金属硫蛋白抑制同型半胱氨酸诱导的大鼠血管平滑肌细胞增殖

目的:研究金属硫蛋白(MT)对同型半胱氨酸(Hcy)诱导大鼠血管平滑肌细胞(vascularsmoothmusclecells,VSMCs)增殖的影响及其作用机制。方法:以³H]-TdR掺入法测定VSMCs增殖程度,免疫沉淀法测定VSMCs内丝裂素活化蛋白激酶(MAPK)活性,¹⁰⁹Cd]-血红蛋白饱和法测定MT含量,硫代巴比妥酸法测定丙二醛(MDA)含量,NADH氧化法测定乳酸脱氢酶(LDH)漏出量。结果:Hcy(10^-6-10^-4mmol/L)呈浓度依赖性的刺激培养大鼠VSMCs³H]-TdR掺入,0.1mmol/LHcy刺激³H]-TdR掺入比对照组高4.2倍(P＜0.01)。Hcy亦呈浓度依赖性地激活VSMCsMAPK活性、增加细胞MDA的生成和LDH的漏出(P均<0.01)。单独MT孵育,对VSMCs的上述指标均无明显影响(P＞0.05)。但MT(10^-6-10^-4mol/L)呈浓度依赖性抑制100μmol/LHcy的促增殖效应(r=0.98,P＜0.01)。MT显著抑制Hcy对VSMCs的MAPK活性、MDA生成和LDH漏出的激活作用(均P＜0.01)。以0.5mmol/LZnCl₂预孵育6h后,VSMCsMT含量比非诱导细胞高5.7倍(P＜0.01),这种内源性MT高表达的细胞,显著抵抗Hcy刺激的-TdR掺入和MAPK激活;抑制Hcy的促细胞MDA生成与LDH漏出效应(均P＜0.01)。结论:MT能有效抑制Hcy促大鼠VMSCs增殖作用,其机制可能与MT拮抗Hcy对MAPK的激活和其抗氧化作用有关。

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM: To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS: VSMCs proliferation was measured by -TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by 109 Cd]-hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS: Hcy(10 -6 -10 -4 mmol/L) stimulated -TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, -TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold ( P< 0 01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release ( P< 0 01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10 -6 -10 -4 mol/L) attenuated significantly Hcy-stimulated proliferation of VSMCs ( P< 0 01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl 2 for 6 h induced an increase cellular MT content by 5.7-fold ( P< 0 01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage ( P< 0 01). CONCLUSION: These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.