中国兽医杂志2026，Vol.62Issue(9)：38-46,9.DOI:10.20157/j.cnki.zgsyzz.2026.sf.067
以鱼藤酮为例分析细胞活力检测法在抗病毒药物评估中的局限性
Analysis of the Limitations of Cell Viability Assays in Antiviral Drug Evaluation Using Rotenone as a Model
摘要
Abstract
To reveal the limitations of conventional cell viability assays in antiviral drug screening and avoid misinterpreting sublethal cytotoxicity as drug safety,thereby affecting the objective evaluation of antiviral efficacy,rotenone,an inhibitor of mitochondrial respiratory chain complex I,was used as a model compound in this study.The antiviral activity of rotenone was evaluated using a recombinant porcine reproductive and respiratory syndrome virus(PRRSV)strain carrying a HiBiT tag.The cytotoxicity of rotenone to African green monkey embryonic kidney cells(MARC-145)was assessed using the cell counting kit-8(CCK-8)assay and lactate dehydrogenase(LDH)release assay.Cellular morphological changes were observed by bright-field microscopy.Furthermore,transcriptome sequencing was performed on rotenone-treated cells(10 μmol/L),PRRSV-infected cells[infected with highly pathogenic PRRSV(HP-PRRSV)strain JXwn06 at a multiplicity of infection(MOI)of 0.1],and rotenone-treated PRRSV-infected cells(infected with JXwn06 at an MOI of 0.1 and treated with 10 μmol/L rotenone)to analyze differentially expressed genes and enriched pathways.The results showed that rotenone exhibited anti-PRRSV activity with a half-maximal inhibitory concentration(IC50)of 0.082 μmol/L.The half-maximal cytotoxic concentrations(CC50)determined by the CCK-8 and LDH assays were 119.71 and 114.75 μmol/L,respectively,resulting in a selectivity index(SI)of 1 399,indicating an apparent"low toxicity and high efficacy"profile.However,bright-field microscopy revealed that rotenone at a concentration of only 10 μmol/L induced obvious cellular abnormalities,including cell rounding,swelling,cytoplasmic vacuolization,and syncytium formation,suggesting severe cellular stress.Transcriptomic analysis showed that rotenone treatment alone significantly upregulated the expression of endoplasmic reticulum stress marker DNA damage-inducible transcript 3(DDIT3),cell cycle inhibitor cyclin-dependent kinase inhibitor 1A(CDKN1A),and lysosome-associated gene cathepsin L(CTSL).These differentially expressed genes were mainly enriched in pathways related to positive regulation of apoptotic processes and responses to external stimuli.In the context of PRRSV infection,rotenone co-treatment significantly downregulated the expression of DNA replication-associated genes proliferating cell nuclear antigen(PCNA)and minichromosome maintenance 10 replication initiation factor(MCM10),resulting in marked inhibition of cell cycle-related pathways.These results indicate that the apparent"antiviral phenotype"induced by rotenone is essentially an accompanying effect of host cell physiological dysfunction and proliferation inhibition,highlighting the potential risk of false-positive results in antiviral drug screening.The mismatch between the targets of conventional cytotoxicity assays and the actual mechanisms underlying drug toxicity may lead to falsely favorable safety evaluations.Therefore,preliminary safety assessment of antiviral candidates should integrate morphological observations with multidimensional physiological indicators.关键词
鱼藤酮/猪繁殖与呼吸综合征病毒(PRRSV)/抗病毒药物筛选/细胞毒性评价/转录组测序/CCK-8局限性Key words
rotenone/porcine reproductive and respiratory syndrome virus(PRRSV)/antiviral drug screening/cytotoxicity evaluation/transcriptome sequencing/limitations of CCK-8 assay分类
农业科技引用本文复制引用
刘天娇,杨汉春,刘瑞,周琼琼,高鹏,张永宁,盖新娜,韩军,郭鑫,周磊..以鱼藤酮为例分析细胞活力检测法在抗病毒药物评估中的局限性[J].中国兽医杂志,2026,62(9):38-46,9.基金项目
"十四五"国家重点研发计划(2023YFD1800801) （2023YFD1800801）
国家现代农业产业技术体系(CARS-35) （CARS-35）
