed by UPLC-MS.The cytotoxicity of BLX in HRT-18 cells and the effect of BLX treatment on the proliferation of HCoV-OC43 in cells were analyzed.Copies of viral gene were detected by real-time PCR.The effect of BLX treatment on the life cycle of HCoV-OC43 was detected by time-of-addition assay.The maximum tolerated dose of BLX and the influences of BLX on the body weight and survival time of suckling mice infected with HCoV-OC43 were determined.The expression of viral protein in the brain and lung tissue was analyzed by immunohistochemistry.Result:There were 11 chemical components identified in BLX by UPLC-MS.BLX showed the 50%cytotoxic concentration(CC50)of(13859.56±319)mg·L^(-1),the median inhibitory concentration(IC50)of(1439.09±200)mg·L^(-1),and the selection index of 8.26-11.44 for HCoV-OC43 in HRT-18 cells.Compared with the cells infected with HCoV-OC43,BLX at the concentrations of 1500,1000,500 mg·L^(-1) inhibited the proliferation of this virus(P<0.05,P<0.01).BLX exhibited antiviral effect in the early stage of virus infection,and the inhibition role in the attachment stage was more significant than that in the entry stage(P<0.05).In the suckling mice infected with HCoV-OC43,BLX at 1200 and 600 mg·kg^(-1)·d^(-1)alleviated the symptoms,prolonged the survival period,reduced the death rate,and down-regulated the mRNA level of nucleocapsid protein in the mice.Moreover,BLX at 1200 mg·kg^(-1)·d^(-1)down-regulated the expression of nucleocapsid protein in the brain(P<0.01)and the lung(P<0.01).Conclusion:BLX contained multiple antiviral ingredients.It inhibited the proliferation of HCoV-OC43 both in vitro and in vivo by interference with viral attachment.This study provides theoretical reference for the treatment of acute respiratory tract infection with HCoV-OC43 and for further development and application of BLX.

关 键 词：百里香药茶 人冠状病毒OC43(HCoV-OC43) 抗病毒活性 细胞模型 小鼠模型 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33