胺碘酮对人脐静脉内皮细胞的损伤作用及机制研究  

Study on the injury and its mechanism of amiodarone on human umbilical vein endothelial cells

作　　者：王娟娟[1] 田继华[2] 亢晶 杨佳[2] 常思佳 冀贺 黄太平 樊卫平[2] 郭锦丽[1] 王艳红[2] Wang Juanjuan;Tian Jihua;Kang Jing;Yang Jia;Chang Sijia;Ji He;Huang Taiping;Fan Weiping;Guo Jinli;Wang Yanhong(Nursing Department,Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Teaching Department of Microbiology and Immunology,Shanxi Medical University,Taiyuan 030001,China;Department ofHematology Oncology,Xi′an Honghui Hospital,Xi′an 710016,China)

机构地区：[1]山西医科大学第二医院护理部,太原030001 [2]山西医科大学微生物学与免疫学教研室,太原030001 [3]西安市红会医院血液肿瘤科,西安710002

出　　处：《药物不良反应杂志》2021年第9期461-467,共7页Adverse Drug Reactions Journal

基　　金：国家自然科学基金青年科学基金(81500529,81500518);山西省自然科学基金(201901D111187,201901D111188)。

摘　　要：目的探讨胺碘酮对人脐静脉内皮细胞(HUVEC)的损伤作用及可能的机制。方法取经过3代培养的HUVEC接种于96孔板,加入0、10、20、30或60μmol/L胺碘酮培养24 h,采用细胞计数试剂盒8(CCK-8)法检测细胞活力,以0μmol/L组细胞活力为100%,计算各加药组的相对细胞活力。选择可将细胞活力降至70%左右的胺碘酮浓度用于后续各项实验。采用CCK-8法检测该浓度胺碘酮作用不同时间(6、12、24、36、48 h)对HUVEC活力的影响。以加入该浓度胺碘酮培养的HUVEC为实验组,不加入者为对照组,采用钙依赖性磷脂结合蛋白V-异硫氰酸荧光素/碘化丙啶双染法流式细胞术检测细胞凋亡率;蛋白质印迹法和实时荧光定量聚合酶链反应法分别检测B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶3(caspase-3)、白细胞介素10(IL-10)、IL-1β、IL-6、肿瘤坏死因子α(TNF-α)蛋白和mRNA表达水平;2',7'-二氯二氢荧光素二乙酸酯荧光探针法检测活性氧(ROS)含量,水溶性四氮唑-1法检测超氧化物歧化酶(SOD)活性,微板法检测还原型谷胱甘肽(GSH)含量。结果经10、20、30、60μmol/L胺碘酮作用24 h的各加药组HUVEC活力与对照组(100%)相比分别为(88.82±2.64)%、(74.96±1.75)%、(64.95±2.10)%和(18.57±0.65)%,各加药组与对照组比较以及加药组之间两两比较均P<0.01。选择30μmol/L胺碘酮用于后续实验。经30μmol/L胺碘酮作用6、12、24、36、48 h的各实验组HUVEC活力与对照组(100%)相比分别为(90.19±1.88)%、(82.81±2.51)%、(75.33±1.37)%、(65.76±1.85)%和(47.01±3.29)%,各实验组与对照组比较以及实验组之间两两比较均P<0.01。实验组细胞凋亡率明显高于对照组(48.59%比16.34%,P<0.01),促凋亡蛋白Bax和caspase-3以及促炎因子IL-1β、IL-6、TNF-α的蛋白和mRNA表达水平均高于对照组(均P<0.01),而抗凋亡蛋白Bcl-2和抗炎因子IL-10的蛋白和mRNA表达水平均低于对照组(P<0.05,P<0.01)。结论胺�Objective To explore the injury effect and its possible mechanism of amiodarone on human umbilical vein endothelial cells(HUVECs).Methods After 3 generations of cultivation,the HUVECs were seeded in 96-well plates and incubated with amiodarone(0,10,20,30,and 60μmol/L)for 24 hours.The cell viability was detected using cell counting kit 8(CCK-8)assay and the relative viability of cells incubated with different concentrations of amiodarone were calculated by taking the cell viability of the 0μmol/L group as 100%.The concentration of amiodarone at which cell viability was reduced to 70%was selected for subsequent experiments.The effect of amiodarone of this concentration on the activity of HUVECs after action for different time(6,12,24,36,and 48 hours)was detected using the CCK-8 assay.HUVECs cultured with amiodarone of this concentration were set as the experimental groups and those without amiodarone were set as the control group.Apoptosis rate of HUVECs was detected by Annexin V-FITC/P flow cytometry;the protein and mRNA expression levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),Caspase-3,interleukin 10(IL-10),IL-1β,IL-6,and tumor necrosis factor alpha(TNF-α)were detected using western blotting and real-time fluorescence quantitative polymerase chain reaction,respectively;the reactive oxygen species(ROS)was detected by DCFH-DA fluorescence probe assay;the superoxide dismutase(SOD)activity was detected by water-soluble tetrazolium-1 assay;the reduced glutathione(GSH)content was detected by microplate assay.Results The viabilities of HUVECs incubated with amiodarone at concentration of 10,20,30,and 60μmol/L for 24 hours were(88.82±2.64)%,(74.96±1.75)%,(64.95±2.10)%,and(18.57±0.65)%,respectively;differences were all significant(all P<0.01)between each experiment group and control group,as well as between each experiment group.Amiodarone at a concentration of 30μmol/L was used for subsequent experiments.After incubating with 30μmol/L amiodarone for 6,12,24,36,and 48 hours,the viabilities

关 键 词：胺碘酮 内皮细胞 静脉炎 细胞凋亡 炎症 氧化应激 

分 类 号：R965[医药卫生—药理学]