丹参酚酸B通过高迁移率族蛋白1抑制氧化应激诱导的内皮细胞焦亡  

Salvianolic acid B inhibits endothelial cell pyroptosis induced by oxidative stress via high mobility group box 1

作　　者：龚明艳 李敏 郭军[1] Gong Mingyan;Li Min;Guo Jun(Department of Cardiology,the First Affiliated Hospital of Jinan University,Guangzhou 510630,China)

机构地区：[1]暨南大学附属第一医院心血管内科,广州510630

出　　处：《中华心血管病杂志（网络版）》2022年第1期252-264,共13页Chinese Video Journal of Cardiology

基　　金：国家自然科学基金(81673635);广东省自然科学基金(2022A1515011133)。

摘　　要：目的探讨丹参酚酸B(Salvianolic acid B,Sal B)在内皮细胞氧化应激损伤中的保护作用。研究对象提取原代人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),将获取的细胞分成7个实验组:(1)对照组,(2)过氧化氢(hydrogen peroxide,H_(2)O_(2))组,(3)H_(2)O_(2)+Sal B组,(4)Sal B组,(5)H_(2)O_(2)+小干扰核糖核酸(small interfering RNA,siRNA)阴性对照(negative control,NC)组,(6)H_(2)O_(2)+高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)siRNA组,(7)H_(2)O_(2)+Sal B+HMGB1 siRNA组。干预措施(1)对照组:予1‰二甲基亚砜(dimethyl sulfoxide,DMSO)处理;(2)H_(2)O_(2)组:予200μmol/L H_(2)O_(2)处理;(3)H_(2)O_(2)+Sal B组:10μmol/L Sal B预处理24 h后再予200μmol/L H_(2)O_(2)处理;(4)Sal B组:10μmol/L Sal B预处理24 h后再予1‰DMSO处理;(5)H_(2)O_(2)+siRNA NC组:siRNA NC预处理24 h后再予200μmol/L H_(2)O_(2)处理;(6)H_(2)O_(2)+HMGB1 siRNA组:HMGB1 siRNA预处理24 h后再予200μmol/L H_(2)O_(2)处理;(7)H_(2)O_(2)+Sal B+HMGB1 siRNA组:10μmol/L Sal B及HMGB1 siRNA预处理24 h后再予200μmol/L H_(2)O_(2)处理。观测指标及测量方法采用荧光探针法检测细胞内活性氧(reactive oxygen species,ROS)水平、细胞线粒体膜电位及三磷酸腺苷(adenosine triphosphate,ATP)水平;采用透射电镜观察细胞结构变化及采用Annexin V/PI染色进行流式分析检测细胞膜破损情况;使用蛋白质印迹(Western blot,WB)法检测氧化应激状态下细胞焦亡相关蛋白核苷酸结合寡聚化结构域样受体蛋白(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)、消皮素D(gasdermin D,GSDMD)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a caspase recruitment domain,ASC)、胱天蛋白酶-1(caspase-1)、白细胞介素-1β(interleukin-1β,IL-1β)蛋白;利用siRNA瞬时敲低细胞中HMGB1,观察有无Sal B预处理细胞中焦亡相关分子NLRP3、GSDMD、ASC、caspase-1、IL-1β蛋白表达情况。实验结果使用GraObjective To explore the protective effects of salvianolic acid B(Sal B)on oxidative stress injury in endothelial cells.Subjects Primary umbilical vein endothelial cells(human umbilical vein endothelial cells,HUVECs)were extracted and assigned to 7 experimental groups:(1)control group;(2)hydrogen peroxide(H_(2)O_(2))group;(3)H_(2)O_(2)+Sal B group;(4)Sal B+H_(2)O_(2)+small interfering RNA(siRNA)negative control(NC)group;(5)H_(2)O_(2)+high mobility group box‑1(HMGB1)siRNA group;(6)H_(2)O_(2)+Sal B+HMGB1 siRNA group.Interventions(1)Control group:treated with 1‰dimethyl sulfoxide(DMSO);(2)H_(2)O_(2) group:treated with 200μmol/L H_(2)O_(2);(3)H_(2)O_(2)+Sal B group:pretreated with 10μmol/L Sal B for 24 h,followed by 200μmol/L H_(2)O_(2) treatment;(4)Sal B group:10μmol/L Sal B pretreated for 24 h,followed by 1‰DMSO treatment;(5)H_(2)O_(2)+siRNA NC group:siRNA pretreated for 24 h,followed by 200μmol/L H_(2)O_(2) treatment;(6)H_(2)O_(2)+HMGB1 siRNA group:HMGB1 siRNA pretreated for 24 h,followed by 200μmol/L H_(2)O_(2) treatment;(7)H_(2)O_(2)+Sal B+HMGB1 siRNA group:10μmol/L Sal B and HMGB1 siRNA pretreated for 24 h,followed by 200μmol/L H_(2)O_(2) treatment.Main Outcomes and Measurements A fluorescence probe was used to measure intracellular reactive oxygen species(ROS)levels,mitochondrial membrane potential,and ATP levels.Changes in cell structure were observed using transmission electron microscopy,and cell membrane damage was examined using Annexin V/PI staining with flow cytometry.Western blot analysis was used to examine the expression of pyroptosis‑related proteins,namely,nucleotide‑binding oligomerization domain‑like receptor protein 3(NLRP3)under oxidative stress,gasdermin D(GSDMD),apoptosis‑associated speck‑like protein containing a caspase recruitment domain(ASC),inflammatory caspase‑1,and interleukin‑1β(IL‑1β).HMGB1 was transiently knocked down in cells using siRNA,and the protein expression of NLRP3,GSDMD,ASC,caspase‑1,and IL‑1βwas detected in Sal B‑pretreated cells.Exp

关 键 词：丹参酚 HMGB1蛋白质 NLR家族 热蛋白结构域包含蛋白3 细胞焦亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]