摘要
为研究大豆皂苷Bc(SS-Bc)对伪狂犬病病毒(PRV)复制的影响,本研究采用CCK-8法测定SS-Bc对Vero E6细胞的半数细胞毒性浓度(CC_(50))为253.6μg/m L,同时测定了SS-Bc对PRV的半数抑制浓度(IC_(50))为21.61μg/m L。根据上述结果,将不同浓度的SS-Bc(15μg/mL、30μg/mL、60μg/mL)与PRV-GFP(MOI 0.01)共处理Vero E6细胞,通过倒置荧光显微镜观察含绿色荧光的细胞数量,结合Image J软件分析荧光强度,并采用Reed-Muech法测定细胞上清中的病毒滴度(TCID_(50));进一步采用不同浓度的SS-Bc(30μg/mL、60μg/mL)分别与不同MOI的PRV-GFP(MOI0.05、MOI 0.1及MOI 0.5)共处理Vero E6细胞,24 h后检测上述指标。结果显示,与阳性对照组相比,SS-Bc处理组细胞的相对荧光强度值和细胞上清中病毒的TCID_(50)均极显著降低(P<0.01),且对病毒的抑制作用呈剂量依赖性,60μg/mL时效果最佳;即使在较高MOI PRV感染下,SS-Bc仍极显著抑制该病毒的增殖(P<0.01)。为了确定SS-Bc的最佳给药方式,本实验于PRV-GFP(MOI 0.01)感染后不同时间,以SS-Bc(60μg/mL)处理Vero E6细胞24 h后检测上述指标。结果显示,所有药物处理组的CPE数量、相对荧光强度值和细胞上清中病毒的TCID_(50)均极显著低于阳性对照组(P<0.01),其中PRV感染后1 h和3 h给药时抑制病毒增殖的效果更佳。进一步体外模拟病毒感染各阶段,将SSBc(60μg/mL)与PRV-GFP(MOI 0.1和1)以不同方式处理后接种Vero E6细胞,采用荧光定量PCR(qPCR)检测病毒吸附、内化及复制阶段gB基因mRNA的相对转录水平,在释放阶段测定细胞上清中病毒的TCID_(50),分析SS-Bc对PRV感染的影响。结果显示SS-Bc在PRV的吸附和复制阶段均显著降低了gB基因mRNA的转录水平(P<0.05),但SS-Bc在病毒的内化和释放阶段均无显著影响。综上所述,本研究首次证实SS-Bc在体外能够显著抑制PRV的增殖,主要作用于病毒的吸附和复制阶段;且无论在病毒接种前、同时或接种后给药,SS-Bc均表现出良好的抗病毒效果,其中在病毒进入细胞后给药效果更佳。本研究为SS-Bc作为潜在的抗PRV药物提供了科学依据,并为SS-Bc的进一步研究和临床应用奠定了基础。
This study investigated the effects of Soybean saponin Bc(SS-Bc)on the replication of pseudorabies virus(PRV).initially,the cytotoxicity of SS-Bc on Vero E6 cells was assessed using the CCK-8 assay,yielding a half-maximal cytotoxic concentration(CC_(50))of 253.6μg/mL.The half-maximal inhibitory concentration(IC_(50))of SS-Bc against PRV was determined to be 21.61μg/mL.Based on these results,Vero E6 cells were co-incubated with varying concentrations of SS-Bc(15μg/mL,30μg/mL,and 60μg/mL)and PRV-GFP(MOI 0.01).After 36 hours,the number of green fluorescent cells was counted using an inverted fluorescence microscope.Fluorescence intensity was analyzed using Image J software.The TCID_(50) of PRV in the cell supernatant was determined using the Reed-Muench method.Further,Vero E6 cells were co-treated with different concentrations of SS-Bc(30μg/mL and 60μg/mL)and PRV-GFP at various MOIs(MOI 0.05,0.1,and 0.5),and the above indicators were measured after 24 hours.Results showed that compared with the positive control,SS-Bc treatment significantly reduced relative fluorescence intensity and viral TCID_(50) in the supernatant(P<0.01),with a dose-dependent inhibitory effect,and 60μg/mL was most effective.Moreover,even at higher MOI,SS-Bc significantly inhibited viral proliferation(P<0.01).To determine the optimal administration strategy of SS-Bc,Vero E6 cells infected with PRV-GFP(MOI 0.01)were treated with SS-Bc(60μg/mL)at different time points post-infection.After 24 hours,the aforementioned indicators were assessed.Results indicated that all SS-Bc treatment groups exhibited significant reductions in CPE,relative fluorescence intensity,and viral TCID_(50)(P<0.01),with the most effective inhibition observed when SS-Bc was administered 1 hour and 3 hours post-infection.To further simulate the different stages of viral infection in vitro,SS-Bc(60μg/mL)and PRV-GFP(MOI 0.1 and 1)were applied to Vero E6 cells using various treatment strategies.Quantitative real-time PCR(qPCR)was used to assess the mRNA transcription levels of the PRV gB gene during the adsorption,internalization,and replication stages,while viral TCID_(50) in the supernatant was measured during the release phase.The results showed that SS-Bc significantly reduced gB gene mRNA transcription levels during the adsorption and replication stages(P<0.05),but had no significant effect during internalization and release stages.In conclusion,SS-Bc effectively inhibited PRV proliferation in vitro,primarily targeting the adsorption and replication stages of the virus.It exhibited robust antiviral efficacy,whether applied before,during,or after viral inoculation,with the most pronounced effects observed when administered after viral entry.These findings provide a scientific rationale for SS-Bc as a potential anti-PRV agent and lay the foundation for future research and clinical applications.
作者
高柯欣
李艳华
张艳禾
汤艳东
安同庆
蔡雪辉
周双海
王淑杰
GAO Ke-xin;LI Yan-hua;ZHANG Yan-he;TANG Yan-dong;AN Tong-qing;CAI Xue-hui;ZHOU Shuang-hai;WANG Shu-jie(College of Animal Medicine,Beijing University of Agriculture,Beijing 102206,China;State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Animal Medicine,Northeast Agricultural University,Harbin 150030,China)
出处
《中国预防兽医学报》
北大核心
2025年第6期545-552,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然基金面上项目(32273018)。
作者简介
高柯欣(1999-),女,河南洛阳人,硕士研究生,主要从事兽医微生物及其分子生物学研究;通信作者:周双海,E-mail:shhaizhou@bua.edu.cn;通信作者:王淑杰。
