转化生长因子-β1预处理调控缺氧诱导因子-1α蛋白表达水平对心肌细胞缺氧/复氧损伤的影响
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摘要
目的探讨转化生长因子-β1(TGF-β1)预处理调控缺氧诱导因子-1α(HIF-1α)的蛋白表达水平是否可以影响心肌细胞缺氧/复氧损伤。方法建立心肌细胞株H9C2缺氧/复氧损伤模型及缺氧预处理模型,将H9C2心肌细胞随机分为5组:正常对照组(Control组)、缺氧复氧组(H/R组)、缺氧预处理组(HPC组),TGF-β1预处理组(TGF-β1-Pre组)、TGF-β1抑制剂组(SB431542组),各组复氧结束后测定培养液乳酸脱氢酶(LDH)活性、HIF-1α蛋白表达水平、心肌细胞凋亡率。结果Control组、H/R组、HPC组、TGF-β1-Pre组、SB431542组的LDH活性(U/L)分别为(201.2±48.9)、(936.6±93.7)、(727.0±75.9)、(747.3±63.1)、(957.3±55.3);心肌细胞凋亡率(%)分别为(25.5±3.2)、(71.8±5.5)、(42.8±6.1)、(54.1±4.1)、(82.8±9.7),与H/R组比较,HPC组、TGF-β1-Pre组的LDH活性、心肌细胞凋亡率显著降低(P<0.05),HIF-1α蛋白表达水平升高(P<0.05);与HPC组比较,TGF-β1-Pre组各项指标差异均无统计学意义(P>0.05)。结论不论在常氧条件还是在缺氧环境下,TGF-β1均可以增加HIF-1α的蛋白表达水平,并且在缺氧复氧诱导所致的心肌细胞损伤中发挥抗凋亡作用,这种作用与调控HIF-1α的蛋白水平有关。
Objective To investigate whether the HIF-1α protein level regulated by TGF-β1 preconditioning can affect the cardiomyocytes hypoxia/reoxgenation-induced injury of cardiomyocytes. Methods H9C2 cardiomyocytes were used to establish hypoxia/reoxgenation-induced injury model. Cardiomyocytes were randomly divided into five groups: control group( Control),hypoxia/reoxgenation group( H/R),hypoxic preconditioning group( HPC),TGF-β1 preconditioning group( TGF-β1-Pre),TGF-β1 inhibitor group( SB431542). Lactic dehydrogenase( LDH) activity,HIF-1α protein content and cardiomyocytes apoptosis rate were measured at the end of the reperfusion. Results The LDH activity of Control group,H/R group,HPC group,TGF-β1-Pre group,SB431542 group were( 201.2±48.9) U/L,( 936.6±93.7) U/L,( 727.0±75.9) U/L,( 747.3± 63.1) U/L,( 957.3± 55.3) U/L,respectively.The cell apoptosis rate of Control group,H/R group,HPC group,TGF-β1-Pre group,SB431542 group were( 25.5±3.2) %,( 71.8±5.5) %,( 42.8±6.1) %,( 54.1±4.1) %,( 82.8± 9.7) %,respectively. The LDH activity and cell apoptosis rate in HPC group and TGF-β1-Pre group were both decreased compared with that in H/R group,meanwhile accompanied by increased HIF-1α protein content( P <0.05). There was no significantly difference between HPC group and TGF-β1-Pre group. Conclusion TGF-β1 could increase the expression level of HIF-1α protein no matter in normoxia or hypoxia circumstance,TGF-β1 exert antiapoptotic effect in hypoxia/reoxgenation-induced injury of cardiomyocytes,which is associated with the modulation of HIF-1α.
Objective To investigate whether the HIF-1α protein level regulated by TGF-β1 preconditioning can affect the cardiomyocytes hypoxia/reoxgenation-induced injury of cardiomyocytes. Methods H9C2 cardiomyocytes were used to establish hypoxia/reoxgenation-induced injury model. Cardiomyocytes were randomly divided into five groups: control group( Control),hypoxia/reoxgenation group( H/R),hypoxic preconditioning group( HPC),TGF-β1 preconditioning group( TGF-β1-Pre),TGF-β1 inhibitor group( SB431542). Lactic dehydrogenase( LDH) activity,HIF-1α protein content and cardiomyocytes apoptosis rate were measured at the end of the reperfusion. Results The LDH activity of Control group,H/R group,HPC group,TGF-β1-Pre group,SB431542 group were( 201.2±48.9) U/L,( 936.6±93.7) U/L,( 727.0±75.9) U/L,( 747.3± 63.1) U/L,( 957.3± 55.3) U/L,respectively.The cell apoptosis rate of Control group,H/R group,HPC group,TGF-β1-Pre group,SB431542 group were( 25.5±3.2) %,( 71.8±5.5) %,( 42.8±6.1) %,( 54.1±4.1) %,( 82.8± 9.7) %,respectively. The LDH activity and cell apoptosis rate in HPC group and TGF-β1-Pre group were both decreased compared with that in H/R group,meanwhile accompanied by increased HIF-1α protein content( P <0.05). There was no significantly difference between HPC group and TGF-β1-Pre group. Conclusion TGF-β1 could increase the expression level of HIF-1α protein no matter in normoxia or hypoxia circumstance,TGF-β1 exert antiapoptotic effect in hypoxia/reoxgenation-induced injury of cardiomyocytes,which is associated with the modulation of HIF-1α.
引文
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[13]Loboda A,Jozkowicz A,Dulak J.HIF-1 and HIF-2 transcription factors similar but not identical[J].Mol Cells,2010,29(5):435-442.
[14]Semenza GL.Oxygen sensing,hypoxia-inducible factors,and disease pathophysiology[J].Annu Rev Pathol,2014,9(1):47-71.
[15]Tirado-Rodriguez B,Ortega E,Segura-Medina P,et al.TGF-β:an important mediator of allergic disease and a molecule with dual activity in cancer development[J].J Immunol Res,2014,2014(5):318481.
[16]Dandapat A,Hu CP,Li D,et al.Overexpression of TGFbeta l by adeno-associated virus type-2 vector protects myocardium fromischemia-reperfusion injury[J].Gene therapy,2008,15(6):415-423.
[17]Frantz S,Hu K,Adamek A,et al.Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction[J].Basic Res Cardiol,2008,103(5):485-492.
[18]Al-Azayzih A,Gao F,Goc A,et al.TGFβ1 induces apoptosis in invasive prostate cancer and bladder cancer cells via AKT-independent,p38 MAPK and JNK/SAPK-mediated activation of caspases[J].Biochem Biophys Res Commun,2012,427(1):165-170.
[19]Wang F,Chen L,Ni H,et al.APRIL depletion induces cell cycle arrest and apoptosis through blocking TGF-βl/ERK signaling pathway in human colorectal cancer cells[J].Mol Cell Biochem,2013,383(1-2):179-189.
[20]Vivar R,Humeres C,Ayala P,et al.TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways[J].Biochim Biophys Acta,2013,1832(6):754-762.
[2]Wright DE,Hunt DP.Perioperative surveillance for adverse myocardial events[J].South Med J,2008,101(1):52-58.
[3]Murry CE,Jennings RB,Reimer KA.Preconditioning with ischemia:a delay of lethal cell injury in ischemic myocardium[J].Circulation,1986,74(5):1124-1136.
[4]Ke Q,Costa M.Hypoxia-inducible factor-1(HIF-1)[J].Mol Pharmacol,2006,70(5):1469-1480.
[5]Sen Banerjee S,Thirunavukkarasu M,Tipu Rishi M,et al.HIF-prolyl hydroxylases and cardiovascular diseases[J].Toxicol Mech Methods,2012,22(5):347-358.
[6]卢向航,冉珂,徐军美,等.延迟相吗啡预处理对兔心肌缺血再灌注损伤的保护作用[J].中国体外循环杂志,2008,6(3):180-182.
[7]Mcmahon S,Charbonneau M,Grandmont S,et al.Transforming growth factor beta1 induces hypoxia-inducible factor-1 stabilization through selective inhibition of PHD2 expression[J].J Biol Chem,2006,281(34):24171-24181.
[8]Watanabe T,Yasue A,Tanaka E.Inhibition of transforming growth factorβ1/Smad3 signaling decreases hypoxia-inducible factor-1αprotein stability by inducing prolyl hydroxylase 2 expression in human periodontal ligament cells[J].J Periodontol,2013,84(9):1346-1352.
[9]Liu L,Wang P,Liu X,et al.Exogenous NAD(+)supplementation protects H9C2 cardiac myoblasts against hypoxia/reoxygenation injury via Sirt1-p53 pathway[J].Fundam Clin Pharmacol,2014,28(2):180-189.
[10]Pan YX,Ren AJ,Zheng J,et al.Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes:role of GRP78[J].Life Sci,2007,81(13):1042-1049.
[11]Eckle T,Kohler D,Lehmann R,et al.Hypoxia-inducible factor-1 is central to cardioprotection:a new paradigm for ischemic preconditioning[J].Circulation,2008,118(2):166-175.
[12]Semenza GL,Shimoda LA,Prabhakar NR.Regulation of gene expression by HIF-1[J].Novartis Found Symp,2006,272(10):2-8.
[13]Loboda A,Jozkowicz A,Dulak J.HIF-1 and HIF-2 transcription factors similar but not identical[J].Mol Cells,2010,29(5):435-442.
[14]Semenza GL.Oxygen sensing,hypoxia-inducible factors,and disease pathophysiology[J].Annu Rev Pathol,2014,9(1):47-71.
[15]Tirado-Rodriguez B,Ortega E,Segura-Medina P,et al.TGF-β:an important mediator of allergic disease and a molecule with dual activity in cancer development[J].J Immunol Res,2014,2014(5):318481.
[16]Dandapat A,Hu CP,Li D,et al.Overexpression of TGFbeta l by adeno-associated virus type-2 vector protects myocardium fromischemia-reperfusion injury[J].Gene therapy,2008,15(6):415-423.
[17]Frantz S,Hu K,Adamek A,et al.Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction[J].Basic Res Cardiol,2008,103(5):485-492.
[18]Al-Azayzih A,Gao F,Goc A,et al.TGFβ1 induces apoptosis in invasive prostate cancer and bladder cancer cells via AKT-independent,p38 MAPK and JNK/SAPK-mediated activation of caspases[J].Biochem Biophys Res Commun,2012,427(1):165-170.
[19]Wang F,Chen L,Ni H,et al.APRIL depletion induces cell cycle arrest and apoptosis through blocking TGF-βl/ERK signaling pathway in human colorectal cancer cells[J].Mol Cell Biochem,2013,383(1-2):179-189.
[20]Vivar R,Humeres C,Ayala P,et al.TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways[J].Biochim Biophys Acta,2013,1832(6):754-762.