摘要
目的:观察异丹叶大黄素(Isorhapontigenin,ISO)对血小板源性生长因子-BB(PDGF-BB)诱导的SD大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响,并探讨其机制。方法:本研究采用20 ng·ml^(-1)PDGF-BB刺激SD大鼠原代PASMCs建立细胞增殖模型,通过不同浓度的ISO(5,10,20μmol·L^(-1))干预,分别在12,24,48 h后通过CCK-8和Brd U试剂盒检测细胞增殖与DNA合成,筛选ISO抑制PASMCs增殖的最适浓度,不同浓度的ISO孵育PASMCs后于12,24,48 h后通过0.4%台盼蓝染色法检测PASMCs存活率;选择20μmol·L^(-1)ISO与PDGF-BB共同孵育PASMCs 24 h后,通过流式细胞仪分析细胞周期,荧光酶标仪检测细胞内活性氧族(ROS)产生,采用超氧化物歧化酶(SOD)活性检测试剂盒测定的SOD的含量,细胞丙二醛(MDA)检测试剂盒检测MDA的含量,实时荧光定量聚合酶链反应(RT-PCR)与Western Blot检测细胞内还原型辅酶Ⅱ氧化酶1(Nox1)、细胞内还原型辅酶Ⅱ氧化酶4(Nox4)m RNA与蛋白的表达。结果:CCK-8和Brd U检测结果表明ISO可浓度及时间依赖性抑制PDGF-BB刺激的PASMCs增殖及DNA合成(P<0.05);流式细胞仪检测检测结果表明ISO可抑制PDGF-BB诱导的PASMCs分裂,阻滞PASMCs细胞周期于G0/G1期(P<0.05);荧光酶标仪检测结果表明ISO可抑制PDGF-BB诱导PASMCs内ROS的产生(P<0.05);SOD和MDA活性检测试剂盒检测结果表明ISO能够拮抗PDGF-BB诱导PASMCs内SOD的表达下调以及MDA的表达上调(P<0.05);RT-PCR与Western Blot显示ISO能够抑制PASMCs内Nox1/4 m RNA和蛋白的表达(P<0.05)。结论:ISO能够抑制PDGF-BB诱导的PASMCs增值,其机制可能与抑制氧化应激有关。
Objective:To investigate the effect of isorhapontigenin(ISO)on platelet-derived growth factor-BB(PDGF-BB)induced proliferation of pulmonary artery smooth muscle cells(PASMCs)in Sprague Dawley(SD)rats,and to explore related mechanisms.Methods:Pre-incubation with various concentrations of isorhapontigenin(5,10,20μmol·L^(-1))was performed in 20 ng·ml^(-1)PDGF-BB-stimulated-PASMCs.The proliferation and DNA synthesis of PASMCs were detected by CCK-8 and Brd U kit at different time points(12,24,48 h).PASMCs were incubated with different concentrations of isorhapontigenin for 12,24 and 48 h,and then the trypan blue exclusion test was used to determine cell viability;PASMCs were co-incubated with 20μmol·L^(-1)isorhapontigenin and 20ng·ml^(-1)PDGF-BB for 24 h,the cell cycle progression was detected byow cytometry,reactive oxygen species(ROS)production was analyzed by a fluorescence microplate reader,SOD and MDA were detected by ELISA kits,and m RNA and protein expression of Nox1/4 was determined by RT-PCR and Western blot.Results:The presented data demonstrated isorhapontigenin inhibited the cell proliferation and DNA synthesis in a dose-and time-dependent manner in PDGF-BB-stimulated PASMCs without cell cytotoxicity(P<0.05).In accordance with the findings,isorhapontigenin increased the proportion of PASMCs in G0/G1 phase,and decreased the proportion of PASMCs in S phase in PDGF-BB-stimulated PASMCs(P<0.05).Further experiments showed that the beneficial effect of isorhapontigenin on blocking the proliferation of PASMCs was related to suppression of oxidative stress,which was associated with the level increase of SOD,as well as the decrease of the generation of ROS and expression of Nox1/4(P<0.05).Conclusion:Isorhapontigenin inhibites PDGF-BB-induced PASMCs proliferation through suppression of oxidative stress.
作者
陈昌贵
易春峰
李立为
张帆
Chen Changgui;Yi Chunfeng;Li Liwei;Zhang Fan(Department of Cardiology,Wuhan No.1 Hospital,Wuhan 430022,China)
出处
《中国药师》
CAS
2021年第6期1040-1045,1056,共7页
China Pharmacist
作者简介
通信作者:陈昌贵,Tel:15926327463,E-mail:chenchanggui2008@aliyun.com。
