1、壓力負荷增加大鼠模型血漿內皮素水平的改變    摘要 目的:觀察壓力負荷增加對大鼠血漿內皮素水平的影響。方法:采用腹主動脈狹窄法所致壓力負荷增加誘發大鼠左室肥厚模型,觀察左室重量指數(LVMI),采用放射免疫分析法檢測血漿內皮素水平。結果:LVMI模型組明顯高于假手術組(P<0.01);血漿內皮素水平模型組較假手術組顯著升高(P<0.01)。結論:腹主動脈縮窄所致壓力負荷增加可誘發大鼠左室肥厚,該作用可能與血漿內皮素水平升高有關。 關鍵詞 慢性心力衰竭; 壓力負荷; 內皮素; 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 心室重塑是導致心力衰竭(heart failure,HF)不斷進展的病理生理學基礎,HF時心室重塑的病理刺激主要來自兩方面:一方面神經內分泌的過度激活,另一方面為血流動力學異常。研究發現,異常升高的血管緊張素(Ang)、去甲腎上腺素(NE)、醛固酮和內皮素(ET)等可直接作用于心肌,促進心肌肥厚、心肌細胞凋亡和間質纖維化1。ET是目前已知最強烈持久的血管收縮因子,不但直接刺激或激活多種激素和細胞因子,而且參與心血管疾病的病理生理過程2。作者通過觀察壓力負荷增加對大鼠血漿ET水平的影響,以探討血漿ET在HF病理生理過程中的作用。 1 材料和方

6、法 1.1 實驗材料 動物:雄性SD大鼠20只,體重180200 g;血漿ET放免藥盒(解放軍總醫院科技開發中心放射免疫所提供)。1.2 方法 1.2.1 造模方法 參考Doering等3的方法,2%戊巴比妥鈉40 mgkg-1腹腔注射麻醉,固定,剃毛,常規消毒,分層打開腹腔,在左腎上緣分開后腹膜等軟組織暴露腹主動脈,并在雙腎動脈上方分離腹主動脈,用銀夾造成腹主動脈狹窄(銀夾內徑0.7 mm),分層關閉腹腔。 1.2.2 實驗分組 所有動物隨機分為模型組和假手術組2組,均常規喂養。假手術組手術時只分離腹主動脈,不用銀夾縮窄,余操作與模型組相同。手術8周后處死,計算左室重量指數(LVMI),利用

7、光鏡觀察左室心肌病理形態。 1.3 觀察指標 1.3.1 LVMI 左室與室間隔的重量(濕重)為左室重量,用上海精科天平廠生產的JA1003型電子天平稱重。左室重量(mg)/體重(g)為LVMI()。 1.3.2 左室心肌病理形態 常規石蠟切片采用HE染色,經過脫蠟、染色、脫水、二甲苯透明及中性樹膠封固等步驟,在光鏡下觀察4。 1.3.3 血漿ET放免測定 腹主動脈取血2 ml,注入含10% EDTA二鈉30 l和抑肽酶40 l的試管中,混勻,4 3 000 rmin-1離心10 min,分離血漿。-70 存放,測定前冷水中復融,再次4 3 000 rmin-1離心5 min,取上清采用非平衡

8、法測定5。將聚苯乙稀試管編號,經過加液程序后4 3 500 rmin-1離心25 min,吸棄上清液,在計數器上測定沉淀cpm數。 1.4 統計學處理 定量資料以±s表示,用SPSS 11.5統計軟件進行統計學處理。模型組和假手術組之間比較采用t檢驗,P<0.05表示差異有統計學意義。 2 結果 2.1 LVMI 壓力負荷增加大鼠造模8周LVMI模型組(2.98±0.36)較假手術組(2.11±0.14)有顯著升高(P<0.05)。 2.2 左室心肌HE染色結果 光鏡下假手術組造模8周后左室心肌HE染色顯示:心肌纖維排列整齊,心肌細胞大小正常;模型組造

9、模8周后左室心肌HE染色發現:心肌纖維排列紊亂,細胞橫徑稍大(圖1、2)。 2.3 血漿ET水平的改變 壓力負荷增加大鼠各組血漿ET水平的改變見圖3,模型組血漿ET水平較假手術組顯著升高(P<0.01)。 3 討論 ET是一族由血管內皮細胞、心肌、平滑肌等合成及分泌的含21個氨基酸的活性多肽,是目前已知最強烈持久的血管收縮因子,可使所有的動、靜脈強烈收縮,其強度比Ang強10倍,比NE強1 000倍,尤其是對冠狀動脈的作用最強;另ET-1還具有刺激心肌細胞增生肥大和促進心臟纖維化等生物學效應6。生理條件下,低濃度的ET對維持血管張力和血壓具有重要意義;在某些病理條件下,ET則過量釋放7。

10、如慢性心力衰竭(CHF)時心輸出量下降,心室壁機械負荷過重,導致組織切應力改變,心肌受損部位周圍神經內分泌激活和組織缺氧可使心肌細胞ET-1過度表達,不僅引起血管痙攣、心肌纖維化和心肌平滑肌細胞裂解與凋亡,同時可直接刺激或激活多種激素和細胞因子,共同參與CHF的病理生理過程8。 ET的釋放受許多因素的調節,如缺氧、機械性應力、神經內分泌激素(如NE、Ang)、細胞因子(包括轉化生長因子-和白介素-1)等均可誘導ET-1的表達。HF時,血中ET-1水平明顯升高,升高的程度與HF的嚴重程度呈正相關關系。隨著年齡的增長,血漿ET-1活性水平隨之上升,而有氧鍛煉可降低血漿ET-1的活性9。最近研究發現

11、,特異性ET-1基因敲除大鼠心肌細胞的凋亡增加,而ET-1可通過FasL/TNF信號通路削弱TNF介導的凋亡10。Khan等11的實驗認為big-ET是進展期HF患者的獨立預后因素;還有一些學者提出了不同意見,但大多數研究者已達成共識,認為ET-1是HF強烈的獨立預后因素12。本研究通過腹主動脈縮窄法制作的大鼠模型,造模8周后LVMI模型組較假手術組有顯著升高(P<0.01);模型組左室心肌經HE染色光鏡下見心肌纖維排列紊亂,細胞橫徑稍大,循環ET水平升高。 由于ET在心血管系統中舉足輕重的作用,近年來對ET的認識也逐漸深刻。研究發現ET-1即ETA和ETB對心臟的作用主要通過內皮素受體

12、(endothelin receptor,ETR)介導,其中以ETA為主。人類ETA受體mRNA主要表達于血管平滑肌細胞,ETB受體mRNA大量表達于血管內皮細胞,也表達于主動脈、肺動脈和冠狀動脈的血管平滑肌細胞。ETA受體選擇性與ET-1結合,而ETB受體與ET的各種異構體均具有相同的親和力,也稱為非特異性受體13。由于ETR的分布具有組織特異性,所以它們發揮的功能也不盡相同,ETA受體主要介導心血管收縮作用,ETB受體通過釋放舒張因子,如前列環素(PGI2)、一氧化氮(NO)而介導心血管舒張作用,ETB受體還有清除循環ET-1作用。最近研究發現,ET-1和ETR結合后通過一系列過程可激活促

13、分裂原活化蛋白激酶(MAPK),使細胞生長、肥大,參與心肌重塑。而ET拮抗劑在慢性心力衰竭中的研究觀察到波生坦可減輕HF病人的臨床癥狀14。由于ET和ETB受體結合后可起到擴血管、增加NO和PGI2濃度、增強肺清除ETA等作用,所以選擇性ETA受體拮抗劑可能比同時抑制ETA和ETB受體的拮抗劑更為有利15。 本實驗運用光鏡觀察左室心肌HE染色結果,同時檢測LVMI、血漿ET水平的改變,證實腹主動脈縮窄8周后大鼠心肌肥厚模型建立成功,符合針對CHF的病理生理改變的要求,為臨床及基礎研究提供新的思路。 參考文獻 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 肖繼明,陳銳華,徐軍,等.急性心肌梗死患者血漿內皮素的動態變化及意義J.醫學研究生學報,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. 參考文獻 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 肖繼明,陳銳華,徐軍,等.急性心肌梗死患者血漿內皮素的動態變化及意義J.醫學研究生學報,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. 參考文獻 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 肖繼明,陳銳華,徐軍,等.急性心肌梗死患者血漿內皮素的動態變化及意義J.醫學研究生學報,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. 參考文獻 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 肖繼明,陳銳華,徐軍,等.急性心肌梗死患者血漿內皮素的動態變化及意義J.醫學研究生學報,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. 參考文獻 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