氯吡格雷对尼古丁诱导的大鼠血管内皮损伤的保护作用
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摘要
目的:通过使用氯吡格雷干预尼古丁诱导的血管内皮损伤的大鼠模型,探讨氯吡格雷对血管内皮细胞损伤的保护作用及机制。
方法:健康雄性Sprague Dawley(SD)大鼠共40只,体重180～220g,随机分为5组,每组8只,分别为:对照组、模型组、氯吡格雷低剂量组、氯吡格雷中剂量组和氯吡格雷高剂量组。模型组每天给予腹腔注射尼古丁(2 mg /kg),连续4周,建立血管内皮损伤模型。氯吡格雷低剂量组在模型组的基础上给予氯吡格雷溶液(3mg/kg)灌胃,氯吡格雷中剂量组在模型组的基础上给予氯吡格雷溶液(10mg/kg)灌胃,氯吡格雷高剂量组在模型组的基础上给予氯吡格雷溶液(30mg/kg)灌胃。在造模前一天及造模4周后,舌下静脉抽血测定大鼠血浆中SOD、ET-1及NO的水平。试验结束后取各组大鼠胸主动脉做成石蜡切片,免疫组织化学SABC法染色,于光镜下观察血管内皮细胞eNOS的表达情况。同时取各组大鼠胸主动脉做成电镜标本,通过扫描电镜观察血管内皮细胞的形态学变化。
结果:(1)造模4周后,与对照组比较,模型组血浆中SOD含量下降(p <0.01);三个剂量治疗组与模型组比较血浆SOD含量明显升高(p <0.01);模型组,造模后与造模前比较,血浆SOD含量降低(p <0.01);低、中剂量组,造模后与造模前比较,血浆SOD含量降低(p <0.05,p <0.01);高剂量组,造模前后,血浆SOD含量的差异没有显著性(p >0.05);
(2)造模4周后,与对照组比较,模型组血浆NO含量降低(p <0.01);三个剂量治疗组与模型组比较血浆NO含量明显升高(p <0.05,p <0.01);模型组,造模后与造模前比较,血浆NO含量降低(p <0.01);低、中剂量组,造模后与造模前比较,血浆NO含量降低(p <0.05,p <0.01);高剂量组,造模前后,血浆NO含量的差异没有显著性(p >0.05);
(3)造模4周后,与对照组比较,模型组血浆ET-1的含量升高(p <0.01);与模型组比较,低剂量组血浆ET-1的含量降低,但差异没有显著性(p >0.05),而中、高剂量氯吡格雷组血浆ET-1的含量下降(p <0.01);模型组,造模后与造模前比较,血浆ET-1含量升高(p <0.01);三个剂量治疗组,造模后与造模前比较,血浆ET-1含量升高(p <0.05,p <0.01);
(4) eNOS免疫组化染色可见eNOS在对照组血管内皮细胞的阳性表达率为(86.50±5.42%),模型组血管内皮细胞的eNOS阳性细胞表达率(32.75±7.086%)较对照组明显减少(p <0.01),低剂量治疗组血管内皮细胞的eNOS阳性细胞表达率(39.50±6.02%)较模型组升高(p <0.05),中、高剂量组血管内皮细胞的eNOS阳性细胞表达率分别为(49.00±5.95%)、(70.25±6.62%)较模型组明显升高(p <0.01);
(5)电镜可见:模型组大鼠胸主动脉内皮出现明显损伤,细胞间隙增大或出现深大裂隙,胞膜表面部分脱落呈大小不等的虫蚀状、火山口状缺损,三个氯吡格雷治疗组损伤明显轻于模型组,内皮细胞呈带状排列,大小形态相似,结构基本正常,呈修复状态,而且随氯吡格雷浓度递增呈现损伤减轻的趋势。
结论:氯吡格雷可能是通过提高血管内皮细胞中eNOS的表达,从而改善尼古丁对血管内皮细胞的损伤。
Objective To reseach the protective effect of clopidogrel on nicotine - induced endothelial dysfunction in rats.
Methods 40 SD rats, Weight 180～220g, were divided into five groups:①normal control group,②nicotine control group (2 mg / kg),③low-dose clopidogrel group (nicotine 2 mg / kg + clopidogrel 3mg/kg),④middle dose clopidogrel group (nicotine 2 mg / kg + clopidogrel 10mg/kg),⑤high-dose clopidogrel group (nicotine 2 mg / kg + clopidogrel 30mg/kg). The day before and 4 weeks later of modeling, sublingual vein blood,to measure plasma SOD, ET-1 and NO levels of the rats. After the test , take each group of rat thoracic aorta to Make of paraffin sections, SABC immunohistochemical staining method ,to observed the expression of eNOS In vascular endothelial cells under light microscope . We observe the morphological changes of vascular endothelial cells by the scanning electron microscopy.
Results (1) The plasma SOD levels of model group were obviously lower than the control group (p <0.01),the plasma SOD levels of three-dose treatment group were obviously higher than the model group 4 weeks after modeling(p <0.01); the plasma SOD levels of model group were obviously lower than the the pre-modeling (p <0.01); the plasma SOD levels of low、middle dose group were obviously lower than the the pre-modeling (p <0.05, p <0.01); the plasma SOD levels of high-dose group were similar to the pre-modeling(p >0.05).(2) The plasma NO levels of model group were obviously lower than the control group (p <0.01),the plasma NO levels of three-dose treatment group were obviously higher than the model group 4 weeks after modeling(p <0.05, p <0.01); the plasma NO levels of model group were obviously lower than the the pre-modeling (p <0.01); the plasma NO levels of low、middle dose group were obviously lower than the the pre-modeling (p <0.05, p <0.01); the plasma NO levels of high-dose group were similar to the pre-modeling(p >0.05). (3) The plasma ET-1 levels of model group were obviously higher lower than the control group (p <0.01),the plasma ET-1 levels of middle、high dose treatment group were obviously lower than the model group 4 weeks after modeling(p <0.05, p <0.01); the plasma ET-1 levels of model group were obviously higher than the the pre-modeling (p <0.01); the plasma ET-1 levels of three-dose treatment group were obviously higher than the the pre-modeling (p <0.05, p <0.01).
(4)The eNOS immunohistochemistry shows the eNOS positive of endothelial cells expression of the control group were(86.50±5.42%). The eNOS positive of endothelial cells expression of the model group (32.75±7.086%) were lower than the control group (p < 0.01). low-dose treatment group(39.50±6.02%) were higher than the model group (p <0.05). compared with model group ,the eNOS positive of endothelial cells expression of middle and high dose group ( 49.00±5.95%), (70.25±6.62%) were significantly higher (p <0.01).
(5)SEM : the thoracic aortic endothelial apparent injury in the model group. Compared with the model group, the thoracic aorta endothelial damage of three-dose treatment group were mitigation.
Conclusion Clopidogrel can significantly relieve the nicotine on vascular endothelial cell damage.
方法:健康雄性Sprague Dawley(SD)大鼠共40只,体重180～220g,随机分为5组,每组8只,分别为:对照组、模型组、氯吡格雷低剂量组、氯吡格雷中剂量组和氯吡格雷高剂量组。模型组每天给予腹腔注射尼古丁(2 mg /kg),连续4周,建立血管内皮损伤模型。氯吡格雷低剂量组在模型组的基础上给予氯吡格雷溶液(3mg/kg)灌胃,氯吡格雷中剂量组在模型组的基础上给予氯吡格雷溶液(10mg/kg)灌胃,氯吡格雷高剂量组在模型组的基础上给予氯吡格雷溶液(30mg/kg)灌胃。在造模前一天及造模4周后,舌下静脉抽血测定大鼠血浆中SOD、ET-1及NO的水平。试验结束后取各组大鼠胸主动脉做成石蜡切片,免疫组织化学SABC法染色,于光镜下观察血管内皮细胞eNOS的表达情况。同时取各组大鼠胸主动脉做成电镜标本,通过扫描电镜观察血管内皮细胞的形态学变化。
结果:(1)造模4周后,与对照组比较,模型组血浆中SOD含量下降(p <0.01);三个剂量治疗组与模型组比较血浆SOD含量明显升高(p <0.01);模型组,造模后与造模前比较,血浆SOD含量降低(p <0.01);低、中剂量组,造模后与造模前比较,血浆SOD含量降低(p <0.05,p <0.01);高剂量组,造模前后,血浆SOD含量的差异没有显著性(p >0.05);
(2)造模4周后,与对照组比较,模型组血浆NO含量降低(p <0.01);三个剂量治疗组与模型组比较血浆NO含量明显升高(p <0.05,p <0.01);模型组,造模后与造模前比较,血浆NO含量降低(p <0.01);低、中剂量组,造模后与造模前比较,血浆NO含量降低(p <0.05,p <0.01);高剂量组,造模前后,血浆NO含量的差异没有显著性(p >0.05);
(3)造模4周后,与对照组比较,模型组血浆ET-1的含量升高(p <0.01);与模型组比较,低剂量组血浆ET-1的含量降低,但差异没有显著性(p >0.05),而中、高剂量氯吡格雷组血浆ET-1的含量下降(p <0.01);模型组,造模后与造模前比较,血浆ET-1含量升高(p <0.01);三个剂量治疗组,造模后与造模前比较,血浆ET-1含量升高(p <0.05,p <0.01);
(4) eNOS免疫组化染色可见eNOS在对照组血管内皮细胞的阳性表达率为(86.50±5.42%),模型组血管内皮细胞的eNOS阳性细胞表达率(32.75±7.086%)较对照组明显减少(p <0.01),低剂量治疗组血管内皮细胞的eNOS阳性细胞表达率(39.50±6.02%)较模型组升高(p <0.05),中、高剂量组血管内皮细胞的eNOS阳性细胞表达率分别为(49.00±5.95%)、(70.25±6.62%)较模型组明显升高(p <0.01);
(5)电镜可见:模型组大鼠胸主动脉内皮出现明显损伤,细胞间隙增大或出现深大裂隙,胞膜表面部分脱落呈大小不等的虫蚀状、火山口状缺损,三个氯吡格雷治疗组损伤明显轻于模型组,内皮细胞呈带状排列,大小形态相似,结构基本正常,呈修复状态,而且随氯吡格雷浓度递增呈现损伤减轻的趋势。
结论:氯吡格雷可能是通过提高血管内皮细胞中eNOS的表达,从而改善尼古丁对血管内皮细胞的损伤。
Objective To reseach the protective effect of clopidogrel on nicotine - induced endothelial dysfunction in rats.
Methods 40 SD rats, Weight 180～220g, were divided into five groups:①normal control group,②nicotine control group (2 mg / kg),③low-dose clopidogrel group (nicotine 2 mg / kg + clopidogrel 3mg/kg),④middle dose clopidogrel group (nicotine 2 mg / kg + clopidogrel 10mg/kg),⑤high-dose clopidogrel group (nicotine 2 mg / kg + clopidogrel 30mg/kg). The day before and 4 weeks later of modeling, sublingual vein blood,to measure plasma SOD, ET-1 and NO levels of the rats. After the test , take each group of rat thoracic aorta to Make of paraffin sections, SABC immunohistochemical staining method ,to observed the expression of eNOS In vascular endothelial cells under light microscope . We observe the morphological changes of vascular endothelial cells by the scanning electron microscopy.
Results (1) The plasma SOD levels of model group were obviously lower than the control group (p <0.01),the plasma SOD levels of three-dose treatment group were obviously higher than the model group 4 weeks after modeling(p <0.01); the plasma SOD levels of model group were obviously lower than the the pre-modeling (p <0.01); the plasma SOD levels of low、middle dose group were obviously lower than the the pre-modeling (p <0.05, p <0.01); the plasma SOD levels of high-dose group were similar to the pre-modeling(p >0.05).(2) The plasma NO levels of model group were obviously lower than the control group (p <0.01),the plasma NO levels of three-dose treatment group were obviously higher than the model group 4 weeks after modeling(p <0.05, p <0.01); the plasma NO levels of model group were obviously lower than the the pre-modeling (p <0.01); the plasma NO levels of low、middle dose group were obviously lower than the the pre-modeling (p <0.05, p <0.01); the plasma NO levels of high-dose group were similar to the pre-modeling(p >0.05). (3) The plasma ET-1 levels of model group were obviously higher lower than the control group (p <0.01),the plasma ET-1 levels of middle、high dose treatment group were obviously lower than the model group 4 weeks after modeling(p <0.05, p <0.01); the plasma ET-1 levels of model group were obviously higher than the the pre-modeling (p <0.01); the plasma ET-1 levels of three-dose treatment group were obviously higher than the the pre-modeling (p <0.05, p <0.01).
(4)The eNOS immunohistochemistry shows the eNOS positive of endothelial cells expression of the control group were(86.50±5.42%). The eNOS positive of endothelial cells expression of the model group (32.75±7.086%) were lower than the control group (p < 0.01). low-dose treatment group(39.50±6.02%) were higher than the model group (p <0.05). compared with model group ,the eNOS positive of endothelial cells expression of middle and high dose group ( 49.00±5.95%), (70.25±6.62%) were significantly higher (p <0.01).
(5)SEM : the thoracic aortic endothelial apparent injury in the model group. Compared with the model group, the thoracic aorta endothelial damage of three-dose treatment group were mitigation.
Conclusion Clopidogrel can significantly relieve the nicotine on vascular endothelial cell damage.
引文
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[21] Shinsuke Miyahara,Junichi Kiryu,Kazuaki Miyamoto,et al. Alteration of Leukocyte–Endothelial Cell Interaction During Aging in Retinal Microcirculation of Hypertensive Rats[J].Jpn J Ophthalmol 2006, 50(6):509–514.
[22]张红旗,徐丹令,都颖,等.用血管紧张素缓释泵制作大鼠血管内皮损伤模型探讨一种内皮早期损伤模型的新方法[J].中国临床医学,2007,14(4):451-454.
[23] Yang LX.Role of TRPC1 and NF-kappaB in mediating angiotensin II-induced Ca2+ entry and endothelial hyperpermeability[J]. Peptides, 2009,30(7):1368-1373.
[24] Zhang H,Schmeisser A,Garlichs CD, et al.Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases[J].Cardiovasc Res. 1999,44(1):215-222.
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[26] Vlassara H , Fuh H , Makita Z , et al . Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals : a model for diabetic and aging complications [J]. Proc Natl Acad Sci USA JT.1992,89(24): 12043-12047.
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