摘要
目的观察利拉鲁肽(LIR)对血小板源性生长因子-BB(PDGF-BB)诱导血管平滑肌细胞(VSMCs)增殖的影响,并对其可能机制进行探讨。方法实验分6组:空白对照组、PDGF-BB组、PDGF-BB+LIR(25、50、100 nM)组、LIR(100 nM)组;细胞接受不同干预因素刺激后,采用锥虫蓝染色检测LIR的细胞毒性;细胞增殖检测试剂盒CCK-8及Brdu检测细胞增殖及DNA合成;流式细胞仪检测细胞周期;实时荧光定量RT-PCR检测调控细胞周期相关蛋白的基因表达;酶标仪检测活性氧(ROS)的产生;蛋白印迹法(Western blot)对可能机制进行探讨。结果①细胞毒性:PDGF-BB和/或LIR(25、50、100 nM)对VSMCs无毒性。②增殖作用:PDGF-BB干预48 h可显著诱导VSMCs增殖及DNA合成(P<0.01),LIR(25、50、100 nM)与PDGF-BB联合干预则可呈浓度依赖性的抑制细胞增殖及DNA合成(P均<0.01);对细胞周期的影响:LIR可阻滞G0/G1期细胞向S期过渡;与对照组相比,PDGF-BB组中细胞周期蛋白依赖性激酶6、4(CDK6,CDK4)、细胞周期蛋白D1、E(CyclinD1,CyclinE)的基因表达均明显增加(P均<0.01),细胞周期蛋白激酶抑制蛋白P27(P27)的表达下调(P<0.01),LIR(100 nM)与PDGF-BB联合干预后可逆转这种现象。③ROS的产生:与对照组相比,PDGF-BB组中ROS的水平显著提高(P<0.01),LIR(25、50、100 nM)与PDGF-BB联合干预后可呈浓度依赖性的抑制ROS的产生(P均<0.01)。④机制探讨:与对照组相比,PDGF-BB组中JNK、ERK1/2及p38的磷酸化水平均显著升高(P均<0.01);与PDGF-BB组相比,LIR(100 nM)与PDGF-BB联合干预后,ERK1/2和p38的磷酸化水平均显著降低(P均<0.01),而JNK的磷酸化水平无显著变化。结论LIR可通过抑制PDGF-BB诱导ROS的产生以及其下游ERK1/2和p38信号通路的活化发挥抑制细胞增殖的作用,为LIR预防和治疗血管重构相关疾病的提供了理论依据。
Objective To investigate the effect of Liraglutide(LIR)on Vascular Smooth Muscle Cells(VSMCs)proliferation in vitro induced by Platelet Derived Growth Factor-BB(PDGF-BB),and to explore its possible mechanism.Methods The experimental samples were divided into 6 groups:blank control group,PDGF-BB group,PDGF-BB+LIR(25,50,100 nM)group,LIR(100 nM)group.After the cells were stimulated by different intervention factors,trypan blue staining was used to detect the cytotoxicity of LIR;cell proliferation detection kit CCK-8 and Brdu were used to detect cell proliferation and DNA synthesis;flow cytometry was used to detect the cell cycle,real-time fluorescence quantitative PCR(RT-PCR)was used to detect the gene expression of cell cycle-related proteins;microplate reader was used to detect the expression level of reactive oxygen species(ROS);Western blot was used to detect the protein expression level.Results(1)Cell cytotoxicity:PDGF-BB and/or LIR(25,50,100 nM)did not induce VSMCs necrosis.(2)Cell proliferation:PDGF-BB intervention 48 h significantly induced VSMCs proliferation and DNA synthesis(P<0.01),while LIR(25,50,100 nM)reversed the effect in a concentration-dependent manner when co-treated with PDGF-BB(all P<0.01).Cell cycle:LIR can block the transition from G0/G1 to S phase.Compared with the control group,the gene expression of cyclin-dependent kinases 6,4(CDK6,CDK4),cyclin D1,E were significantly increased(all P<0.01),cyclin kinase inhibitor P27(P27)was decreased(P<0.01),while LIR(100 nM)and PDGF-BB combined intervention can reverse this phenomenon.(3)ROS expression:compared with the control group,ROS level was significantly increased in the PDGF-BB group(P<0.01),while LIR(25,50,100 nM)significantly reversed the effect in a concentration-dependent manner when co-treated with PDGF-BB(all P<0.01).(4)Signaling pathway:Compared with the control group,the phosphorylation levels of JNK,ERK1/2 and p38 in the PDGF-BB group were significantly increased(all P<0.01).Compared with the PDGF-BB group,after LIR(100 nM)and PDGF-BB combined intervention,the phosphorylation levels of ERK1/2 and p38 were significantly reduced(both P<0.01),while the phosphorylation level of JNK did not change significantly.Conclutions LIR can inhibit the proliferation of ROS induced by PDGF-BB and the activation of its downstream ERK1/2 and p38 signaling pathways,and it can provide a theoretical basis for the prevention and treatment of vascular remodeling-related diseases by LIR.
作者
聂晓东
刘源
申娟
祁华琪
Nie Xiaodong;Liu Yuan;Shen Juan;Qi Huaqi(Department of cardiology,Affiliated Hospital of Henan Medical College,Zhengzhou 451191,China;不详)
出处
《中国循证心血管医学杂志》
2020年第10期1218-1222,共5页
Chinese Journal of Evidence-Based Cardiovascular Medicine
基金
2017年河南省科技厅科技攻关项目(172102310531)。
关键词
利拉鲁肽
血管平滑肌细胞
信号通路
增殖
Liraglutide
Vascular smooth muscle cells
Signal pathway
Proliferation
