牦牛血低聚肽对缺氧介导的H9c2心肌细胞损伤的保护及其作用机制
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摘要
体外培养H9c2心肌细胞建立缺氧损伤模型,随机分为5组(n=6):正常对照组、缺氧组、牦牛血低聚肽低、中、高剂量组。结果表明,缺氧组的细胞存活率显著降低(P<0.01)、细胞凋亡率显著升高(P<0.01)、LDH释放水平显著升高(P<0.01),线粒体膜电位极显著降低(P<0.01),表明缺氧对H9c2心肌细胞产生了损伤。与缺氧组相比,牦牛血低聚肽干预组的细胞存活率增加,细胞凋亡减少以及细胞中LDH的释放水平下降,并呈良好的剂量依赖关系。同时,牦牛血低聚肽能够抑制线粒体膜电位下降,提高细胞T-AOC水平,降低TNF-α水平,抑制缺氧介导的细胞色素C的释放,上调抗凋亡蛋白(Survivin、p-Akt、Akt)的表达,降低促凋亡蛋白(Caspase-3/9)的活性,且牦牛血低聚肽也影响编码上述蛋白的m RNA水平。以上结果表明;牦牛血低聚肽对H9c2心肌细胞的保护与抑制PI3K-Akt信号转导通路、保护细胞线粒体结构功能完整性有密切关系。
To investigate the protective effect of yak blood oligopeptides on hypoxic injury of H9 c2 cardiomyocytes and its mechanism.The hypoxia injury model was established by culturing H9 c2 cardiomyocytes in vitro,randomly divided into 5 groups(n=6):normal control group,hypoxia group,low-dose yak blood oligopeptide group,middle-dose yak blood oligopeptide group and high-dose yak blood oligopeptide group.The results showed that:the cell survival rate of hypoxia group was significantly lower than that of control group(P<0.01),the rate of apoptosis was significantly increased(P<0.01),the release level of LDH was significantly increased(P<0.01),mitochondrial membrane potential decreased significantly(P<0.01),which showed that hypoxia could cause damage to H9 c2 cardiomyocytes.At the same time,compared with the anoxic group,yak blood oligopeptides could inhibit mitochondrial membrane potential decline,improved the T-AOC level of cells,reduced the level of TNF-α,inhibited the release of cytochrome C,upregulated the expression of anti-apoptotic proteins(Survivin,p-Akt,Akt),decreased the activity of apoptosis protein(Caspase-3/9),and affected the level of mRNA encoding of the above protein.The above results showed that the protective effect of yak blood oligopeptides on H9 c2 cardiomyocytes was closely related to the inhibition of PI3 K-Akt signal transduction pathway and the protection of mitochondrial structural and functional integrity.
To investigate the protective effect of yak blood oligopeptides on hypoxic injury of H9 c2 cardiomyocytes and its mechanism.The hypoxia injury model was established by culturing H9 c2 cardiomyocytes in vitro,randomly divided into 5 groups(n=6):normal control group,hypoxia group,low-dose yak blood oligopeptide group,middle-dose yak blood oligopeptide group and high-dose yak blood oligopeptide group.The results showed that:the cell survival rate of hypoxia group was significantly lower than that of control group(P<0.01),the rate of apoptosis was significantly increased(P<0.01),the release level of LDH was significantly increased(P<0.01),mitochondrial membrane potential decreased significantly(P<0.01),which showed that hypoxia could cause damage to H9 c2 cardiomyocytes.At the same time,compared with the anoxic group,yak blood oligopeptides could inhibit mitochondrial membrane potential decline,improved the T-AOC level of cells,reduced the level of TNF-α,inhibited the release of cytochrome C,upregulated the expression of anti-apoptotic proteins(Survivin,p-Akt,Akt),decreased the activity of apoptosis protein(Caspase-3/9),and affected the level of mRNA encoding of the above protein.The above results showed that the protective effect of yak blood oligopeptides on H9 c2 cardiomyocytes was closely related to the inhibition of PI3 K-Akt signal transduction pathway and the protection of mitochondrial structural and functional integrity.
引文
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[22]ZHANG Xiao-li,YAN Zhen-wen,SHENG Wei-wen,et al.Activation of hypoxia-inducible factor-1ameliorates postischemic renal injury via inducible nitric oxide synthase[J].Molecular&Cellular Biochemistry,2011,358(1/2):287-295.
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[26]郑晓佳,余婷,张华,等.表没食子儿茶素没食子酸酯对心肌细胞缺氧/复氧损伤的抑制作用及其机制[J].中国药理学通报,2017,33(11):1 584-1 588.
[27]庞晓斌,谢欣梅,赵清辉,等.红景天苷对人肝癌HepG2细胞增殖及凋亡的影响[J].中国药学杂志,2014,49(3):195-198.
[28]LIN Yi-huang,FANG Lian-hua,DU Guan-hua,et al.Progress in regulation of RhoA in myocardial ischemia/reperfusion[J].Chin Pharmacol Bull,2015,31(10):1 336-1 339.
[29]ANJANA B,BHARAT B.Aggarwal.Receptor-Mediated Choreography of Life and Death[J].Journal of Clinical Immunology,2003,23(5):317-332.
[30]BARFOLOMEEV E,GONCHAROV T,FEDOROVA AV,et al.c-IAP and c-IAP2are critical mediators of tumor necrosis factorα(TNFα)induced NFκB activation[J].Proc Natl Acad Sci USA,2008,105(34):12 429-12 434.
[31]RAUERT H,WICOVSKY A,MLLER N,et al.Membrane Tumor Necrosis Factor(TNF)Induces p100Processing via TNF Receptor-2(TNFR2)[J].The Journal of Biological Chemistry,2010,285(10):7 394-7 404.
[2]张才骏,马凤莲,李黎,等.互助白牦牛血液蛋白质多态性的研究[J].青海畜牧兽医杂志,1996,26(3):9-11.
[3]魏稚萍.大通家牦牛及含野血牦牛血清运铁蛋白多态性的比较研究[J]..甘肃畜牧兽医,1997,27(5):18-19.
[4]杜昕.菌酶联合制备牦牛血抗氧化肽及其分离纯化的研究[D].雅安:四川农业大学,2016:2-4.
[5]姚星辰,许博林,铁军,等.牦牛活性蛋白对小鼠抗缺氧抗疲劳能力及血糖的影响[J].时珍国医国药,2014,25(11):2 637-2 639.
[6]BOUNDA G A,ZHOU Wang,WANG Dan-dan,et al.Rhein elicits in titro cytotoxicity in primary human liver HL-7702cells by inducing apoptosis through mitochondria-mediated pathway[J].Evidence-Based Complementary and Alternative Medicine,DOI:10.1155/2015/329831.
[7]ZANELLI S A,TRIMMER P A,SOLENSKI N J.Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia[J].Journal of Neurochemistry,2006,97(3):724-736.
[8]HERRMANN J M,RIEMER J.The intermembrane space of mitochondria[J].Antioxidants and Redox Signaling,2010,13(9):1 341-1 358.
[9]JEZEK P,PLECITA-HLAVATA L.Mitochondrial reticulum network dynamics in relation to oxidatve stress,redox regulation,and hypoxia[J].International Journal of Biochemistry and Cell Biology,2009,41:1 790-1 804.
[10]ZWINGMANN C,BUTTERWORTH R.An update on the role of brain glutamine synthesis and its relation to cell-specific energy metabosism in the hyperammonemic brain:Further studies using NMR spectroscopy[J].Neurochemistry International,2005,47(1/2):19-30.
[11]HEISKANEN K M,BHAT M B,WANG Hsing-wen,et al.Mitochondrial depolarization accompanies cytochrome Crelease during apoptosis in PC6 Cells[J].Journal of Biological Chemistry,1999,274(9):5 654-5 658.
[12]ADRAIN C,MARTIN S J.The mitochondrial apoptosome:A killer unleashed by the cytochrome seas[J].Trends in Biochemical Sciences,2001,26(6):390-397.
[13]VARA JF,CASADO E,DE CASTRO J,et al.PI3K/Akt signalling pathway and cancer[J].Cancer Treatment Reviews,2004,30(2):193-204.
[14]LIAO Ping,SUN Gui-bo,ZHANG Chan,et al.Bauhinia championii flavone attenuates hypoxia-reoxygenation induced apoptosis in H9c2cardiomyocytes by improving mitochondrial dysfunction[J].Molecules,2016,21(11):1 469.
[15]COIMBRA-COSTA D,ALVA N,DURAN M,et al.Oxidative stress and apoptosis after acute respiratory hypoxia and reoxygenation in rat brain[J].Redox Biology,2017,12:216-225.
[16]ALTRERI D C.Targeting survivin in cancer[J].Cancer Letters,2013,332(2SI):225-228
[17]CONWAY E M,ZWERTS F,VAN EYGEN V,et al.Survivin-depengdent angiogenesis in is chemicbrain:Molecular mechanisms of hyoxia-induced up-regulation[J].The American Journal of Pathology,2003,163(3):935-946.
[18]HSU S Y,HSUEH A.Tissue-specific Bcl-2 protein partners in apoptosis:An ovarian paradigm[J].Physiological Reviews,2000,80(2):593-614.
[19]DANIAL N N.BAD:undertaker by night,candyman by day[J].Oncogene,2008,27:S53-S70.
[20]PARIKH N,KOSHY C,DHAYABARAN V,et al.The N-terminus and alpha-5,alpha-6helices of the pro-apoptotic protein Bax,modulate functional interactions with the antiapoptotic protein Bcl-XL[J].BMC Cell Biolgy,2007,8(16):1-16.
[21]KE Qing-dong,COSTA M.Hypoxia-inducible factor-1(HIF-1)[J].Molecular pharmacology,2006,70(5):1 469-1 480.
[22]ZHANG Xiao-li,YAN Zhen-wen,SHENG Wei-wen,et al.Activation of hypoxia-inducible factor-1ameliorates postischemic renal injury via inducible nitric oxide synthase[J].Molecular&Cellular Biochemistry,2011,358(1/2):287-295.
[23]SEMENZA G L.Oxygen-regulated transcription factors and their role in pulmonary disease[J].Respiratory Research,2000,1(3):159-162.
[24]SEMENZA G L.Hypoxia-inducible factor 1:Oxygen homeostasis and disease pathophysiology[J].Trends in Molecular Medicine,2001,7(8):345-350.
[25]楚秉泉.西藏芜菁的抗缺氧功能成分分离及其作用机制研究[D].杭州:浙江大学,2017:65-67.
[26]郑晓佳,余婷,张华,等.表没食子儿茶素没食子酸酯对心肌细胞缺氧/复氧损伤的抑制作用及其机制[J].中国药理学通报,2017,33(11):1 584-1 588.
[27]庞晓斌,谢欣梅,赵清辉,等.红景天苷对人肝癌HepG2细胞增殖及凋亡的影响[J].中国药学杂志,2014,49(3):195-198.
[28]LIN Yi-huang,FANG Lian-hua,DU Guan-hua,et al.Progress in regulation of RhoA in myocardial ischemia/reperfusion[J].Chin Pharmacol Bull,2015,31(10):1 336-1 339.
[29]ANJANA B,BHARAT B.Aggarwal.Receptor-Mediated Choreography of Life and Death[J].Journal of Clinical Immunology,2003,23(5):317-332.
[30]BARFOLOMEEV E,GONCHAROV T,FEDOROVA AV,et al.c-IAP and c-IAP2are critical mediators of tumor necrosis factorα(TNFα)induced NFκB activation[J].Proc Natl Acad Sci USA,2008,105(34):12 429-12 434.
[31]RAUERT H,WICOVSKY A,MLLER N,et al.Membrane Tumor Necrosis Factor(TNF)Induces p100Processing via TNF Receptor-2(TNFR2)[J].The Journal of Biological Chemistry,2010,285(10):7 394-7 404.