PPARγ过表达与激活对心肌细胞凋亡的影响

为了探讨心肌细胞受损及凋亡与过氧化物增殖受体(PPARγ)的关系。本研究采用Western blotting检测高脂饮食小鼠心肌组织中PPARγ2蛋白的表达。再购买质粒PCMV6-PPARγ2并进行扩增及空白载体的构建。在小鼠心肌细胞株中转染目标质粒后,加入10μmol/L PPARγ激动剂罗格列酮、抑制剂GW9662化合物处理24 h后,通过tunnel检测和细胞流式计算检测细胞凋亡的情况。结果显示:与低脂对照组比较,高脂饮食组心肌组织内PPARγ2蛋白的表达显著增高;利用XhoⅠ和SmaⅠ酶对PCMV6-PPARγ2质粒酶切,T4连接酶连接从而获取PCMV6空白质粒;转染质粒过表达PPARγ2,tunnel检测结果和流式技术检测显示细胞凋亡增加,而加入PPARγ抑制剂凋亡得到抑制。由此得出结论,PPARγ2的过表达及激活对心肌细胞凋亡的增加。     

凋亡诱导因子介导缺氧/复氧致肥大心肌细胞凋亡的作用

心肌细胞凋亡导致心肌组织合胞体功能丧失,最终使代偿性心肌肥大向心力衰竭转化。过去的研究已经确认天门冬氨酸特异性半胱氨酸蛋白酶(caspartate-specificcysteinylproteinase,caspase)依赖机制在心肌细胞凋亡中的作用,但对caspase非依赖机制即凋亡诱导因子(apoptosis-inducingfactor,AIF)在心肌细胞凋亡中的作用尚不明确。本研究应用血管紧张素Ⅱ(0.1μmol/L培养12h)诱导培养的小鼠肥大心肌细胞,利用三气孵箱建立缺氧/复氧模型以模拟缺血再灌注。应用RT-PCR、Westernblot、siRNA基因转染、Hoechst33258染色法检测AIF在mRNA和蛋白质水平的表达及细胞凋亡的变化,分析AIF在缺氧/复氧致肥大心肌细胞凋亡中的意义。结果如下:(1)与对照组比较,缺氧8h组(H8h)和缺氧12h组(H12h)AIFmRNA及蛋白表达水平均显著升高(mRNA:0.52±0.04及0.85±0.10vs0.29±0.08,P<0.05;蛋白质:2.07±0.15和3.12±0.19vs0.29±0.04,P<0.05),即随缺血时间的延长,AIFmRNA及蛋白表达水平均显著增加。(2)与对应单纯缺氧组比较,缺氧后给予复氧刺激,H8h/R组和H12h/R组AIFmRNA及蛋白表达水平均显著升高(mRNA:1.09±0.12和1.41±0.23,P<0.05;蛋白质:4.57±0.25和5.71±0.27,P<0.05)。仅在H8h/R及H12h/R组,可见AIF核转位显著增加。(3)AIFsiRNA转染可显著抑制肥大心肌细胞AIF的表达,对缺氧时细胞凋亡无明显影响(P>0.05),但可显著降低缺氧/复氧诱导的肥大心肌细胞凋亡率(P<0.05)。同时抑制AIF及caspase-3活性,可显著加强单一抑制剂对缺氧/复氧诱导的肥大心肌细胞凋亡的抑制作用。(4)抑制caspase-3活性对缺氧/复氧诱导的AIF核转位无明显影响。上述结果提示,缺氧/复氧时AIFmRNA、蛋白表达和核转位均显著增加,且在缺氧/复氧诱导肥大心肌细胞凋亡中具有重要的作用。     

microRNA-21在大鼠心肌缺氧复氧损伤中的作用及其对细胞自噬的影响

本文应用大鼠心肌细胞缺氧/复氧损伤模型,探讨microRNA-21(miR-21)在大鼠心肌缺氧复氧损伤中的作用及其对细胞自噬的影响.缺氧复氧后,RT-PCR检测发现心肌miR-21表达上调(P0.05),流式细胞术检测表明细胞凋亡增加,RT-PCR及蛋白质印迹(Western blot)检测发现p62显著下调而beclin-1显著上调(P0.05),提示缺氧复氧诱导心肌细胞凋亡和自噬异常.脂质体转染miR-21 mimic后,细胞凋亡显著增加(P0.05),p62显著上调而beclin-1显著下调(P0.05),而转染miR-21抑制剂引起相反结果,提示miR-21在心肌缺氧复氧损伤中具有促进细胞凋亡、抑制细胞自噬的作用.生物信息学预测显示,Rab11a的3′-UTR含有miR-21的结合位点,双荧光素酶基因报告系统及Rab11a过表达实验表明Rab11a是miR-21的靶基因之一.心肌过表达Rab11a能减少缺氧复氧后miR-21介导的细胞凋亡及自噬.由此表明,在大鼠心肌缺氧复氧损伤中,miR-21可能通过负调控Rab11a促进心肌细胞凋亡,抑制心肌细胞自噬.本研究可能为预防和治疗心肌缺血再灌注损伤提供新策略.     

虎杖苷对急性心肌梗死所致心脏损伤的保护作用

为了考察虎杖苷对急性心肌梗死所致心脏损伤的保护作用,本研究对H9c2大鼠心肌细胞进行缺氧诱导来模拟急性心肌梗死中心肌细胞的变化。然后用200μmol/L的虎杖苷处理心肌细胞12 h。考察虎杖苷对心肌细胞活力、细胞凋亡及相关蛋白(caspase-3, Bcl-2)和ROS生成的应用,并用小干扰RNA敲低Nrf2,考察敲低Nrf2对心肌细胞的影响。研究显示,缺氧处理可显著降低心肌细胞活力并增加细胞凋亡率,而虎杖苷可抑制缺氧诱导的细胞活力降低和细胞凋亡。虎杖苷可显著抑制缺氧诱导的caspase-3的下调并抑制缺氧诱导的Bcl-2的上调。虎杖苷可显著抑制缺氧诱导的Nrf2和HO-1的下调。敲低Nrf2可降低H9c2心肌细胞活力并增加细胞凋亡率。敲低Nrf2可上调caspase-3表达,并下调Bcl-2和Nrf2/HO-1信号通路的表达。缺氧可诱导H9c2细胞中ROS的产量升高,虎杖苷可抑制ROS的生成。然而,敲低Nrf2可导致细胞中ROS产量再次升高。虎杖苷具有抑制缺氧诱导的心肌细胞凋亡的作用,并且虎杖苷可通过抗氧化作用来减轻急性心肌梗死所致的心脏损伤。虎杖苷的抗氧化和心肌保护作用部分依赖于Nrf2/HO-1信号通路。     

玉郎伞查尔酮激活Nrf2/ARE信号通路减轻缺氧/复氧所致的H9c2细胞凋亡及氧化应激损伤

研究玉郎伞查尔酮(YLSC)对缺氧/复氧所致的H9c2细胞损伤的影响及可能的作用机制。缺氧12 h、复氧24 h建立心肌细胞缺氧/复氧模型,并对细胞凋亡、氧化应激相关指标和Nuclear-Nrf2等蛋白表达进行了检测。结果显示,YLSC可明显增加细胞生存率,提高SOD、GSH-Px水平,降低细胞凋亡率和LDH、ROS、MDA水平。使Nuclear-Nrf2、HO-1、Bcl-2蛋白表达增高而Cleaved caspase 3、Bax蛋白表达减少,联合应用Nrf2抑制剂可抑制YLSC作用效果。结果表明,YLSC可以减轻缺氧/复氧所致的心肌细胞凋亡及氧化应激损伤,其机制可能与激活Nrf2/ARE信号通路有关。     

黄芪甲苷后处理对乳鼠心肌细胞缺氧复氧损伤的作用研究

目的:观察黄芪甲苷(AstragalosideⅣ,AsⅣ)后处理对缺氧复氧损伤(simulated ischemia reperfusion injury,SI/RI)的SD乳鼠心肌细胞是否具有保护作用。方法:将乳鼠原代心肌细胞平均分为五组,即空白对照组(Control)、缺氧复氧处理组(SI/RI)、黄芪甲苷预处理(5,10、20μM)+SI/RI组(AsIV+SI/RI)。各组细胞经处理后,四氮唑溴盐比色法(MTT)检测各组细胞存活率;TUNEL染色法测定各组细胞凋亡率;SOD测试盒检测培养液中超氧化物歧化酶(SOD)含量,总嘌呤氧化酶(XOD)测试盒检测丙二醛(MDA)含量。Western blot法检测各组细胞抗凋亡蛋白Bcl-2和促凋亡蛋白Caspase-3的表达。结果:与空白组相比,缺氧复氧损伤组细胞活力显著下降(P0.05),凋亡率显著上升(P0.05),其培养液中SOD水平显著降低(P0.05),MDA水平显著升高。而不同浓度AsⅣ后处理组的心肌细胞存活率显著上升,凋亡率显著下降,培养液中SOD水平显著上升,MDA水平显著下降(P0.05),且呈浓度呈依赖性。Western blot结果显示AsⅣ后处理组细胞中的Bcl-2表达明显上升,Caspase-3明显下降。结论:黄芪甲苷后处理对缺氧复氧诱导的乳鼠心肌细胞损伤具有显著的保护作用,能够显著上调抗凋亡蛋白Bcl-2的表达,下调促凋亡蛋白Caspase-3的表达。     

银杏叶提取物对新生SD 乳鼠心肌细胞缺氧损伤的影响

目的:研究银杏叶提取物对缺氧状态下新生SD乳鼠心肌细胞的影响及其可能机制。方法:新生1天SD乳鼠心肌细胞原代培养并利用氮气培养箱模拟低氧构建乳鼠心肌缺氧体外模型。分为3组处理:对照组,缺氧组,缺氧+药物拮抗组。缺氧时间为12 h,通过免疫组化等检测方法,观察各组心肌细胞的损伤情况及心肌Bcl-2、Bax蛋白表达情况。结果:缺氧可以造成新生SD乳鼠心肌细胞凋亡的发生(hypoxia:75.21%±1.21%,control:1.38%±0.45%,P<0.05,n=20),并导致其表达凋亡抑制因子Bcl-2蛋白水平的显著降低(0.125 fold VS control group,P<0.05),促细胞凋亡因子Bax蛋白水平显著升高(3.011fold VS control group,P<0.05);而银杏叶提取物作用后可明显逆转新生SD乳鼠心肌细胞凋亡的发生(EGb761:23.17%±0.43%,hypoxia:73.13%±1.22%,P<0.05,n=20),并明显逆转Bcl-2(5.716 fold VS hypoxia group,P<0.05)、Bax(0.273fold VS hypoxia group,P<0.05)等蛋白的表达水平。结论:凋亡相关因子Bcl-2和Bax等参与缺氧致心肌损伤过程,导致心肌细胞凋亡,银杏叶提取物能降低心肌Bax表达,提高Bcl-2表达,从而保护心肌细胞,抑制凋亡。     

丹参酮ⅡA调节microRNA-1保护缺氧心肌细胞的作用研究

目的:研究丹参酮Ⅱ A(TanshinoneⅡA)通过调节microRNA-1抗心肌细胞缺氧损伤的作用。方法:原代培养新生大鼠心肌细胞,建立心肌细胞缺氧模型。MTT法检测心肌细胞存活率(%);TUNEL、流式细胞术测心肌细胞凋亡率;激光共聚焦检测心肌细胞内钙离子[Ca2+]i浓度的变化情况。结果:MTT结果显示丹参酮ⅡA对缺氧心肌细胞及过表达miR-1引起心肌细胞损伤具有保护作用。丹参酮ⅡA增加了缺氧心肌细胞的存活率(P0.05),同时给予丹参酮ⅡA和miR-1组与单独miR-1损伤组相比较,存活率也明显升高,呈现剂量依赖性。TUNEL结果显示丹参酮ⅡA可以抑制缺氧诱导的细胞凋亡,丹参酮ⅡA可以明显降低由缺氧导致的细胞凋亡率(P0.05)。共聚焦检测结果显示,缺氧损伤的心肌细胞内[Ca2+]i显著升高1322.72±5.16(vs正常对照组,P0.05),丹参酮ⅡA则有效抑制由缺氧引起过高的[Ca2+]i。miR-1诱导的细胞内[Ca2+]i升高至1349.33±62.63,约为正常对照组的1.96倍,而丹参酮ⅡA则有效抑制胞内过高的[Ca2+]i,从而发挥心肌保护作用。结论:丹参酮ⅡA可能是通过抑制胞内miR-1的表达,参与对钙离子浓度的调控,发挥其对心肌细胞的保护作用。     

脂联素对乳鼠心肌细胞缺氧/复氧损伤的保护作用

本研究通过在大鼠乳鼠心室肌细胞上建立缺氧/复氧(hypoxia/reoxygenation,H/R)模型,模拟在体心肌缺血/再灌注损伤,观察脂联素(adiponectin,APN)对心肌细胞H/R损伤的影响,并探讨其作用机制。采用胰蛋白酶消化法原代培养乳鼠心室肌细胞,α-肌动蛋白免疫荧光法进行鉴定。选用培养72h的单层心肌细胞进行实验,随机分为5组:对照组、单纯H/R组、H/R+APN组、H/R+APN+腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)特异性抑制剂阿糖胞苷(AraA)组、H/R+AraA组。观察各组心肌细胞形态及自发搏动频率,用琼脂糖凝胶电泳和流式细胞术检测各组心肌细胞凋亡情况,并测定细胞丙二醛(MDA)含量及培养液中超氧化物歧化酶(SOD)活性,激光共聚焦显微镜观察心肌细胞内钙荧光强度,Western blot检测各组心肌细胞AMPK磷酸化水平。结果显示,与对照组相比,单纯H/R组细胞生长状态较差,搏动频率减慢甚至消失,DNA电泳呈凋亡特征性的梯状条带,细胞凋亡率显著增加,胞浆MDA水平增高,上清液中SOD活性下降,胞内钙荧光强度明显增高,AMPK磷酸化水平升高(P0.05)。与H/R组细胞相比,APN预处理后再进行H/R的心肌细胞搏动频率较快,凋亡率明显减少,MDA水平明显下降,SOD活性明显升高,心肌细胞AMPK磷酸化水平明显增高(P0.05)。AraA可以阻断APN的上述保护作用。以上结果表明,APN可减轻H/R导致的心肌细胞凋亡,减轻脂质过氧化及细胞内钙超载,这一保护作用可能与AMPK途径激活有关。     

番茄红素对大鼠乳鼠心肌细胞缺氧复氧的保护作用以及其机制

目的：探讨番茄红素对心肌细胞缺氧复氧的保护作用以及其分子机制。方法：采用原代培养心肌细胞建立缺氧/复氧损伤模型,实验分8组：正常对照组,H/R组,H/R＋番茄红素（1,2,4,8,16,32μmol/L）剂量组。观察各组细胞经H/R损伤后,细胞内天冬氨酸氨基转移酶（AST）、肌酸激酶（CK）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量的变化情况,选择正常对照组,H/R组,最佳番茄红素剂量组做MTT分析细胞凋亡,Western检测TRL 4以及NF-κB的表达。结果：番茄红素（16,8,4,2μmol/L）剂量组可显著降低缺氧/复氧损伤心肌细胞内AST、CK、LDH释放量及MDA的生成,并能提高SOD活性。此外番茄红素可减少心肌细胞缺氧/复氧损伤后的心肌凋亡,减少TRL 4受体以及NF-κB的表达。结论：番茄红素具有抗缺氧/复氧损伤,保护心肌细胞的作用,其机制可能是通过抑制TRL 4通路来实现的。     

AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress

Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.     

Activation of the prolyl-hydroxylase oxygen-sensing signal cascade leads to AMPK activation in cardiomyocytes

The proline hydroxylase domain-containing enzymes (PHD) act as cellular oxygen sensors and initiate a hypoxic signal cascade to induce a range of cellular responses to hypoxia especially in the aspect of energy and metabolic homeostasis regulation. AMP-activated protein kinase (AMPK) is recognized as a major energetic sensor and regulator of cardiac metabolism. However, the effect of PHD signal on AMPK has never been studied before. A PHD inhibitor (PHI), dimethyloxalylglycine and PHD2-specific RNA interference (RNAi) have been used to activate PHD signalling in neonatal rat cardiomyocytes. Both PHI and PHD2-RNAi activated AMPK pathway in cardiomyocytes effectively. In addition, the increased glucose uptake during normoxia and enhanced myocyte viability during hypoxia induced by PHI pretreatment were abrogated substantially upon AMPK inhibition with an adenoviral vector expressing a dominant negative mutant of AMPK-α1. Furthermore, chelation of intracellular Ca2+ by BAPTA, inhibition of calmodulin-dependent kinase kinase (CaMKK) with STO-609, or RNAi-mediated down-regulation of CaMKK α inhibited PHI-induced AMPK activation significantly. In contrast, down-regulation of LKB1 with adenoviruses expressing the dominant negative form did not affect PHI-induced AMPK activation. We establish for the first time that activation of PHD signal cascade can activate AMPK pathway mainly through a Ca(2+)/CaMKK-dependent mechanism in cardiomyocytes. Furthermore, activation of AMPK plays an essential role in hypoxic protective responses induced by PHI.     

大鼠心肌细胞凋亡的保护作用

为探讨p53上调凋亡调制物(p53 up-regulated modulator of apoptosis, PUMA)在大鼠心肌细胞缺氧/复氧（hypoxia/reoxygenatio, H/R）损伤中的作用,本 研究将靶向PUMA的siRNA（si-PUMA）转染大鼠心肌细胞以建立PUMA沉默表达模型,观察其对心肌细胞H/R损伤的影响.RT-PCR和Western印迹结果表明,最适转染浓度50 nmol/L si-PUMA能靶向抑制H/R损伤心肌细胞的PUMA表达;MTT法检测心肌细胞存活率及培养基乳酸脱氢酶（lactate dehydrogenase, LDH）活性测定结果发现,si-PUMA 组细胞存活率较H/R 6 h模型组明显提高,培养液中LDH活性显著降低（P<0.01）;分光光度法及Annexin V-FITC/PI联合染色流式细胞凋亡检测结果显示,si-PUMA组caspase-3活性较H/R 6h组明显下调,细胞凋亡率明显降低（P <0.01）;RT-PCR结果 提示,与H/R 6 h组相比,si-PUMA组Bax及Bcl-2表达分别出现显著下调及上调（P <0.05）.以上结果表明,靶向PUMA的siRNA转染能明显增强心肌细胞耐受H/R损伤的能力,对心肌细胞具有较好的保护作用;PUMA介导H/R诱导的心肌细胞凋亡,是心肌缺血/再灌注损伤基因治疗的一个潜在靶点.     

线粒体ATP敏感钾通道对大鼠肺动脉平滑肌细胞低氧诱导因子-1α表达及细胞增殖的作用

本文旨在探讨线粒体ATP敏感钾（mitochondrial ATP-sensitive K＋,MitoKATP）通道对大鼠肺动脉平滑肌细胞低氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）表达和细胞增殖的影响。原代培养大鼠肺动脉平滑肌细胞,分为常氧对照组、常氧＋diazoxide（MitoKATP通道的选择性开放剂）组、常氧＋5-hydroxydecanoate（5-HD,MitoKATP通道的选择性阻断剂）组、低氧对照组、低氧＋diazoxide组、低氧＋5-HD组,共6组,分别应用罗丹明123荧光技术检测各组大鼠肺动脉平滑肌细胞的线粒体膜电位,免疫组化检测HIF-1α的表达及酶联免疫检测仪检测细胞增殖的变化。结果显示,常氧＋ diazoxide组与常氧对照组比较,罗丹明123荧光、HIF-1α表达及细胞增殖明显增强（P〈0．05）;低氧＋diazoxide组与低氧对照组比较,罗丹明123荧光、HIF-1α表达及细胞增殖明显增强（P〈0.05）：常氧＋5-HD组与常氧对照组比较,罗丹明123荧光、HIF-1α表达、细胞增殖没有明显变化（P〉0．05）;但低氧＋5-HD组与低氧对照组比较,罗丹明123荧光明显减弱、HIF-1α表达及细胞增殖有所减弱（P〈0．05）。结果提示：MitoKATP通道的开放能引起大鼠肺动脉平滑肌细胞线粒体膜去极化,并可以促进HIF-1α的表达及细胞增殖。     

Role of adiponectin receptors in endothelin-induced cellular hypertrophy in cultured cardiomyocytes and their expression in infarcted heart

Adiponectin, an adipocyte-derived protein, has cardioprotective actions. We elucidated the role of the adiponectin receptors AdipoR1 and AdipoR2 in the effects of adiponectin on endothelin-1 (ET-1)-induced hypertrophy in cultured cardiomyocytes, and we examined the expression of adiponectin receptors in normal and infarcted mouse hearts. Recombinant full-length adiponectin suppressed the ET-1-induced increase in cell surface area and [(3)H]leucine incorporation into cultured cardiomyocytes compared with cells treated with ET-1 alone. Transfection of small interfering RNA (siRNA) specific for AdipoR1 or AdipoR2 reversed the suppressive effects of adiponectin on ET-1-induced cellular hypertrophy in cultured cardiomyocytes. Adiponectin induced phosphorylation of AMP-activated protein kinase (AMPK) and inhibited ET-1-induced ERK1/2 phosphorylation, which were also reversible by transfection of siRNA for AdipoR1 or AdipoR2 in cultured cardiomyocytes. Transfection of siRNA for alpha(2)-catalytic subunits of AMPK reduced the inhibitory effects of adiponectin on ET-1-induced cellular hypertrophy and ERK1/2 phosphorylation. Effects of globular adiponectin were similar to those of full-length adiponectin, and siRNA for AdipoR1 reversed the actions of globular adiponectin. Compared with normal left ventricle, expression levels of AdipoR1 mRNA and protein were decreased in the remote, as well as the infarcted, area after myocardial infarction in mouse hearts. In conclusion, AdipoR1 and AdipoR2 mediate the suppressive effects of full-length and globular adiponectin on ET-1-induced hypertrophy in cultured cardiomyocytes, and AMPK is involved in signal transduction through these receptors. AdipoR1 and AdipoR2 might play a role in the pathogenesis of ET-1-related cardiomyocyte hypertrophy after myocardial infarction.     

Recombinant PTD-Cu/Zn SOD attenuates hypoxia–reoxygenation injury in cardiomyocytes

Abstract

Background. Oxidative stress plays a pivotal role in myocardial ischemia–reperfusion injury. Increasing the protein expression of intracellular Cu/Zn SOD, which is the major endogenous antioxidant enzyme, may attenuate or prevent hypoxia–reoxygenation injury (HRI) in cultured cardiomyocytes. However, ectogenic Cu/Zn-SOD can hardly be transferred into cells to exert biological effects. In this study, we constructed PTD-Cu/Zn SOD plasmid with a kind of translocation structure-Protein transduction domain (PTD) and detected its transmembrane ability and antioxidant effects in H9c2 rat cardiomyocytes subjected to hypoxia/reoxygenation injury (HRI). Methods. We constructed the pET-PTD-Cu/Zn SOD (CDs) prokaryotic expression vectors in plasmid that were inserted into E. coli BL21 to induce the protein expression of PTD-Cu/Zn SOD. H9c2 cardiomyocyte HRI was achieved by exposing cardiomyocytes to 12 h hypoxia followed by 2 h reoxygenation. Protein expression of PTD-Cu/Zn SOD in cardiomyocytes was assayed by Western blot and their enzyme activities were investigated by immunohistochemistry and flow cytometry. Results. In cultured cardiomyocytes hypoxia–reoxygenation injury model, exogenous PTD-Cu/Zn SOD could penetrate cell membrane to clear superoxide anion and decrease hydrogen peroxide level in H9c2 cardiomyocytes subjected to HRI. The level of mitochondrial membrane potential was restored to normal, and the cell apoptosis was reduced in cardiomyocytes with PTD-Cu/Zn SOD treatment during HRI. Conclusion. Recombinant PTD-Cu/Zn SOD could scavenge intracellular-free superoxide anion, protect mitochondria from damages, and attenuate the hypoxia–reoxygenation injury in cultured cardiomyocytes.     

Small interference RNA against PTP-1B reduces hypoxia/reoxygenation induced apoptosis of rat cardiomyocytes

Objective To investigate the effect of siRNA against PTP-1B on neonatal rat cardiac myocyte apoptosis induced by hypoxia-reoxygenation (H/R) and its molecular mechanisms. Methods Isolated neonatal and adult rat cardiac myocytes were cultured for 24 h after PTP-1B siRNA transfection, and with 2, 4 and 6 h of hypoxia followed by 6 h of reoxygenation (H/R). The cardiac myocyte apoptosis induced by the treatments was assessed by TUNEL staining. Levels of PTP-1B and phospho-Akt were determined by Western blot, colorimetric assay kits were used to measure activities of caspase-3 and 8, and co-immunoprecipitation was used to check the amount of PTP-1B bound to FasR. Sodium orthovanadate, a general pharmacological phosphatase blocker and LY294002, an inhibitor of PI3-kinase/Akt pathway, were respectively used to inhibit PTP-1B and Akt activity. Results H/R resulted in severe injury in cultured rat cardiomyocytes and upregulated PTP-1B expression. However, siRNA against PTP-1B significantly decreased the number of apoptotic cardiomyocytes induced by 4H/6R as compared with cells without siRNA treatment (Apoptotic index: 12.1 ± 1.4% vs. 23.2 ± 1.6%, P < 0.05), along with greater phosphorylation of Akt, reduced activities of caspase-3 and 8, and the lower association of PTP-1B with FasR. Vanadate and LY294002 also partly reduced apoptosis of cardiomyocytes induced by 4H/6R. Conclusions PTP-1B is a key regulator of apoptosis of cardiomyocytes induced by H/R, and siRNA against PTP-1B effectively protects cardiomyocytes against H/R injury, the mechanisms of which might be associated with Akt activation, the reduction of both caspase-3 and 8 activities, and the lower amount of PTP-1B bound to FasR.     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。