李婷;夏勇;
LI Ting;XIA Yong;Institute of Cardiovascular Disease,Xuzhou Medical College;Department of Cardiology,the Affiliated Hospital of Xuzhou Medical College;

• 1:徐州医学院心血管病研究所
• 2:徐州医学院附属医院心内科

摘要(Abstract)：

目的:探讨大蒜素(allicin)抑制胰岛素(insulin)诱导的血管平滑肌细胞(VSMCs)增殖和迁移及其作用机制。方法:采用组织贴块法培养大鼠的VSMCs,并进行平滑肌α-肌动蛋白(α-SMA)免疫荧光鉴定,取3⁵代细胞进行实验。建立胰岛素刺激的VSMCs模型。实验分5组:对照组、胰岛素组、大蒜素组、ERK抑制剂PD98059组、大蒜素+PD98059组。噻唑蓝(CCK8)比色法检测VSMCs的增殖;细胞计数法检测VSMCs的迁移;免疫印迹实验(Western blotting)测定total ERK,PhosphoERK(p-ERK),PCNA蛋白表达的情况。结果:原代培养的VSMCs生长良好,光镜下呈长梭形及"峰与谷"样生长。α-SMA免疫荧光显示培养的细胞具有典型的VSMCs特征。胰岛素能刺激VSMCs的增殖和迁移,且以100 nmol·L-1浓度时效果最明显。大蒜素预处理能显著抑制胰岛素刺激的VSMCs的增殖和迁移,且呈剂量依赖性。PD98059、大蒜素+PD98059预处理亦能显著抑制胰岛素刺激的VSMCs的增殖和迁移。胰岛素可明显促进VSMCs表达p-ERK,PCNA蛋白。大蒜素能显著抑制VSMCs表达p-ERK,PCNA蛋白,且呈剂量依赖性。结论:大蒜素能显著抑制胰岛素诱导的VSMCs的增殖与迁移,其作用机制可能是抑制ERK信号通路的激活。
Objective: To investigate the effect of allicin in inhibiting insulin-induced vascular smooth muscle cell( VSMC)proliferation and migration. Method: The tissue explant method was adopted to culture rat' s VSMCs,and the immunofluorescence method was used to identify α-SMA. Cells from the third to fifth generations were selected in the experiment. The insulin-induced VSMC model was established. The experiment was carried out in five groups: the control group,the insulin group,the allicin group,the ERK inhibitors PD98059 group( 20 μmol·L- 1) and the PD98059 + allicin group. VSMCs' proliferation was determined by CCK8 colorimetric method,while its migration was detected by cell counting; The western blotting was used to detect total ERK,PhosphoERK,PCNA protein's expression. Result: Primary cultured VSMCs grew well in the spindle shape under the lightmicroscope,with peak and valley. α-SMA immunofluorescence results showed that the cultured cells had typical VSMCs' features. Insulin could stimulate VSMCs' proliferation and migration,with the best effect at the concentration of 100 nmol · L- 1. The pretreatment with allicin could significantly inhibit VSMCs' proliferation and migration induced by insulin in a dose-dependent manner. The pretreatment with PD98059 and allicin + PD98059 could inhibit VSMCs' proliferation and migration induced by insulin remarkably as well. Insulin could significantly accelerate VSMCs' expression of such proteins as p-ERK,PCNA. Contrarily,allicin could notably inhibit VSMCs' expression of such proteins as p-ERK,PCNA in a dose-dependent manner. Conclusion: Allicin could significantly inhibit VSMCs' proliferation and migration induced by insulin,which may be related to the inhibition of the activation of ERK signal path.

关键词(KeyWords)： 血管平滑肌细胞;胰岛素;大蒜素;增殖;迁移
vascular smooth muscle cell;insulin;allicin;proliferation;migration

Abstract:

Keywords:

基金项目(Foundation):

作者(Authors): 李婷;夏勇;
LI Ting;XIA Yong;Institute of Cardiovascular Disease,Xuzhou Medical College;Department of Cardiology,the Affiliated Hospital of Xuzhou Medical College;

参考文献(References)：

• [1]Li P,Zhu N,Yi B,et al.MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation[J].Circ Res,2013,113(10):1117.
• [2]Li Y,Song L Q,Chen M Q,et al.Low strength static magnetic field inhibits the proliferation,migration,and adhesion of human vascular smooth muscle cells in a restenosis model through mediating integrinsβ1-FAK,Ca2+signaling pathway[J].Ann Biomed Eng,2012,40(12):2611.
• [3]Chen D,Liu J,Rui B,et al.GSTpi protects against angiotensin II-induced proliferation and migration of vascular smooth muscle cells by preventing signal transducer and activator of transcription3 activation[J].Biochim Biophys Acta,2013,1843(2):454.
• [4]Abhijit S,Bhaskaran R,Narayanasamy A,et al.Hyperinsulinemia-induced vascular smooth muscle cell(VSMC)migration and proliferation is mediated by converging mechanisms of mitochondrial dysfunction and oxidative stress[J].Mol Cell Biochem,2013,373(1/2):95.
• [5]Breen D M,Giacca A.Effects of insulin on the vasculature[J].Curr Vasc Pharm,2011,9(3):321.
• [6]He R,Qu A J,Mao J M,et al.Synergistic proliferation induced by insulin and glycated serum albumin in rat vascular smooth muscle cells[J].Acta Physiol Sin,2007,59(1):1.
• [7]杨静,刘德山,高伟,等.大蒜素对兔血管平滑肌细胞增殖及c-myc基因表达的影响[J].中国老年学杂志,2011(31):622.
• [8]Golovchenko I,Yang C H,Goalstone M L,et al.Garlic extract methylallyl thiosulfinate blocks insulin potentiation of platelet-derived growth factor-stimulated migration of vascular smooth muscle cells[J].Metabolism,2003,52(2):254.
• [9]Borisov N,Aksamitiene E,Kiyatkin A,et al.Systems-leve linteractions between insulin-EGF networks amplify mitogenicsignaling[J].Mol Syst Biol,2009,5:256.
• [10]Izawa Y,Yoshizumi M,Fujita Y,et al.ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells[J].Exp Cell Res,2005,308(2):291.
• [11]Zhong X,Wang H,Jian X.Insulin augments mechanical straininduced ERK activation and cyclooxygenase-2 expression in MG63 cells through integrins[J].Exp Therap Med,2013,7(1):295.
• [12]Cha J H,Choi Y J,Cha S H,et al.Allicin inhibits cell growth and induces apoptosis in U87MG human glioblastoma cells through an ERK-dependent pathway[J].Oncol Rep,2012,28(1):41.
• [13]Elkayam A,Peleg E,Grossman E,et al.Effects of allicin on cardiovascular risk factors in spontaneously hypertensive rats[J].Isr Med Assoc J,2013,15(3):170.