SARS-CoV-2刺突糖蛋白对神经细胞的损伤效应及机制  

Damage effect and mechanism of SARS-CoV-2 spike protein on nerve cells

作　　者：王娇 李佳佳 肖文一 韦冬晖 蒋宁 周文霞 WANG Jiao;LI Jiajia;XIAO Wenyi;WEI Donghui;JIANG Ning;ZHOU Wenxia(Shaanxi Provincial University Engineering Research Center of Traditional Chinese Medicine Brain Health Industry,Key Laboratory of Pharmacodynamic Mechanism and Material Basis of Traditional Chinese Medicine,Shaanxi Provincial Administration of Traditional Chinese Medicine,School of Pharmacy,Shaanxi University of Traditional Chinese Medicine,Xianyang 712046,China;National Key Laboratory of National Security Specially Needed Medicines,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]陕西中医药大学药学院,陕西省中医药管理局中药药效机制与物质基础重点研究室,中医药脑健康产业陕西省高校工程研究中心,陕西咸阳712046 [2]军事科学院军事医学研究院国家安全特需药品全国重点实验室,北京100850

出　　处：《中国药理学与毒理学杂志》2024年第5期375-383,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家重点研发计划课题(2022YFC3500304);省部共建组分中药国家重点实验室开放课题(CBCM2023101)。

摘　　要：目的探讨严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突糖蛋白(S蛋白)对人神经母细胞瘤细胞(SH-SY5Y)的损伤效应及其机制。方法用S蛋白0(细胞对照组),25,50,75和100 mg·L^(-1)处理SH-SY5Y 24 h,CCK-8法检测细胞活力;比色法检测乳酸脱氢酶(LDH)释放率;EdU试剂盒检测细胞增殖;荧光素酶发光法检测细胞内ATP含量;JC-1荧光探针法检测细胞线粒体膜电位(MMP);Seahorse XF检测细胞糖酵解及线粒体氧化磷酸化水平。结果与细胞对照组相比,S蛋白25,50,75和100 mg·L^(-1)组细胞活力显著降低(P<0.01),半数抑制浓度(IC_(50))为65.05 mg·L^(-1);LDH释放率显著增加(P<0.01);EdU阳性细胞比例显著降低(P<0.01);S蛋白75和100 mg·L^(-1)组细胞内ATP含量显著降低(P<0.01);S蛋白50和75 mg·L^(-1)组细胞内MMP显著降低(P<0.05,P<0.01);S蛋白50 mg·L^(-1)组基础糖酵解水平和糖酵解能力的最大值显著升高(P<0.05,P<0.01),S蛋白25和50 mg·L^(-1)组呼吸能力最大值显著升高(P<0.05,P<0.01)。SH-SY5Y细胞活力与细胞内ATP含量和MMP均呈正相关(r^(2)=0.9209,P=0.001;r^(2)=0.6170,P=0.0025);与反映细胞糖酵解水平的细胞基础糖酵解水平和糖酵解能力最大值呈负相关(r^(2)=0.5194,P=0.0285;r^(2)=0.6664,P=0.0073),与反映线粒体氧化磷酸化水平的ATP生成能力呈负相关(r^(2)=0.8204,P=0.0008)。结论S蛋白使SH-SY5Y细胞活力下降,抑制细胞增殖,其机制可能与干扰神经细胞内能量代谢密切相关。OBJECTIVE To investigate the damage effect and potential toxic mechanism of SARS-CoV-2 spike protein(S protein)on human neuroblastomacells(SH-SY5Y).METHODS SH-SY5Y were treated with S protein at concentrations of 25,50,75,and 100 mg·L^(-1) for 24 h.Cell viability of SH-SY5Y was detected using the CCK-8 assay.The cytotoxic lactate dehydrogenase(LDH)detection kit was used to measure the release rate of LDH,and the 5-ethynyl-2′-deoxyuridine(EdU)-488 cell prolifera-tion kit was used to assess cell proliferation.The ATP detection kit was used to measure intracellular ATP content.The JC-1 fluorescent probe method was employed to detect the mitochondrial membrane potential(MMP)of cells.Seahorse XF was used to measure mitochondrial respiratory and glycolytic capacity.RESULTS Compared with the cell control group,cell viability was significantly reduced in S protein 25,50,75 and 100 mg·L^(-1) groups(P<0.01),and the half-inhibition concentration(IC_(50))was 65.05 mg·L^(-1).The LDH release rate wassignificantly increased(P<0.01)and the proportion of EdU positive cellswas significantly reduced(P<0.01)in S protein 25,50,75 and 100 mg·L^(-1) groups.S protein signifi-cantly reduced intracellular ATP content(P<0.01)at the concentrations of 75 and 100 mg·L^(-1),while significantly reduced intracellular MMP(P<0.05,P<0.01)at the concentrations of 50 and 75 mg·L^(-1).S protein 50 mg·L^(-1) increased the maximum value of basal glycolysis levels and glycolytic capacity(P<0.05,P<0.01),and S protein 25 and 50 mg·L^(-1) increased the maximum value of respiration capacity(P<0.05,P<0.01).SH-SY5Y cell viability was positively correlated with the intracellular ATP content and the MMP level(r^(2)=0.9209,P=0.001;r^(2)=0.6170,P=0.0025),and negatively correlated with the maximum level of basal glycolysis and glycolytic capacity(r^(2)=0.5194,P=0.0285;r^(2)=0.6664,P=0.0073),and nega-tively correlated with ATP production capacity(r^(2)=0.8204,P=0.0008).CONCLUSIONS protein decreases the viability of SH-SY5Y cells and inhibited cell prolifer

关 键 词：严重急性呼吸综合征冠状病毒2(SARS-CoV-2) 刺突糖蛋白 人神经母细胞瘤细胞 神经损伤 糖酵解 能量代谢 

分 类 号：R99[医药卫生—毒理学] R373[医药卫生—药学]