天然小分子药物小檗胺影响牛冠状病毒复制的研究  

Study on the effect of natural small molecule drug berbamine on bovine coronavirus replication

作　　者：李泽宇 王俊 刘芯怡 刘心愿 杨莉 史慧君[1,3] 常继涛 尹鑫 付强 LI Ze-yu;WANG Jun;LIU Xin-yi;LIU Xin-yuan;YANG Li;SHI Hui-jun;CHANG Ji-tao;YIN Xin;FU Qiang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Xinjiang Key Laboratory of New Drug Study and Creation for Herbivorous Animals,Urumqi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]中国农业科学研究院哈尔滨兽医研究所,黑龙江哈尔滨150069 [3]新疆草食动物新药研究与创制重点实验室,新疆乌鲁木齐830052

出　　处：《中国预防兽医学报》2024年第12期1230-1235,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：新疆维吾尔自治区自然科学基金面上项目(2021D01A77);新疆农业大学研究生科研创新计划项目(XJAUGRI2024029)。

摘　　要：为研究小檗胺(BBM)是否具有抑制牛冠状病毒(BCoV)复制的作用,本研究采用不同浓度BBM(0、1μmol/L、5μmol/L、8μmol/L、10μmol/L、15μmol/L、20μmol/L、40μmol/L、60μmol/L)处理人回盲肠癌细胞(HRT-18G),于处理后不同时间(0、12 h、24 h、36 h、48 h)采用CCK-8法分析BBM对HRT-18G细胞存活率的影响,以确定BBM对HRT-18G细胞的毒性作用。BBM预处理HRT-18G细胞后,将100 TCID_(50)牛冠状病毒(BCoV)感染细胞,24 h和48 h后收集细胞,采用荧光定量PCR(qPCR)检测BCoV N基因mRNA的转录水平,同时采用western blot检测BCoV N蛋白表达水平、经倒置显微镜观察细胞病变(CPE)情况、Reed-Meuench法检测病毒滴度等方法检测BBM预处理对BCoV N基因mRNA的转录水平、BCoV N蛋白表达水平、致CPE作用、子代病毒滴度等的影响。结果显示,低浓度BBM短时间处理对HRT-18G细胞存活率的抑制作用较低,当处理时间达到24 h时15μmol/L、20μmol/L、40μmol/L和60μmol/L BBM均极显著降低细胞存活率(P<0.01);因此采用5μmol/L、10μmol/L、20μmol/L、40μmol/L和60μmol/L浓度的BBM作用HRT-18 G细胞进行后续研究。qPCR结果显示,与NC组相比20μmol/L BBM预处理HRT-18G细胞24 h即可极显著降低BCoV N基因mRNA的转录水平(P<0.01);western blot结果显示,与NC组相比,40μmol/L BBM预处理HRT-18G细胞48 h可完全抑制BCoV N蛋白的表达;CPE观察结果显示,与NC组相比,40μmol/L和60μmol/L BBM预处理24 h和48 h可减轻BCoV感染引起的CPE;子代病毒滴度检测结果显示,与NC组相比,40μmol/L和60μmol/L BBM可极显著降低BCoV子代病毒滴度(P<0.01)。综上所述,本研究首次证实BBM具有抑制BCoV复制的作用,为防控BCoV的感染提供了新方法,同时为开发BBM作为抗病毒药物提供参考依据。In order to investigate whether Berbamine(BBM)can inhibit the replication of bovine coronavirus(BCoV).In this study,different concentrations of BBM(0,1μmol/L,5μmol/L,8μmol/L,10μmol/L,15μmol/L,20μmol/L,40μmol/L and 60μmol/L)were used to treat Human Rectal Tumor-18(HRT-18G)cells at different times(0,12 hours,24 hours,36 hours and 48 hours).Subsequently,the effect of BBM on the survival rate of HRT-18G was detected.HRT-18G cells were pretreated with BBM and infected with 100TCID_(50)BCoV for 24 hours and 48 hours.Fluorescent quantitative PCR(qPCR)was used to detect the mRNA transcription level of BCoV N gene,while western blot was used to detect the expression level of BCoV N protein.Viral infection was detected by an inverted microscope for cytopathic lesions,and viral titer was detected using a TCID_(50)assay calculated by the Reed-Meuench method.The effect of BBM pre-treatment on the mRNA transcription level of the BCoV N gene,the expression level of BCoV N protein,the cytopathic effect,and the viral titer of progeny were detected.The results showed that short-term treatment with a low concentration of berberine had little impact on inhibiting the survival rate of HRT-18G cells.In contrast,treatment time reached 24 hours at concentrations of 15μmol/L,20μmol/L,40μmol/L and 60μmol/L significantly inhibited the survival rate of HRT-18G cells(P<0.01).Therefore,HRT-18G cells were treated with BBM at concentration of 5μmol/L,10μmol/L,20μmol/L,40μmol/L,and 60μmol/L for subsequent research.The qPCR results showed that HRT-18G cells pretreated with 20μmol/L BBM for 24 hours significantly decreased the mRNA transcription level of BCoV N gene(P<0.01).Western blot results showed that pre-treatment of HRT-18G cells with 40μmol/L BBM for 24 hours completely inhibited the expression of BCoV N protein to compare with NC.CPE results showed that 40μmol/L and 60μmol/L BBM pre-treatment for 24 hours and 48 hours could reduce the cytopathia induced by BCoV infection to compare with NC.The results showed that 40μ

关 键 词：天然小分子药物 小檗胺 牛冠状病毒 病毒复制 病毒滴度 

分 类 号：S852.65[农业科学—基础兽医学]