摘要
目的探讨丹参酮ⅡA磺酸钠(STS)对过氧化氢(H_(2)O_(2))诱导后人脐静脉内皮细胞(HUVECs)焦亡的影响及其可能机制。方法2021年11月至2022年9月,采用无锡市第九人民医院保存的HUVECs细胞株作为研究对象,实验分为4组:空白对照组(正常条件)、空白+STS组、H_(2)O_(2)组、H_(2)O_(2)+STS组。待细胞长至80%融合度时,H_(2)O_(2)组和H_(2)O_(2)+STS组加入500.00μmol/L H_(2)O_(2)诱导3 h,去除含500.00μmol/L H_(2)O_(2)的培养基之后,空白+STS组和H_(2)O_(2)+STS组加入5.00μg/ml STS与HUVECs共培养24 h。通过CCK-8实验检测不同浓度(0、0.05、0.50、5.00、50.00、500.00μg/ml)的STS对HUVECs增殖的影响,TUNEL染色检测DNA损伤阳性细胞,实时荧光定量PCR(RT-PCR)检测炎症小体核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)的表达来确定H_(2)O_(2)诱导焦亡的最适浓度。检测试剂盒检测H_(2)O_(2)诱导后活性氧自由基(ROS)表达。细胞划痕实验和基质胶体外成管实验探究STS对焦亡状态下HUVECs迁移能力和成管能力的影响。RT-PCR和Western blotting检测NLRP3、Caspase-1、白细胞介素-18、白细胞介素-1β的基因和蛋白表达。不同时点浓度比较采用重复测量方差分析,2组间数据比较采用t检验,多组间数据比较采用单因素方差分析,P<0.05为差异有统计学意义。结果50.00μg/ml以下的STS对HUVECs的增殖无影响,500.00μmol/L H_(2)O_(2)诱导HUVECs焦亡效果最佳。TUNEL染色显示,与空白对照组相比,H_(2)O_(2)组TUNEL阳性细胞率显著升高,差异有统计学意义(P<0.01),与H_(2)O_(2)+STS组TUNEL阳性细胞率的差异无统计学意义(P>0.05)。ROS检测结果显示,与H_(2)O_(2)组比,H_(2)O_(2)+STS组细胞内ROS明显降低,差异有统计学意义(P<0.01)。细胞划痕和体外成管实验显示,与空白对照组相比,H_(2)O_(2)组细胞迁移率和成管能力明显减弱,差异均有统计学意义(P均<0.01),与H_(2)O_(2)+STS组差异均无统计学意义(P均>0.05)。RT-PCR和We
Objective To investigate the effect of sodium tanshinoneⅡA sulfonate(STS)on pyroptosis of human umbilical vein endothelial cells(HUVECs)induced by H_(2)O_(2) and its possible mechanism.Methods From November 2021 to September 2022,HUVECs were used as the research subjects at Wuxi Ninth People’s Hospital.The experiment was divided into four groups:the blank control group(normal condition),blank+STS group,H_(2)O_(2) group and H_(2)O_(2)+STS group.When the cells reached 80%fusion,500.00μmol/L of H_(2)O_(2) was added to H_(2)O_(2) group and H_(2)O_(2)+STS group for 3 hours,and then the medium containing 500.00μmol/L H_(2)O_(2) was removed.After that,the blank+STS group and the H_(2)O_(2)+STS group were each supplemented with 5.00μg/ml of STS and co-cultured with HUVECs for 24 hours.CCK-8 was used to assess the impact of STS at various concentrations(0.00,0.05,0.50,5.00,50.00,500.00μg/ml)on the proliferation of HUVECs.DNA damage-positive cells were detected with TUNEL staining.The expression of NOD-like receptor protein 3(NLRP3)was detected using real-time PCR(RT-PCR)to investigate the optimal concentration of pyroptosis induced by H_(2)O_(2).A detection kit was used to measure the expression of reactive oxygen species(ROS)induced by H_(2)O_(2).The effect of STS on the migration and tube formation of HUVECs during pyroptosis was examined using a cell scratch test and a matrix gel tube formation test.The expressions of NLRP3,caspase-1,interleukin-18,and interleukin-1βwere detected using RT-PCR and Western blotting.Repeated measures ANOVA was used to compare the concentrations at different time points,t-tests were used to compare data between two groups,and one-way ANOVA was used to compare data between multiple groups.P<0.05 was considered statistically significant.Results STS below 50.00μg/ml had no effect on the proliferation of HUVECs,while 500.00μmol/L H_(2)O_(2) had the most significant effect on inducing pyroptosis in HUVECs.TUNEL staining showed that compared with the control group,the number of TUNEL-
作者
潘筱云
陶先耀
糜菁熠
毛栋
汪凯
芮永军
Pan Xiaoyun;Tao Xianyao;Mi Jingyi;Mao Dong;Wang Kai;Rui Yongjun(Institute of Orthopedics,Wuxi Ninth Hospital Affiliated to Soochow University,Wuxi 214063,China;Department of Hand Surgery,Wuxi Ninth Hospital Affiliated to Soochow University,Wuxi 214063,China;Department of Sport Medicine,Wuxi Ninth Hospital Affiliated to Soochow University,Wuxi 214063,China;Department of Traumatic Orthopedics,Wuxi Ninth Hospital Affiliated to Soochow University,Wuxi 214063,China)
出处
《中华整形外科杂志》
CSCD
北大核心
2024年第3期307-317,共11页
Chinese Journal of Plastic Surgery
基金
无锡市"太湖人才计划"顶尖医疗团队项目(TTPJY2020)
无锡市卫生健康委员会青年项目(Q202046)。
关键词
人脐静脉内皮细胞
细胞焦亡
丹参酮ⅡA磺酸钠
血管生成
Human umbilical vein endothelial cell
Pyroptosis
Sodium tanshinoneⅡA sulfonate
Angiogenesis
