Autocamtide 2-相关抑制肽对心肌成纤维细胞分泌细胞因子及基质金属蛋白酶表达的抑制研究

摘要/Abstract

摘要： 目的: 观察钙-钙调素依赖性蛋白激酶(CaMK)Ⅱ抑制剂(autocamtide 2-相关抑制肽, AIP)在大鼠心肌成纤维细胞增殖中的作用及其分子机制。方法: 培养新生1～3 d 乳鼠心肌成纤维细胞(3代),分为正常对照组(CON),血管紧张素Ⅱ(AngⅡ,0.1 umol/L)组,AngⅡ(0.1 μmol/L)+AIP(0.2 μmol/L)组, AngⅡ(0.1 μmol/L)+AIP(0.5 μmol/L)组,AngⅡ(0.1 μmol/L)+AIP组(1.0 μmol/L);MTT法及细胞记数法分别测定心肌成纤维细胞增殖;ELISA法测定细胞因子(TGF-β₁, TNF-a);RT-PCR检测基质金属蛋白酶-2,9(MMP-2,9)mRNA水平;免疫印记法检测MMP-2,9的蛋白表达。结果: AIP(0.5 μmol/L,1.0 μmol/L)抑制AngII引起的心肌成纤维细胞增殖,并呈剂量依赖;AIP(0.5 μmol/L,1.0 μmol/L) 抑制AngII引起的细胞因子分泌,并呈剂量依赖AIP(0.5 μmol/L,1.0 μmol/L)预防 0.1 μmol/L AngⅡ引起的MMP-2,9 mRNA及蛋白的表达增加。结论: AIP(0.5 μmol/L,1.0 μmol/L)对AngⅡ诱导的心肌纤维化具有保护作用,其机制可能与AIP预防心肌成纤维细胞增殖、细胞因子分泌以及对基质金属蛋白酶的调节有关。

关键词: 心肌成纤维细胞, 血管紧张素Ⅱ, 钙-钙调素依赖性蛋白激酶Ⅱ, autocamtide 2-相关抑制肽, 基质金属蛋白酶-2,9

Abstract: AIM: To observe the role of calcium calmodulin dependent protein Kinase(CaMK) Ⅱinhibitor(autocamtide 2-related inhibitory peptide, AIP) in rat cardiac fibroblasts proliferation and their molecular mechanism. METHODS: Using cultured cardiac fibroblasts from neonatal 1-3 days rat (3 generation); This cells were divided into five groups as follows :control group, AngⅡ group(0.1 μmol/L), AngⅡ(0.1 μmol/L)+AIP group(0.2 μmol/L), AngⅡ(0.1 μmol/L))+AIP group(0.5 μmol/L), AngⅡ(0.1 μmol/L)+AIP group(1.0 μmol/L); The cardiac fibroblasts proliferation was detected by cell counting and MTT; The cytokines excreting(TGF-β₁, TNF-a) was investigated by ELISA. The expression of MMP-2,9 mRNA was detected by RT-PCR. RESULTS: AIP (0.5 μmol/L,1.0 μmol/L) could prevent cardiac fibroblasts proliferation induced by angiotensin Ⅱin a dose-dependent manner; AIP (0.5 μmol/L,1.0 μmol/L) could prevent cytokines excreting induced by angiotensin Ⅱin a dose-dependent manner; AIP (0.5 μmol/L, 1.0 μmol/L) could prevent the increasing expression of MMP-2,9 mRNA and MMP-2,9 protein expression induced by 0.1 μmol/L AngⅡ. CONCLUSION: AIP(0.5 μmol/L, 1.0 μmol/L) could prevent myocardial fibrosis induced by angiotensin Ⅱ; The probably mechanism was that: AIP could prevent cardiac fibroblasts proliferation,its cytokines excreting and regulate extracellular matrix(MMP-2、9).

Key words: Cardiac fibroblasts, Angiotensin Ⅱ, Calcium calmodulin dependent protein KinaseⅡ, autocamtide 2-related inhibitory peptide, Matrix metalloproteinases-2,9

中图分类号:

R542.2
R969

钟拥军, 陈东芹, 姚小燕. Autocamtide 2-相关抑制肽对心肌成纤维细胞分泌细胞因子及基质金属蛋白酶表达的抑制研究[J]. 中国临床药理学与治疗学, 2010, 15(4): 422-428.

ZHONG Yong-jun, CHEN Dong-qin, YAO Xiao-yan. Study of autocamtide 2-related inhibitory peptide prevent cardiac fibroblasts excreting cytokines and expressing matrix metalloproteinases[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2010, 15(4): 422-428.

参考文献

[1] Goldsmith EC, Hoffman A, Morales MO, et al. Organization of fibroblasts in the heart[J]. Dev Dyn,2004,230(4):787-794.
[2] Beltrami CA, Finato N, Rocco M, et al. Structural basis of end-stage failure inischemic cardiomyopathy in humans[J]. Circulation ,1994,89(1): 151-163.
[3] Carroll EP, Janicki JS, Pick R, et al. Myocardial stiffness and reparative fibrosis following coronary embolisation in the rat[J]. Cardiovasc Res,1989,23(8): 655-661.
[4] Jalil JE, Doering CW, Janicki JS, et al. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle[J]. Circ Res, 1989,64(6):1041-1050.
[5] Erickson JR, Joiner ML, Guan X, et al. A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation[J]. Cell, 2008,133(2):462-474.
[6] Illario M, Monaco S, Cavallo AL, et al. Calcium-calmodulin-dependent kinase II (CaMKII) mediates insulin-stimulated proliferation and glucose uptake[J]. Cell Signal, 2009,21(5):786-792.
[7] Li N, Wang C, Wu Y, et al. Ca(2+)/calmodulin-dependent protein kinase II promotes cell cycle progression by directly activating MEK1 and subsequently modulating p27 phosphorylation[J]. J Biol Chem, 2009, 284(5):3021-3027.
[8] Zhu W, Woo AY, Yang D, et al. Activation of CaMKIIdeltaC is a common intermediate of diverse death stimuli-induced heart muscle cell apoptosis[J]. J Biol Chem, 2007,282(14):10833-10839.
[9] Wu Y, Gao Z, Chen B, et al. In the cover: Calmodulin kinase II is required for fight or flight sinoatrial node physiology[J]. Proc Natl Acad Sci, 2009,106(14):5972-5977.
[10] Zhang T Brown JH. Role of Ca²⁺/calmodulin-dependent protein kinase II in cardiac hypertrophy and heart failure[J]. Cardiol Res, 2004,63(3):476-486.
[11] Zhang R, Khoo M, Wu Y, et al. Calmodulin kinase II inhibition protects against structural heart disease[J]. Nature Medicine, 2005,4(11):409-417.
[12] Hempel P, Hoch B, Bartel S, et al. Hypertrophic phenotype of cardiac calcium/calmodulin-dependent protein kinase II is reversed by angiotensin converting enzyme inhibition[J]. Basic Res Cardiol, 2002,97(1):96-101.
[13] Lijnen PJ, Petrov VV.Role of intracardiac renin-angiotensin aldo-sterone system in extracellularmatrix remodeling[J]. Methods Find Exp Clin Pharmacol, 2003,25(7):541-564.
[14] Pauschinger M, Knopf D, Petschauer S, et al. Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio[J]. Circulation, 1999,99(21):2750-2756.
[15] Schultz JEJ, Witt SA, Glascock BJ, et al. TGF-β₁ mediates the hypertrophic cardio myocyte growth induced by angiotensin II[J]. J Clin Invest, 2002,109(6):787-796.
[16] Janczewski AM, Kadokami T, Lemster B, et al. Morphological and functional changes in cardiac myocytes isolated from mice overexp-ressing TNF-alpha[J]. Am J Physiol Heart CircPhysiol, 2003,284(3):H960-H969.
[17] Schnee JM,Hsueh WA. Angiotensin II, adhesion, and cardiac fibrosis[J]. Cardiovasc Res, 2000,46(2):264-268
[18] 董六一,陈志武.心肌缺血/再灌注损伤与炎症反应[J]. 中国临床药理学与治疗学, 2008,13(5):582-588.
[19] Ishida A, Shigeri Y, Taniguchi T, et al. Protein phosphatases that regulate multifunctional Ca²⁺/calmodulin-dependent protein kinases: from biochemistry to pharmacology[J]. Pharmacol Ther, 2003,100(3):291-305.
[20] Colomer JM, Mao L, Rockman HA, et al. Pressure overload selectively up-regulates Ca²⁺/calmodulin-dependent protein kinase II in vivo[J]. Mol Endocrinol, 2003,17(2):183-192.
[21] Jorge A, Spadaccio GC , Langer J, et al. Electrostimulation induces cardiomyocyte predifferentiation of fibroblasts[J]. Biochem Biophys Res Commun, 2008,370(3):450-455.