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

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

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 ,[1 0 9Cd] -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(1 0 - 6 - 1 0 - 4mmol/L)呈浓度依赖性的刺激培养大鼠VSMCs [3H] -TdR掺入 ,0 1mmol/LHcy刺激 [3H] -TdR掺入比对照组高 4 2倍 (P <0 0 1 )。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 0 1 )。单独MT孵育 ,对VSMCs的上述指标均无明显影响 (P >0 0 5)。但MT(1 0 - 6 - 1 0 - 4mol/L)呈浓度依赖性抑制 1 0 0 μmol/LHcy的促增殖效应 (r=0 98,P <0 0 1 )。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 0 1 )。以 0 5mmol/LZnCl2 预孵育 6h后 ,VSMCsMT含量比非诱导细胞高 5 7倍 (P <0 0 1 ) ,这种内源性MT高表达的细胞 ,显著抵抗Hcy刺激的 [3H] -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 0 1 )。结论

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.