高同型半胱氨酸血症大鼠MMP-2及MMP-3的表达
摘要
目的：利用蛋氨酸负荷建立大鼠高同型半胱氨酸(hyperhomoeysteine, HHcy)血症模型,测定建模前后血中hcy、基质金属蛋白酶-2及基质金属蛋白酶-3(matrixmetallopoeinases, MMPs)的表达,探讨同型半胱氨酸诱发动脉粥样硬化的相关机制。
方法：取健康雄性Wistar大鼠30只,随机分为对照组与蛋氨酸组及干预组,每组10只。对照组给予普通饲料,蛋氨酸组普通饲料中添加2%蛋氨酸喂养,干预组普通饲料中添加2%蛋氨酸喂养同时每只鼠每日给予叶酸0.5mg,维生素B12 (VitB12) 25ug灌胃,实验期为8周。第8周末,禁食12h,25%乌拉坦腹腔注射麻醉,颈总动脉取血5mL,3000r/min,离心10min,分离血清,-20℃冻存,采用ELISA法批量测定指标,所得数据进行组间比较。用HE染色法对颈总动脉进行病理学检查。
结果：(1)蛋氨酸组中Hcy (20.57±4.34μmol/L)、MMP-2(5470.77±1023.11pg/ml)比对照组Hcy (11.63±4.36μmol/L) MMP-2 (3227.87±746.66pg/ml)浓度升高,P<0.001,差异有统计学意义。MMP-3(2.91±0.46ng/ml)也较对照组中MMP-3浓度(2.03±0.51ng/ml)升高,P<0.05,差异有统计学意义。
(2)干预组中Hcy (16.21±4.41 umol/L)、MMP-2(4030.96±848.32pg/ml)及MMP-3浓度(2.46±0.39ng/ml)比蛋氨酸组降低,P<0.05,差异有统计学意义。
(3)镜下观蛋氨酸组颈总动脉中膜平滑肌细胞增生排列紊乱,弹力纤维融合。干预组与对照组颈总动脉血管壁未见异常。结论：(1)在蛋氨酸饮食喂养8周后,大鼠血清Hcy浓度比正常饮食时升高,颈动脉切片可见标本镜下观察平滑肌层明显厚于对照组,并侵入内皮下间隙,表明高同型半胱氨酸血症大鼠模型制作成功。
(2)蛋氨酸组中Hcy、MMP-2及MMP-3的浓度较正常饮食时升高,结合蛋氨酸组血管镜下表现,说明高同型半胱氨酸血症可以影响血清基质金属蛋白酶的表达,从而促使细胞外基质降解及血管壁重塑,进而促进了动脉粥样硬化。
(3)干预组中Hcy、MMP-2及MMP-3蛋氨酸组浓度降低,说明可以通过补充VitB12和叶酸来有效改善高同型半胱氨酸血症,从而预防动脉粥样硬化。
Objective:To establish a rat model of hyperhomoeysteine (Hhcy) and investigate the expression of blood homoeysteine (Hcy)、matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-3(MMP-3) before and after modelling to explore how Hhcy induces atherosclerosis.
Methods:30 healthy male Wistar rats were selected and randomly divided into three groups: control group、methionine group and intervention group. Control group was fed on ordinary feeding. Methionine group was fed on ordinary feeding plus 2% methionine. Intervention group was fed on ordinary feeding plus 2% methionine, as well as 0.5 mg folic acid and 25ug vitamin B12 (VitB12) by gastric perfusion every day. The rats were treated for 8 weeks. At the end of 8 weeks, absolute diet for 12h was given to the rats and subject to intraperitoneal injection with 25% urethane.5ml blood was extracted from common carotid artery and centrifuged at 3000r/min for 10 minutes. The serum was collected and stored in-20℃. We measured the concentration of Hcy、MMP-2 and MMP-3 by enzyme-linked immuno sorbent assay (ELISA), then compared these data. HE staining was performed to observe the pathology of common carotid artery.
Results:
(1) The levels of Hcy and MMP-2 in methionine group were 20.57±4.34μmol/L and 5470.77±102 3.11pg/ml, which were significantly higher than those in control group, P<0.001 (Hcy:11.63±4.36μmol/L and MMP-2:3227.87±746.66 pg/ml). The level of MMP-3 in methionine group was 2.91±0.46ng/ml, which was significantly higher than that in control group, P<0.05(MMP-3:2.03±0.51ng/ml).
(2) The levels of Hcy、MMP-2 and MMP-3 in intervention group were 16.21±4.41μmol/L, 4030.96±848.32pg/ml and 2.460.39ng/ml respectively. The differences of the three data in interven-tion group and methionine group were statistically significant, P<0.05.
(3)The proliferation of the smooth muscle cells was observed by microscope and was found that in midlayer of common carotid artery in methionine group were irregularly organized, and the elastic fiber was fusied. The common carotid artery walls in intervention group and control group were normal.
Conclusion:
(1) The level of Hcy in rats fed on methionine feed for 8 weeks is higher than that on ordinary feeding. Smooth muscle layer is much thicker in rats with methionine feeding than those in control group, which suggests that the model of rats with Hhcy is successful.
(2) The levels of Hcy、MMP-2 and MMP-3 in rats fed on methionine feeding are were higher than those on ordinary feeding. In addition, We conclud that Hhcy can promote atherosclerosis by influencing the expression of MMP to cause the degradation of extracellular matrix and the remodelling of blood vessel wall.
(3) The levels of Hcy、MMP-2 and MMP-3 in intervention group are lowerer than those in methionine group, suggesting that VitB12 and folic acid could prevent atherosclerosis by reducing the level of Hcy.
方法：取健康雄性Wistar大鼠30只,随机分为对照组与蛋氨酸组及干预组,每组10只。对照组给予普通饲料,蛋氨酸组普通饲料中添加2%蛋氨酸喂养,干预组普通饲料中添加2%蛋氨酸喂养同时每只鼠每日给予叶酸0.5mg,维生素B12 (VitB12) 25ug灌胃,实验期为8周。第8周末,禁食12h,25%乌拉坦腹腔注射麻醉,颈总动脉取血5mL,3000r/min,离心10min,分离血清,-20℃冻存,采用ELISA法批量测定指标,所得数据进行组间比较。用HE染色法对颈总动脉进行病理学检查。
结果：(1)蛋氨酸组中Hcy (20.57±4.34μmol/L)、MMP-2(5470.77±1023.11pg/ml)比对照组Hcy (11.63±4.36μmol/L) MMP-2 (3227.87±746.66pg/ml)浓度升高,P<0.001,差异有统计学意义。MMP-3(2.91±0.46ng/ml)也较对照组中MMP-3浓度(2.03±0.51ng/ml)升高,P<0.05,差异有统计学意义。
(2)干预组中Hcy (16.21±4.41 umol/L)、MMP-2(4030.96±848.32pg/ml)及MMP-3浓度(2.46±0.39ng/ml)比蛋氨酸组降低,P<0.05,差异有统计学意义。
(3)镜下观蛋氨酸组颈总动脉中膜平滑肌细胞增生排列紊乱,弹力纤维融合。干预组与对照组颈总动脉血管壁未见异常。结论：(1)在蛋氨酸饮食喂养8周后,大鼠血清Hcy浓度比正常饮食时升高,颈动脉切片可见标本镜下观察平滑肌层明显厚于对照组,并侵入内皮下间隙,表明高同型半胱氨酸血症大鼠模型制作成功。
(2)蛋氨酸组中Hcy、MMP-2及MMP-3的浓度较正常饮食时升高,结合蛋氨酸组血管镜下表现,说明高同型半胱氨酸血症可以影响血清基质金属蛋白酶的表达,从而促使细胞外基质降解及血管壁重塑,进而促进了动脉粥样硬化。
(3)干预组中Hcy、MMP-2及MMP-3蛋氨酸组浓度降低,说明可以通过补充VitB12和叶酸来有效改善高同型半胱氨酸血症,从而预防动脉粥样硬化。
Objective:To establish a rat model of hyperhomoeysteine (Hhcy) and investigate the expression of blood homoeysteine (Hcy)、matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-3(MMP-3) before and after modelling to explore how Hhcy induces atherosclerosis.
Methods:30 healthy male Wistar rats were selected and randomly divided into three groups: control group、methionine group and intervention group. Control group was fed on ordinary feeding. Methionine group was fed on ordinary feeding plus 2% methionine. Intervention group was fed on ordinary feeding plus 2% methionine, as well as 0.5 mg folic acid and 25ug vitamin B12 (VitB12) by gastric perfusion every day. The rats were treated for 8 weeks. At the end of 8 weeks, absolute diet for 12h was given to the rats and subject to intraperitoneal injection with 25% urethane.5ml blood was extracted from common carotid artery and centrifuged at 3000r/min for 10 minutes. The serum was collected and stored in-20℃. We measured the concentration of Hcy、MMP-2 and MMP-3 by enzyme-linked immuno sorbent assay (ELISA), then compared these data. HE staining was performed to observe the pathology of common carotid artery.
Results:
(1) The levels of Hcy and MMP-2 in methionine group were 20.57±4.34μmol/L and 5470.77±102 3.11pg/ml, which were significantly higher than those in control group, P<0.001 (Hcy:11.63±4.36μmol/L and MMP-2:3227.87±746.66 pg/ml). The level of MMP-3 in methionine group was 2.91±0.46ng/ml, which was significantly higher than that in control group, P<0.05(MMP-3:2.03±0.51ng/ml).
(2) The levels of Hcy、MMP-2 and MMP-3 in intervention group were 16.21±4.41μmol/L, 4030.96±848.32pg/ml and 2.460.39ng/ml respectively. The differences of the three data in interven-tion group and methionine group were statistically significant, P<0.05.
(3)The proliferation of the smooth muscle cells was observed by microscope and was found that in midlayer of common carotid artery in methionine group were irregularly organized, and the elastic fiber was fusied. The common carotid artery walls in intervention group and control group were normal.
Conclusion:
(1) The level of Hcy in rats fed on methionine feed for 8 weeks is higher than that on ordinary feeding. Smooth muscle layer is much thicker in rats with methionine feeding than those in control group, which suggests that the model of rats with Hhcy is successful.
(2) The levels of Hcy、MMP-2 and MMP-3 in rats fed on methionine feeding are were higher than those on ordinary feeding. In addition, We conclud that Hhcy can promote atherosclerosis by influencing the expression of MMP to cause the degradation of extracellular matrix and the remodelling of blood vessel wall.
(3) The levels of Hcy、MMP-2 and MMP-3 in intervention group are lowerer than those in methionine group, suggesting that VitB12 and folic acid could prevent atherosclerosis by reducing the level of Hcy.
引文
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[3]YaoJ, XiongS, KlosK, et al..MultiPle signaling pathways involved in activation of matrix metalloproteinase29(MMP29)by heregulin2betal in human breast cancer cells. Oneogene, 2001,20(56):8066-8074
[4]ReynoldsM A, Kirchick H J, Dahlen J R, etal. Early biomarkers of stroke.[J]. Clin Chem, 2003,49 (10):1733-1739.
[5]KalelaA, Koivu TA, Sisto T, et al. Serum matrixmetalloproteinase-9 concentration in angiographically assessed coronary artery disease [J]. Scand J Clin Lab Invest,2002,62 (5):337-342.
[6]孟冬梅 孙根义 同型半肤氨酸对人血管内皮细胞基质金属蛋白酶表达的影响：[硕士学位论文].天津：天津医科大学,2006
[7]黎明,陈健,李裕舒等,叶酸降低高同型半胱氨酸血症大鼠主动脉单核细胞趋化蛋白-1的表达.[J]中国病理生理杂志2007,23(4)：678-68
[8]MalinowMR, Bostom AG, KraussRM. Homocysteine, diet ancardiovascular diseases:a statenent for healthcare professionafrom theNutrition Committee, American HeartAssociation[J].Circulation,1999,99(2):178-182.
[9]苏化庆,杨期明,杨期东.高同型半胱氨酸血症与脑血管病发病机制研究进展[J].脑与神经疾病杂志,2005,13(5)：397.
[10]Guthikonda S, Haynes W G. Homocysteine as a novel risk factor for atherosclerosis [J]. Curr Opin Cardiol,1999,14(4):283.
[11]Morita T, Mitsialis SA, KoikeH, et a.l Carbon monoxide con-trols the proliferation of hypoxic vascular smooth muscle cells[J]. J BiolChem,1997,272(52):32804-32809.
[12]Kuzuya M,Lguchi A.Role of matrix metalloproteinases in vascular remodeling.[J] Atheroscler Thromb,2003,10:275-282
[13]AIMES R T, QUIGLEY J P. Matrixmetalloproteinase-2 is an intersti-tial collagenase. Inhibitor-free enzyme catalyzes the cleavage ofcollagenfibrils and soluble native type I collagen generating the specific 3/4-and 1/4-length fragments[J]. JBiolChem,1995,270(11):5872-5876.
[14]GROTE K, FLACH I, LUCHTEFELD M,et al. Mechanical stretchenhancesmRNA expression and proenzyme release ofmatrix metallo-proteinase-2 (MMP-2) viaNAD(P)H oxidase-derived reactive oxygenspecies[J]. Circ Res,2003,92(11):e80-e86.
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