线粒体融合素2通过Ras-PI3K-Akt信号途径抑制缺血再灌注诱导的乳鼠心肌细胞凋亡

摘要/Abstract

摘要： 目的研究线粒体融合素2(Mfn2)基因对缺血再灌注诱导的乳鼠心肌细胞凋亡的影响及其相关的信号通路。方法乳鼠心肌细胞经缺氧/复氧(H/Re)处理模拟心肌缺血再灌注损伤。用Mfn2基因的重组腺病毒(Adv-Mfn2)感染经缺氧复氧处理的乳鼠心肌细胞。采用TUNEL染色、ELISA、流式细胞术等方法检测Mfn2对缺血再灌注诱导的乳鼠心肌细胞凋亡的影响。Western blot分析线粒体凋亡路径中Bcl-2蛋白、Bax蛋白、Caspase-9以及磷酸化蛋白激酶B(p-Akt)的表达变化。结果 TUNEL染色发现Adv-Mfn2感染乳鼠心肌细胞后,细胞凋亡较H/Re组和Adv-LacZ组显著减少。ELISA和流式细胞仪检测结果表明,Adv-Mfn2组心肌细胞凋亡较H/Re组及Adv-LacZ组明显减少,且这一作用呈时间依赖性。Western blot结果显示,Adv-Mfn2组中Bcl-2蛋白表达较H/Re组和Adv-LacZ感染组上升,Bax蛋白表达下降,各组中Caspase-9的表达变化与Bax相同,Adv-Mfn2组的p-Akt蛋白表达水平则较H/Re组和Adv-LacZ感染组明显上升。结论 Mfn2基因主要通过正向调控Ras-PI3K-Akt信号通路,促进Akt的磷酸化水平,使Bcl-2蛋白表达量增加,Bax蛋白表达量降低,抑制Caspase-9活化,从而抑制缺血再灌注诱导的乳鼠心肌细胞凋亡。

关键词: 线粒体融合素2, 腺病毒, 细胞凋亡, 缺血再灌注损伤

Abstract: AIM: To investigate the effect of Mfn2 gene transfer on inhibiting the apoptosis of cardiomyocytes induced by ischemia/reperfusion. METHODS: Treating neonatal rat cardiomyocytes by hypoxia/reoxygenation to simulating myocardial ischemia/reperfusion injury. And then the cardiomyocytes were infected by adenovirus-mediated Mfn2 (Adv-Mfn2). TUNEL staining, cell death ELISA and FACS analysis were used to investigate the role of Adv-Mfn2 gene transfer on neonatal rat cardiomyocytes apoptosis. Western blot was used to analyze the protein expression of Bcl-2, Bax, cleaved caspase-9 and p-Akt. RESULTS: TUNEL staining evidenced that there were less positive-apoptoic cardiomyocytes in Adv-Mfn2 group than in H/Re group and Adv-LacZ group (P<0.01). ELISA and FACS results showed that cardiomyocytes apoptosis in Adv-Mfn2 group was significantly reduced compared with H/Re group and Adv-LacZ group, and this effect was time-dependent (P<0.01). The protein expression of Bcl-2 and phosphorylated Akt were significantly upregulated in Adv-Mfn2 group (P<0.05), whereas Bax and cleaved caspase-9 protein expressions were significantly downregulated in Adv-Mfn2 group (P<0.05). CONCLUSION: Mfn2 gene promotes the protein expression of Bcl-2 and phosphorylated Akt, supppres the protein expression of Bax and cleaved caspase-9 via activation of the Ras-PI3K-Akt signaling pathway to inhibite neonatal rat cardiomyocytes apoptosis induced by ischemia/reperfusion.

Key words: Mfn2, adenovirus, cell apoptosis, ischemia/reperfusion injury

中图分类号:

R965.2

周杰, 严凌, 黄多新. 线粒体融合素2通过Ras-PI3K-Akt信号途径抑制缺血再灌注诱导的乳鼠心肌细胞凋亡[J]. 中国临床药理学与治疗学, 2014, 19(9): 973-978.

ZHOU Jie, YAN Ling, HUANG Duo-xin. Mfn2 gene transfer inhibites neonatal rat cardiomyocytes apoptosis induced by ischemia/reperfusion via activation of the Ras-PI3K-Akt signaling pathway[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(9): 973-978.

参考文献 26

[1]	Colucci WS.Apoptosis in the heart[J]. N Engl J Med, 1996, 335(16): 1224-1226.
[2]	Nakamura T, Mizuno S, Matsumoto K, et al.Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF[J]. J Clin Invest, 2000, 106(12): 1511-1519.
[3]	Chen KH LN, Zhou AR.The role of hypertension-related gene in aortic vascular smooth muscle cells from mice and rats[J]. Chin Med J (Engl), 2001, 114(8): 833-836.
[4]	Eura Y, Ishihara N, Yokota S, et al.Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion[J]. J Biochem, 2003, 134(3): 333-344.
[5]	Ishihara N, Eura Y, Mihara K.Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity[J]. J Cell Sci, 2004, 117(Pt 26): 6535-6546.
[6]	Tanner FC, Boehm M, Akyurek LM, et al.Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation[J]. Circulation, 2000, 101(17): 2022-2025.
[7]	Harbour JW, Dean DC.Rb function in cell-cycle regulation and apoptosis[J]. Nat Cell Biol, 2000, 2(4): E65-E67.
[8]	Miele A, Braastad CD, Holmes WF, et al.HiNF-P directly links the cyclin E/CDK2/p220NPAT pathway to histone H4 gene regulation at the G1/S phase cell cycle transition[J]. Mol Cell Biol, 2005, 25(14): 6140-6153.
[9]	康品方, 高琴, 王晓梅. 线粒体融合素2在糖尿病大鼠心肌损伤中的变化[J]. 中国临床药理学与治疗学, 2012, 17(4): 398-402.
[10]	Zhang C, Meng X, Zhu Z, et al.Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro[J]. Cell Biol Int, 2004, 28(12): 863-873.
[11]	Koyama T, Temma K, Akera T.Reperfusion-induced contracture develops with a decreasing [Ca2+]i in single heart cells[J]. Am J Physiol, 1991, 261(4 Pt 2): H1115-1122.
[12]	Peyton KJ, Reyna SV, Chapman GB, et al.Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth[J]. Blood, 2002, 99(12): 4443-4448.
[13]	Gottlieb RA, Burleson KO, Kloner RA, et al.Reperfusion injury induces apoptosis in rabbit cardiomyocytes[J]. J Clin Invest, 1994, 94(4): 1621-1628.
[14]	Fliss H, Gattinger D.Apoptosis in ischemic and reperfused rat myocardium[J]. Circ Res, 1996, 79(5): 949-956.
[15]	Bialik S, Geenen DL, Sasson IE, et al.Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53[J]. J Clin Invest, 1997, 100(6): 1363-1372.
[16]	Mattson MP, Kroemer G.Mitochondria in cell death: novel targets for neuroprotection and cardioprotection[J]. Trends Mol Med, 2003, 9(5): 196-205.
[17]	Datta SR, Brunet A, Greenberg ME.Cellular survival: a play in three Akts[J]. Genes Dev, 1999, 13(22): 2905-2927.
[18]	Datta SR, Dudek H, Tao X, et al.Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery[J]. Cell, 1997, 91(2): 231-241.
[19]	Cantley LC.The phosphoinositide 3-kinase pathway[J]. Science, 2002, 296(5573): 1655-1657.
[20]	Nagata S.DNA degradation in development and programmed cell death[J]. Annu Rev Immunol, 2005, 23: 853-875.
[21]	Wajant H.The Fas signaling pathway: more than a paradigm[J]. Science, 2002, 296(5573): 1635-1636.
[22]	Jiang X, Wang X.Cytochrome C-mediated apoptosis[J]. Annu Rev Biochem, 2004, 73: 87-106.
[23]	Ong SB, Subrayan S, Lim SY, et al.Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury[J]. Circulation, 2010, 121(18): 2012-2022.
[24]	Zhou L, Ma W, Yang Z, et al.VEGF165 and angiopoietin-1 decreased myocardium infarct size through phosphatidylinositol-3 kinase and Bcl-2 pathways[J]. Gene Ther, 2005, 12(3): 196-202.
[25]	Renatus M, Stennicke HR, Scott FL, et al.Dimer formation drives the activation of the cell death protease caspase 9[J]. Proc Natl Acad Sci USA, 2001, 98(25): 14250-14255.
[26]	Acehan D, Jiang X, Morgan DG, et al.Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation[J]. Mol Cell, 2002, 9(2): 423-432.