压力负荷增加大鼠模型血浆内皮素水平的改变

1、压力负荷增加大鼠模型血浆内皮素水平的改变    摘要 目的:观察压力负荷增加对大鼠血浆内皮素水平的影响。方法:采用腹主动脉狭窄法所致压力负荷增加诱发大鼠左室肥厚模型,观察左室重量指数(LVMI),采用放射免疫分析法检测血浆内皮素水平。结果:LVMI模型组明显高于假手术组(P<0.01);血浆内皮素水平模型组较假手术组显著升高(P<0.01)。结论:腹主动脉缩窄所致压力负荷增加可诱发大鼠左室肥厚,该作用可能与血浆内皮素水平升高有关。 关键词 慢性心力衰竭; 压力负荷; 内皮素; 大鼠 Changes of the plasma endothel

2、in in rats with pressure overloadAbstract Objective: To observe the effects of pressure overload on level of plasma endothelin, and study the possible mechanism of left ventricular remodeling. Methods: left ventricular mass index(LVMI) and plasma endothelin were measured in the rat models with myoca

3、rdial hypertrophy of chronic heart failure and pressure overload by abdominal aortic constriction by radioimmunoassay. Results: LVMI in the model group was obviously higher than that of the sham group(P<0.01). The plasma endothelin levels in the model group were obviously higher than those of sha

4、m group(P<0.01). Conclusion: pressure overload by abdominal aortic constriction can induce left ventricular remodeling in chronic heart failure, the mechanism of left ventricular remodeling might be related to the upregulated endothelin levels in plasma. Key words chronic heart failure; pressure

5、overload; endothelin; rats 心室重塑是导致心力衰竭(heart failure,HF)不断进展的病理生理学基础,HF时心室重塑的病理刺激主要来自两方面:一方面神经内分泌的过度激活,另一方面为血流动力学异常。研究发现,异常升高的血管紧张素(Ang)、去甲肾上腺素(NE)、醛固酮和内皮素(ET)等可直接作用于心肌,促进心肌肥厚、心肌细胞凋亡和间质纤维化1。ET是目前已知最强烈持久的血管收缩因子,不但直接刺激或激活多种激素和细胞因子,而且参与心血管疾病的病理生理过程2。作者通过观察压力负荷增加对大鼠血浆ET水平的影响,以探讨血浆ET在HF病理生理过程中的作用。 1 材料和方

6、法 1.1 实验材料 动物:雄性SD大鼠20只,体重180200 g;血浆ET放免药盒(解放军总医院科技开发中心放射免疫所提供)。1.2 方法 1.2.1 造模方法 参考Doering等3的方法,2%戊巴比妥钠40 mgkg-1腹腔注射麻醉,固定,剃毛,常规消毒,分层打开腹腔,在左肾上缘分开后腹膜等软组织暴露腹主动脉,并在双肾动脉上方分离腹主动脉,用银夹造成腹主动脉狭窄(银夹内径0.7 mm),分层关闭腹腔。 1.2.2 实验分组 所有动物随机分为模型组和假手术组2组,均常规喂养。假手术组手术时只分离腹主动脉,不用银夹缩窄,余操作与模型组相同。手术8周后处死,计算左室重量指数(LVMI),利用

7、光镜观察左室心肌病理形态。 1.3 观察指标 1.3.1 LVMI 左室与室间隔的重量(湿重)为左室重量,用上海精科天平厂生产的JA1003型电子天平称重。左室重量(mg)/体重(g)为LVMI()。 1.3.2 左室心肌病理形态 常规石蜡切片采用HE染色,经过脱蜡、染色、脱水、二甲苯透明及中性树胶封固等步骤,在光镜下观察4。 1.3.3 血浆ET放免测定 腹主动脉取血2 ml,注入含10% EDTA二钠30 l和抑肽酶40 l的试管中,混匀,4 3 000 rmin-1离心10 min,分离血浆。-70 存放,测定前冷水中复融,再次4 3 000 rmin-1离心5 min,取上清采用非平衡

8、法测定5。将聚苯乙稀试管编号,经过加液程序后4 3 500 rmin-1离心25 min,吸弃上清液,在计数器上测定沉淀cpm数。 1.4 统计学处理 定量资料以±s表示,用SPSS 11.5统计软件进行统计学处理。模型组和假手术组之间比较采用t检验,P<0.05表示差异有统计学意义。 2 结果 2.1 LVMI 压力负荷增加大鼠造模8周LVMI模型组(2.98±0.36)较假手术组(2.11±0.14)有显著升高(P<0.05)。 2.2 左室心肌HE染色结果 光镜下假手术组造模8周后左室心肌HE染色显示:心肌纤维排列整齐,心肌细胞大小正常;模型组造

9、模8周后左室心肌HE染色发现:心肌纤维排列紊乱,细胞横径稍大(图1、2)。 2.3 血浆ET水平的改变 压力负荷增加大鼠各组血浆ET水平的改变见图3,模型组血浆ET水平较假手术组显著升高(P<0.01)。 3 讨论 ET是一族由血管内皮细胞、心肌、平滑肌等合成及分泌的含21个氨基酸的活性多肽,是目前已知最强烈持久的血管收缩因子,可使所有的动、静脉强烈收缩,其强度比Ang强10倍,比NE强1 000倍,尤其是对冠状动脉的作用最强;另ET-1还具有刺激心肌细胞增生肥大和促进心脏纤维化等生物学效应6。生理条件下,低浓度的ET对维持血管张力和血压具有重要意义;在某些病理条件下,ET则过量释放7。

10、如慢性心力衰竭(CHF)时心输出量下降,心室壁机械负荷过重,导致组织切应力改变,心肌受损部位周围神经内分泌激活和组织缺氧可使心肌细胞ET-1过度表达,不仅引起血管痉挛、心肌纤维化和心肌平滑肌细胞裂解与凋亡,同时可直接刺激或激活多种激素和细胞因子,共同参与CHF的病理生理过程8。 ET的释放受许多因素的调节,如缺氧、机械性应力、神经内分泌激素(如NE、Ang)、细胞因子(包括转化生长因子-和白介素-1)等均可诱导ET-1的表达。HF时,血中ET-1水平明显升高,升高的程度与HF的严重程度呈正相关关系。随着年龄的增长,血浆ET-1活性水平随之上升,而有氧锻炼可降低血浆ET-1的活性9。最近研究发现

11、,特异性ET-1基因敲除大鼠心肌细胞的凋亡增加,而ET-1可通过FasL/TNF信号通路削弱TNF介导的凋亡10。Khan等11的实验认为big-ET是进展期HF患者的独立预后因素;还有一些学者提出了不同意见,但大多数研究者已达成共识,认为ET-1是HF强烈的独立预后因素12。本研究通过腹主动脉缩窄法制作的大鼠模型,造模8周后LVMI模型组较假手术组有显著升高(P<0.01);模型组左室心肌经HE染色光镜下见心肌纤维排列紊乱,细胞横径稍大,循环ET水平升高。 由于ET在心血管系统中举足轻重的作用,近年来对ET的认识也逐渐深刻。研究发现ET-1即ETA和ETB对心脏的作用主要通过内皮素受体

12、(endothelin receptor,ETR)介导,其中以ETA为主。人类ETA受体mRNA主要表达于血管平滑肌细胞,ETB受体mRNA大量表达于血管内皮细胞,也表达于主动脉、肺动脉和冠状动脉的血管平滑肌细胞。ETA受体选择性与ET-1结合,而ETB受体与ET的各种异构体均具有相同的亲和力,也称为非特异性受体13。由于ETR的分布具有组织特异性,所以它们发挥的功能也不尽相同,ETA受体主要介导心血管收缩作用,ETB受体通过释放舒张因子,如前列环素(PGI2)、一氧化氮(NO)而介导心血管舒张作用,ETB受体还有清除循环ET-1作用。最近研究发现,ET-1和ETR结合后通过一系列过程可激活促

13、分裂原活化蛋白激酶(MAPK),使细胞生长、肥大,参与心肌重塑。而ET拮抗剂在慢性心力衰竭中的研究观察到波生坦可减轻HF病人的临床症状14。由于ET和ETB受体结合后可起到扩血管、增加NO和PGI2浓度、增强肺清除ETA等作用,所以选择性ETA受体拮抗剂可能比同时抑制ETA和ETB受体的拮抗剂更为有利15。 本实验运用光镜观察左室心肌HE染色结果,同时检测LVMI、血浆ET水平的改变,证实腹主动脉缩窄8周后大鼠心肌肥厚模型建立成功,符合针对CHF的病理生理改变的要求,为临床及基础研究提供新的思路。 参考文献 1 VON-LUEDER T G, KJEKSHUS H, EDVARDSEN T,

14、et al. Mechanisms of elevated plasma endothelin-1 in CHF: congestion increases pulmonary synthesis and secretion of endothelin-1J. Cardiovasc Res,2004,63(1):41-50. 2 YAMAMOTO S, MATSUMOTO N, KANAZAWA M, et al. Different contributions of endothelin-A and endothelin-B receptors in postischemic cardiac

15、 dysfunction and norepinephrine overflow in rat hearts J. Circulation,2005,111(3):302-309. 3 DOERING C W, JALIL J E, JANICKI J S, et al. Collagen network remodeling and diastolic stiffness of the rat left ventricle with pressure overload hypertrophyJ. Cardiovasc Res,1998,22:686-694. 4 HIGASHIYAMA H,

16、 SUGAI M, INOUE H, et al. Histopathological study of time course changes in inter-renal aortic banding-induced left ventricular hypertrophy of miceJ. Int J Exp Path, 2007,88(1):31-38. 5 BREDIN F, FRANCO-CERECEDA A. Reversed remodeling in dilated cardiomyopathy by passive containment surgery is assoc

17、iated with decreased circulating levels of endothelin-1J. Eur J Cardiothoracic Surg,2006,29(3):299-303. 6 CLOZEL M, SALLOUKH H. Role of endothelin in fibrosis and antifibrotic potential of bosentanJ. Ann Med,2005,37:2-12. 7 肖继明,陈锐华,徐军,等.急性心肌梗死患者血浆内皮素的动态变化及意义J.医学研究生学报,2007,20(3):331-332. 8 SHAH R. En

18、dothelins in health and disease J. Eur J Inter Med,2007,18:272-282. 9 van GUILDER G P, WESTBY C M, GREINER J J, et al. Endothelin-1 vasoconstrictor tone increases with age in healthy men but can be reduced by regular aerobic exerciseJ. Hypertension,2007,50:403-409. 10 ZHAO X S, PAN W T, BEKEREDJIAN

19、R, et al. Endogenous endothelin-1 is required for cardiomyocyte survival in vivoJ. Circulation,2006,114:830-837. 11 KHAN S Q, DHILLON O, STRUCK J, et al. C-terminal pro-endothelin-1 offers additional prognostic information in patients after acute myocardial infarction: leicester acute myocardial inf

20、arction peptide(LAMP) studyJ.Am Heart J,2007,154:736-742. 12 YIP H K, WU C J, CHANG H W, et al. Prognostic value of circulating levels of endothelin-1 in patients after acute myocardial infarction undergoing primary coronary angioplastyJ. Chest,2005,127:1491-1497. 13 LAFLAMME K, ROBERGE C J, GERMAIN

21、 L, et al. Tissue-engineered human vascular media with a functional endothelin systemJ. Circulation,2005,111(4):459-464. 14 PACKER M, McMURRAY J, MASSIE B M, et al. Clinical effects of endothelin receptor antagonism with bosentan in patients with severe chronic heart failure: results of a pilot stud

22、yJ. J Card Fail,2005,11(1):12-20. 15 COWBURNA P J, CLELANDB J G F, McDONAGH T A, et al. Comparison of selective ETA and ETB receptor antagonists in patients with chronic heart failureJ. Eur J Heart Fail,2005,7:37-42. 参考文献 1 VON-LUEDER T G, KJEKSHUS H, EDVARDSEN T, et al. Mechanisms of elevated plasm

23、a endothelin-1 in CHF: congestion increases pulmonary synthesis and secretion of endothelin-1J. Cardiovasc Res,2004,63(1):41-50. 2 YAMAMOTO S, MATSUMOTO N, KANAZAWA M, et al. Different contributions of endothelin-A and endothelin-B receptors in postischemic cardiac dysfunction and norepinephrine ove

24、rflow in rat hearts J. Circulation,2005,111(3):302-309. 3 DOERING C W, JALIL J E, JANICKI J S, et al. Collagen network remodeling and diastolic stiffness of the rat left ventricle with pressure overload hypertrophyJ. Cardiovasc Res,1998,22:686-694. 4 HIGASHIYAMA H, SUGAI M, INOUE H, et al. Histopath

25、ological study of time course changes in inter-renal aortic banding-induced left ventricular hypertrophy of miceJ. Int J Exp Path, 2007,88(1):31-38. 5 BREDIN F, FRANCO-CERECEDA A. Reversed remodeling in dilated cardiomyopathy by passive containment surgery is associated with decreased circulating le

26、vels of endothelin-1J. Eur J Cardiothoracic Surg,2006,29(3):299-303. 6 CLOZEL M, SALLOUKH H. Role of endothelin in fibrosis and antifibrotic potential of bosentanJ. Ann Med,2005,37:2-12. 7 肖继明,陈锐华,徐军,等.急性心肌梗死患者血浆内皮素的动态变化及意义J.医学研究生学报,2007,20(3):331-332. 8 SHAH R. Endothelins in health and disease J.

27、Eur J Inter Med,2007,18:272-282. 9 van GUILDER G P, WESTBY C M, GREINER J J, et al. Endothelin-1 vasoconstrictor tone increases with age in healthy men but can be reduced by regular aerobic exerciseJ. Hypertension,2007,50:403-409. 10 ZHAO X S, PAN W T, BEKEREDJIAN R, et al. Endogenous endothelin-1 i

28、s required for cardiomyocyte survival in vivoJ. Circulation,2006,114:830-837. 11 KHAN S Q, DHILLON O, STRUCK J, et al. C-terminal pro-endothelin-1 offers additional prognostic information in patients after acute myocardial infarction: leicester acute myocardial infarction peptide(LAMP) studyJ.Am Hea

29、rt J,2007,154:736-742. 12 YIP H K, WU C J, CHANG H W, et al. Prognostic value of circulating levels of endothelin-1 in patients after acute myocardial infarction undergoing primary coronary angioplastyJ. Chest,2005,127:1491-1497. 13 LAFLAMME K, ROBERGE C J, GERMAIN L, et al. Tissue-engineered human

30、vascular media with a functional endothelin systemJ. Circulation,2005,111(4):459-464. 14 PACKER M, McMURRAY J, MASSIE B M, et al. Clinical effects of endothelin receptor antagonism with bosentan in patients with severe chronic heart failure: results of a pilot studyJ. J Card Fail,2005,11(1):12-20. 1

31、5 COWBURNA P J, CLELANDB J G F, McDONAGH T A, et al. Comparison of selective ETA and ETB receptor antagonists in patients with chronic heart failureJ. Eur J Heart Fail,2005,7:37-42. 参考文献 1 VON-LUEDER T G, KJEKSHUS H, EDVARDSEN T, et al. Mechanisms of elevated plasma endothelin-1 in CHF: congestion i

32、ncreases pulmonary synthesis and secretion of endothelin-1J. Cardiovasc Res,2004,63(1):41-50. 2 YAMAMOTO S, MATSUMOTO N, KANAZAWA M, et al. Different contributions of endothelin-A and endothelin-B receptors in postischemic cardiac dysfunction and norepinephrine overflow in rat hearts J. Circulation,

33、2005,111(3):302-309. 3 DOERING C W, JALIL J E, JANICKI J S, et al. Collagen network remodeling and diastolic stiffness of the rat left ventricle with pressure overload hypertrophyJ. Cardiovasc Res,1998,22:686-694. 4 HIGASHIYAMA H, SUGAI M, INOUE H, et al. Histopathological study of time course chang

34、es in inter-renal aortic banding-induced left ventricular hypertrophy of miceJ. Int J Exp Path, 2007,88(1):31-38. 5 BREDIN F, FRANCO-CERECEDA A. Reversed remodeling in dilated cardiomyopathy by passive containment surgery is associated with decreased circulating levels of endothelin-1J. Eur J Cardio

35、thoracic Surg,2006,29(3):299-303. 6 CLOZEL M, SALLOUKH H. Role of endothelin in fibrosis and antifibrotic potential of bosentanJ. Ann Med,2005,37:2-12. 7 肖继明,陈锐华,徐军,等.急性心肌梗死患者血浆内皮素的动态变化及意义J.医学研究生学报,2007,20(3):331-332. 8 SHAH R. Endothelins in health and disease J. Eur J Inter Med,2007,18:272-282. 9

36、van GUILDER G P, WESTBY C M, GREINER J J, et al. Endothelin-1 vasoconstrictor tone increases with age in healthy men but can be reduced by regular aerobic exerciseJ. Hypertension,2007,50:403-409. 10 ZHAO X S, PAN W T, BEKEREDJIAN R, et al. Endogenous endothelin-1 is required for cardiomyocyte surviv

37、al in vivoJ. Circulation,2006,114:830-837. 11 KHAN S Q, DHILLON O, STRUCK J, et al. C-terminal pro-endothelin-1 offers additional prognostic information in patients after acute myocardial infarction: leicester acute myocardial infarction peptide(LAMP) studyJ.Am Heart J,2007,154:736-742. 12 YIP H K,

38、WU C J, CHANG H W, et al. Prognostic value of circulating levels of endothelin-1 in patients after acute myocardial infarction undergoing primary coronary angioplastyJ. Chest,2005,127:1491-1497. 13 LAFLAMME K, ROBERGE C J, GERMAIN L, et al. Tissue-engineered human vascular media with a functional en

39、dothelin systemJ. Circulation,2005,111(4):459-464. 14 PACKER M, McMURRAY J, MASSIE B M, et al. Clinical effects of endothelin receptor antagonism with bosentan in patients with severe chronic heart failure: results of a pilot studyJ. J Card Fail,2005,11(1):12-20. 15 COWBURNA P J, CLELANDB J G F, McD

40、ONAGH T A, et al. Comparison of selective ETA and ETB receptor antagonists in patients with chronic heart failureJ. Eur J Heart Fail,2005,7:37-42. 参考文献 1 VON-LUEDER T G, KJEKSHUS H, EDVARDSEN T, et al. Mechanisms of elevated plasma endothelin-1 in CHF: congestion increases pulmonary synthesis and se

41、cretion of endothelin-1J. Cardiovasc Res,2004,63(1):41-50. 2 YAMAMOTO S, MATSUMOTO N, KANAZAWA M, et al. Different contributions of endothelin-A and endothelin-B receptors in postischemic cardiac dysfunction and norepinephrine overflow in rat hearts J. Circulation,2005,111(3):302-309. 3 DOERING C W,

42、 JALIL J E, JANICKI J S, et al. Collagen network remodeling and diastolic stiffness of the rat left ventricle with pressure overload hypertrophyJ. Cardiovasc Res,1998,22:686-694. 4 HIGASHIYAMA H, SUGAI M, INOUE H, et al. Histopathological study of time course changes in inter-renal aortic banding-in

43、duced left ventricular hypertrophy of miceJ. Int J Exp Path, 2007,88(1):31-38. 5 BREDIN F, FRANCO-CERECEDA A. Reversed remodeling in dilated cardiomyopathy by passive containment surgery is associated with decreased circulating levels of endothelin-1J. Eur J Cardiothoracic Surg,2006,29(3):299-303. 6

44、 CLOZEL M, SALLOUKH H. Role of endothelin in fibrosis and antifibrotic potential of bosentanJ. Ann Med,2005,37:2-12. 7 肖继明,陈锐华,徐军,等.急性心肌梗死患者血浆内皮素的动态变化及意义J.医学研究生学报,2007,20(3):331-332. 8 SHAH R. Endothelins in health and disease J. Eur J Inter Med,2007,18:272-282. 9 van GUILDER G P, WESTBY C M, GREINE

45、R J J, et al. Endothelin-1 vasoconstrictor tone increases with age in healthy men but can be reduced by regular aerobic exerciseJ. Hypertension,2007,50:403-409. 10 ZHAO X S, PAN W T, BEKEREDJIAN R, et al. Endogenous endothelin-1 is required for cardiomyocyte survival in vivoJ. Circulation,2006,114:8

46、30-837. 11 KHAN S Q, DHILLON O, STRUCK J, et al. C-terminal pro-endothelin-1 offers additional prognostic information in patients after acute myocardial infarction: leicester acute myocardial infarction peptide(LAMP) studyJ.Am Heart J,2007,154:736-742. 12 YIP H K, WU C J, CHANG H W, et al. Prognosti

47、c value of circulating levels of endothelin-1 in patients after acute myocardial infarction undergoing primary coronary angioplastyJ. Chest,2005,127:1491-1497. 13 LAFLAMME K, ROBERGE C J, GERMAIN L, et al. Tissue-engineered human vascular media with a functional endothelin systemJ. Circulation,2005,

48、111(4):459-464. 14 PACKER M, McMURRAY J, MASSIE B M, et al. Clinical effects of endothelin receptor antagonism with bosentan in patients with severe chronic heart failure: results of a pilot studyJ. J Card Fail,2005,11(1):12-20. 15 COWBURNA P J, CLELANDB J G F, McDONAGH T A, et al. Comparison of sel

49、ective ETA and ETB receptor antagonists in patients with chronic heart failureJ. Eur J Heart Fail,2005,7:37-42. 参考文献 1 VON-LUEDER T G, KJEKSHUS H, EDVARDSEN T, et al. Mechanisms of elevated plasma endothelin-1 in CHF: congestion increases pulmonary synthesis and secretion of endothelin-1J. Cardiovas

50、c Res,2004,63(1):41-50. 2 YAMAMOTO S, MATSUMOTO N, KANAZAWA M, et al. Different contributions of endothelin-A and endothelin-B receptors in postischemic cardiac dysfunction and norepinephrine overflow in rat hearts J. Circulation,2005,111(3):302-309. 3 DOERING C W, JALIL J E, JANICKI J S, et al. Col

51、lagen network remodeling and diastolic stiffness of the rat left ventricle with pressure overload hypertrophyJ. Cardiovasc Res,1998,22:686-694. 4 HIGASHIYAMA H, SUGAI M, INOUE H, et al. Histopathological study of time course changes in inter-renal aortic banding-induced left ventricular hypertrophy of miceJ. Int J Exp Path, 2007,88(1):31-38. 5 BREDIN F, FRANCO-CERECEDA A. Reversed remodeling in dilated cardiomyopathy by passive containment surgery is assoc