PKCε信号通路介导的葛根素抗心肌细胞缺氧/复氧损伤作用

目的探讨葛根素(puerarin,Pue)抗心肌细胞缺氧/复氧(A/R)损伤保护作用与PKCε蛋白表达的关系。方法培养新生SD大鼠原代心肌细胞,分为正常对照(Con)组、A/R组、葛根素+A/R(Pue+A/R)组、葛根素+抑制剂+A/R(Pue+εV1-2+A/R)组、抑制剂+A/R(εV1-2+A/R)组。Western blot测定PKCε蛋白表达水平,MTT法检测细胞存活率,比色法测定培养液肌酸磷酸酶(CPK)、乳酸脱氢酶(LDH)活性、细胞内丙二醛(MDA)的含量和超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性,流式细胞仪测定线粒体膜电位和细胞凋亡。结果葛根素预处理24h后,心肌细胞PKCε表达呈剂量依赖性上调(P<0.01);培养液CPK、LDH活性降低,细胞内SOD、GSH-Px活性升高,MDA含量减少,细胞存活率升高,细胞凋亡减少(P<0.01);PKCε抑制剂εV1-2则可明显消弱葛根素的上述心肌保护作用。结论葛根素的抗心肌缺血/再灌注损伤保护作用涉及PKCε信号通路,至少部分依赖于其对PKCε表达水平的上调。     

Bcl-2/Bad/mPTP通路介导14-3-3γ对抗脂多糖所致心肌损伤

目的探讨Bcl-2/Bad/mPTP通路在14-3-3γ对抗脂多糖所致心肌损伤中的作用。方法构建pFLAG-14-3-3γ重组质粒,转染至原代乳鼠心肌细胞中,然后行LPS损伤处理。处理结束后,取培养液检测LDH活性,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡,线粒体肿胀实验检测mPTP开放,Western blot检测14-3-3γ、Bad、phospho-Bad以及线粒体Bcl-2蛋白表达。结果 LPS损伤使心肌细胞LDH活性升高、细胞存活率下降、凋亡细胞增加、mPTP开放加剧,转染pFLAG-14-3-3γ重组质粒后再行LPS损伤,则LDH活性下降、细胞存活率升高、mPTP开放与细胞凋亡减少,同时phospho-Bad蛋白表达增加,线粒体Bcl-2蛋白表达增加。结论 pFLAG-14-3-3γ能够对抗脂多糖所致的心肌细胞损伤,其机制可能与磷酸化Bad,释放Bcl-2至线粒体,抑制mPTP的开放有关。     

五甲基槲皮素预处理对大鼠心肌细胞缺氧复氧损伤保护作用的机制研究

目的 探讨五甲基槲皮素（PMQ）预处理对大鼠心肌细胞缺氧/复氧（A/R）损伤的保护作用及其线粒体功能的影响。方法 原代培养SD大鼠乳鼠心肌细胞,经终浓度分别为10,30,100 μmol·L-1 PMQ预处理24 h后,制作A/R损伤,检测培养液中乳酸脱氢酶（LDH） 活性、四唑盐（MTT）比色法检测细胞存活率、流式细胞法检测线粒体膜电位和细胞凋亡情况、线粒体肿胀法检测各组心肌细胞线粒体mPTP开放情况。结果 不同剂量PMQ（10, 30,100 μmol·L-1）预处理24 h后可剂量依赖性的降低LDH活性、增加细胞存活率、减少细胞凋亡（P 〈0.05或P 〈0.01）;30, 100 μmol·L-1 PMQ预处理24 h后,线粒体膜电位更为稳定、mPTP开放减少（P 〈0.05或P 〈0.01）。结论 PMQ预处理24 h后,可产生药理性延迟保护作用,机制与其稳定线粒体膜电位、抑制mPTP开放,进而减少细胞凋亡有关。     

p38MAPK/14-3-3γ通路在LPS预处理对抗心肌细胞缺氧/复氧损伤中的作用

目的研究p38MAPK/14-3-3γ通路在LPS预处理对抗缺氧/复氧(A/R)损伤中的作用。方法采用原代培养乳鼠心肌细胞,建立A/R损伤模型及APC、LPS预处理保护模型。实验结束后测定p38MAPK磷酸化蛋白表达及14-3-3γ蛋白表达,同时检测培养液中乳酸脱氢酶(LDH)活性、四唑盐(MTT)比色试验测定细胞存活率、TUNEL法检测细胞凋亡、线粒体肿胀实验检测线粒体通透性转换孔(mPTP)开放、流式细胞仪检测线粒体膜电位。结果 LPS预处理对随后心肌A/R损伤有类似APC的保护作用,同时伴随p38MAPK的磷酸化水平升高和14-3-3γ蛋白表达升高;给予SB203850特异性阻断p38MAPK通路,则14-3-3γ蛋白表达明显下调,并且心肌细胞LDH值升高,细胞存活率下降,心肌细胞的凋亡指数升高,mPTP开放加剧,线粒体膜电位降低。结论 LPS预处理对心肌细胞A/R损伤有保护作用,其机制可能是通过激活p38 MAPK,上调14-3-3γ的表达实现。     

Bcl-2介导的芹菜素抗心肌细胞缺氧/复氧损伤作用

目的探讨芹菜素(apigenin,Api)在心肌细胞缺氧/复氧损伤中的作用,并阐明Api对心肌损伤的保护作用是否主要由Bcl-2介导。方法培养H9c2心肌样细胞;随机分为正常对照(Cont)组、缺氧/复氧(Anoxia/Reoxygenation,A/R)组、Api预处理组、Api+ABT-737组。MTT法检测细胞存活率;Western blot法检测Bcl-2表达;比色法检测培养液LDH活性、细胞SOD及GSH-Px活性、MDA含量;流式细胞仪检测心肌细胞ROS含量、线粒体膜电位及细胞凋亡。结果 Api预处理25 h后,心肌细胞Bcl-2表达呈剂量依赖性上调(P<0.01);细胞存活率升高,培养液LDH活性降低,细胞SOD、GSH-Px活性升高,MDA含量与ROS生成减少,线粒体膜电位更为稳定,细胞凋亡减少(P<0.01);Bcl-2抑制剂ABT-737则可取消Api的上述心肌保护作用。结论Api抗心肌A/R损伤作用涉及Bcl-2信号通路,至少部分依赖于其对Bcl-2表达水平的上调。     

阿魏酸钠预处理对大鼠心肌细胞缺氧／复氧损伤线粒体的延迟保护作用

目的：研究阿魏酸钠（SF）预处理对心肌细胞缺氧／复氧（A／R）损伤的延迟保护作用的机制。方法：采用MTT比色法检测细胞存活率，生化检测细胞LDH活性、MDA含量、SOD活性和GPx活性．Western Blotting法检测SOD、GPx、HSP70的表达、TUNEL法与PI-Annexin V法测定细胞凋亡、流式细胞仪测定线粒体膜电位（△ψm）及ROS生成、分光光度法测定mPTP开放情况等指标，用终浓度为3．36mmol·L-1的SF预处理原代培养的大鼠乳鼠心肌细胞3h，24h后观察其对A／R损伤的保护作用及其ERKl／2抑制剂PDl84352（终浓度为0．5μmol·L-1）与基因转录抑制剂Act D（终浓度为5μmol·L-1）影响。结果：SF预处理能显著提高心肌细胞存活率、降低LDH活性、MDA含量减少；SOD、GPx活性增加，但其蛋白表达量却无明显差异；HSP70表达量显著上凋；ROS生成显著减少、mPTP开放与线粒体膜电位（△ψm）稳定、凋亡细胞比率减少。PDl84352、Act D均能取消SF预处理的上述作用。结论：SF预处理对心肌细胞A／R损伤延迟保护作用的机制可能与ERKl／2激活、上调HSP70蛋白表达、维护SOD、GPx等蛋白正常结构与功能，使得ROS生成减少、mPTP关闭、线粒体膜电位（△ψm）稳定、凋亡细胞减少有关。     

AE3通过NO通路参与心肌细胞缺氧预适应的保护作用

目的从细胞水平探讨阴离子交换蛋白3(AE3)在心肌细胞缺氧预处理(APC)保护作用中的意义以及与NO通路的关系。方法以原代培养的SD新生乳鼠心肌细胞为研究对象,建立缺氧/复氧(A/R)损伤和缺氧预适应保护模型。心肌细胞随机分为4组,Control组、A/R组、APC组和NO合成酶抑制剂L-NAME组。RT-PCR和Western blot法分别测定AE3的mRNA和蛋白表达,MTT法检测细胞存活率,生化自动分析仪测定LDH、CPK活性。结果 A/R处理后,心肌细胞AE3 mRNA转录及蛋白表达较Control组略增加。APC组AE3 mRNA转录及蛋白表达不仅均较Control组转录明显上调,而且较A/R组也明显上调。但预先加入NO合成酶抑制剂L-NAME则可抑制APC处理时AE3的表达增强,并取消APC的心肌保护作用。结论 AE3可能通过NO通路参与了心肌细胞APC的保护作用。     

线粒体通透性转换孔对红景天苷减轻心肌缺血再灌注损伤的作用

目的探讨线粒体通透性转换孔(mPTP)在红景天苷减轻大鼠心肌缺血再灌注损伤(MIRI)中的作用和机制。方法使用Langendorff装置建立大鼠离体心脏缺血/再灌注损伤模型。将60只SPF级健康雄性成年SD大鼠随机分为假手术组(Sham组)、缺血/再灌注组(I/R组)、红景天苷组(Sal组)和环孢菌素A组(CsA组)。用Western Blot法检测细胞色素C(Cyt-c)分别在细胞浆和线粒体中的表达情况;用免疫组化法检测活化型半胱天冬酶-9(cleaved caspase-9)和活化型半胱天冬酶-3(cleaved caspase-3)在各组心肌组织的表达水平;用线粒体肿胀实验法评估各组心肌的mPTP开放程度;酶联免疫分析(Elisa)法测定各组大鼠心肌组织中线粒体细胞色素C氧化酶(COX)和琥珀酸脱氢酶(SDH)的活性。结果与Sham组比较,I/R组心肌组织中大量Cyt-c由线粒体释放到细胞浆中,cleaved caspase-9和cleaved caspase-3的表达水平均显著上升,线粒体吸光度下降率明显升高,COX和SDH活性降低(P<0.05);与I/R组比较,Sal组和CsA组Cyt-c释放和线粒体吸光度下降均受抑制,cleaved caspase-9和cleaved caspase-3的表达水平均下降,COX和SDH活性均升高(P<0.05)。结论红景天苷可通过抑制mPTP开放、保护线粒体功能来发挥心肌保护作用,其具体机制与减少Cyt-c的释放、抑制caspase-9和caspase-3的激活及提高COX和SDH的活性有关。     

氯离子对心肌细胞缺氧/复氧损伤的影响

目的　研究氯离子对心肌细胞缺氧/复氧损伤的影响。方法　利用原代培养的心肌细胞缺氧/复氧损伤模型,去除细胞外的氯离子或采用氯通道阻断剂DIDS、SITS进行干预,检测细胞存活率及LDH、MDA、SOD、GXH Px活性变化。结果　缺氧/复氧损伤组与对照组比,LDH、MDA活性升高,细胞存活率、SOD、GXH Px降低; Cl- free+A R组、SITS+A R组处理后较缺氧/复氧组LDH、MDA活性低,细胞存活率、SOD、GXH Px升高;DIDS+A R组的上述指标与缺氧/复氧组比无差异。结论　①氯离子在心肌细胞缺氧/复氧损伤中起了重要作用。②氯通道阻断剂SITS对抗心肌细胞缺氧/复氧损伤有明显的保护作用,而DIDS无保护作用。     

细胞色素C在Pim-3抗心肌急性损伤中的作用

目的:探讨原癌基因Pim-3对抗心肌急性损伤的作用是否与线粒体信号转导途径介导的细胞色素C(Cyt C)的释放有关.方法:采用大鼠乳鼠心肌细胞,随机分为4组,对照组;缺氧复氧(A/R)组,建立A/R损伤模型;pcDNA3.1+A/R组,将pcDNA3.1转染入心肌细胞,24 h后进行A/R损伤;pcDNA3.1/Pim-3+A/R组,将Pim-3重组质粒(pcDNA3.1/Pim-3)转染入心肌细胞,24 h后进行A/R损伤.实验结束后,检测培养液中乳酸脱氢酶(LDH) 活性,四唑盐(MTT)比色试验测定细胞存活率,TUNEL法检测细胞凋亡,蛋白印迹检测Pim-3 蛋白表达水平,线粒体、胞浆Cyt C动态变化和caspase-3活性.结果:与对照组比较,A/R组细胞存活率明显下降,LDH值和凋亡指数明显升高,差异有统计学意义(P ＜ 0.01);与A/R组比较,pcDNA3.1/Pim-3+A/R组细胞存活率明显升高,LDH值和凋亡指数明显下降,并且Cyt C由线粒体向胞浆的释放明显被抑制,同时caspase-3活性也下降,差异有统计学意义(P ＜ 0.01).结论:Pim-3对抗心肌急性损伤的机制是通过抑制Cyt C易位释放入胞浆激活caspase-3而致.     

Salvianolic acid A protects H9C2 cardiomyocytes against hypoxia/reoxygenation injuryby regulating VDAC1

OBJECTIVE To investigate the role of voltage-dependent anion channel 1(VDAC1) in the protective effects of Salvianolic acid A(SAA) on H9C2 cardiomyocytes subjected to hypoxia/reoxygenation(H/R) injury.METHODS H9C2 cardiomyocytes were subjected to 4 h of hypoxia(1% O_2, 94% N_2, 5% CO_2, 37℃) followed by 4 h of reoxygenation(95% air, 5% CO_2, 37℃). SAA(1 umol·L~(-1))was administrated at the same time of reoxygenation. To examine whether the expression of VDAC1 is regulated by SAA, H9C2 cardiomyocytes were respectively transfected with VDAC1 si RNA and VDAC1 over expression plasmid. The viability of H9C2 cardiomyocytes was detected by Cell Counting kit-8 assay and further confirmed by detecting the level of intracel ular lactate dehydrogenase.Apoptosis were evaluated using an Annexin Ⅴ-FITC/propidiumiodide staining kit detected by flow cytometry.The level of mitochondrial ROS was measured using the fluorescent probe Mito SOX. The mitochondrial Ca~(2+) signal was monitored with a FRET-based Ca~(2+) indicator. Western blotting and q RT-PCR analysis were used to observe the protein and m RNA levels of VDAC1, and we use luciferase reporter assay to further explore whether SAA has a potential binding sites on VDAC1 promoter to perform the direct regulation on VDAC1. The oligomeric state of VDAC1 was explored by BRET2 assay. RESULTS H/R injury significantly increased the expression of VDAC1, and induced VDAC1 oligomerization. VDAC1 silencing could increase the viability of H/R-treated cardiomyocytes, and attenuate apoptosis as well as the increase on the level of mitochondrial ROS and Ca~(2+) uptake against H/R injury,which performed the same protective effects with SAA.Meanwhile, VDAC1 overexpression could aggravate cell injury. Taken together, VDAC1 was involved in the mechanisms of H/R-induced cell injury. SAA could significantly inhibit the expression of VDAC1 compared with H/R group, we also found that SAA exhibited a direct regulative effect in 3′-UTR of VDAC1 gene. Moreover, SAA could inhibit the oligomerization of VDAC1. Al these protective effects of SAA were abolished in H9C2 cardiomyocytes transfected with VDAC1 overexpression plasmid.CONCLUSION Our findings indicate that SAA can inhibit apoptosis and improve mitochondrial function by regulating the expression and oligomerization of VDAC1, which revealed VDAC1 as a novel potential target and mechanism for the cardioprotective effects of SAA.     

脑源性神经营养因子可减轻 H9c2 心肌细胞缺氧/复氧损伤

目的 探讨脑源性神经营养因子（BDNF）预处理对 H9c2 心肌细胞缺氧/复氧（H/R）损伤的影响及其作用机制。方法 体外培养 H9c2 心肌细胞并以缺氧 （95%N2+5%CO2） 培养 4 h 后复氧 （95%O2+5%CO2） 培养 12 h 建立 H/R 模型, 并分为 Control 组、 H/R 组、 不同浓度 （1、 10、 100 μg/L） BDNF 预处理后 H/R 组、 酪氨酸蛋白激酶受体 B （TrkB）inhibitor 组 （同时加入 100 μg/L BDNF 和 1∶1 000 的 TrkB inhibitor 预处理后 H/R 组）。利用 MTT 方法检测各组心肌细胞的细胞存活率; 并测定各组心肌细胞 H/R 损伤后乳酸脱氢酶（LDH）、 肌酸激酶（CK）、 丙二醛（MDA）、 超氧化物歧化酶（SOD）含量及活性; 流式细胞术测定各组心肌细胞的凋亡率; Western-blot 检测 TrkB、 Bcl-2、 Bax 蛋白表达。结果 与 Control 组相比, H/R 组细胞存活率明显下降, LDH、 CK 和 MDA 含量升高, SOD 活性降低, 细胞凋亡率升高, 抗凋亡 Bcl-2 蛋白表达下降, 促凋亡 Bax 蛋白表达升高（均 P < 0.05）。与 H/R 组相比, 不同浓度 BDNF 预处理H9c2 心肌细胞后 H/R, 各组细胞存活率均明显上升, CK、 LDH、 MDA 含量逐渐下降, SOD 活性升高, 细胞凋亡率下降, TrkB 和 Bcl-2 蛋白表达升高, 而 Bax 蛋白表达降低, 但 BDNF 的作用均受到 TrkB inhibitor 的抑制。结论 BDNF预处理能够提高 H9c2 心肌细胞 H/R 损伤的细胞存活率, 通过降低细胞凋亡率和提升细胞抗氧化能力而发挥保护作用, 并与 BDNF-TrkB 信号通路有关。     

内质网应激相关蛋白 1过表达对缺氧 /复氧诱导的 H9c2心肌细胞凋亡的影响

目的 研究内质网应激相关蛋白1(SERP1)过表达对缺氧/复氧(H/R)诱导的大鼠胚胎H9c2心肌细胞凋亡的影响.方法 2018年1—12月将中国科学院上海细胞库大鼠H9c2心肌细胞系建立H/R损伤大鼠心肌细胞模型,采用pCMV6质粒为载体构建内质pCMV6-网应激相关蛋白1(pCMV6-SERP1)过表达重组质粒,分别设H9c2心肌细胞为正常对照组(NC组),H/R损伤(缺氧4 h/复氧4 h)H9c2心肌细胞为H/R组,H/R损伤H9c2心肌细胞转染pCMV6空质粒为H/R+pCMV6组,H/R损伤H9c2心肌细胞转染pCMV6-SERP1过表达重组质粒为H/R+pCMV6-SERP1组,采用噻唑蓝(MTT)法检测各种H9c2心肌细胞存活率,采用Annexin V-FITC/PI双染技术检测各组H9c2心肌细胞凋亡率,采用实时定量PCR(RT-qPCR)和免疫印迹法分别检测各组细胞SERP1蛋白及凋亡相关蛋白的表达水平.结果 与NC组[(1.06±0.21)、(0.41±0.07)]比较,H/R组、H/R+pCMV6组H9c2心肌细胞SERP1 mRNA[(0.47±0.09)、(0.48±0.08)]及蛋白表达水平[(0.25±0.04)、(0.26±0.05)]显著降低,H/R+pCMV6-SERP1组H9c2心肌细胞SERP1 mRNA(2.94±0.52)及蛋白表达水平(0.83±0.12)显著增加(P<0.05),与H/R组、H/R+pCMV6组比较,H/R+pCMV6-SERP1组H9c2心肌细胞中内质网应激相关蛋白1微小RNA(SERP1 mRNA)及蛋白水平显著升高(P<0.05).与NC组[(100.00±00.00)％、(4.45±0.62)％]比较,H/R组、H/R+pCMV6组、H/R+pCMV6-SERP1组H9c2心肌细胞存活率[(49.86±5.14)％、(48.75±5.23)％、(89.72±8.61)％]、B淋巴细胞瘤-2(Bcl-2)蛋白水平显著降低(P<0.05),H9c2心肌细胞凋亡率[(35.84±4.13)％、(33.66±4.07)％、(10.96±1.74)％]、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(caspase-3)蛋白表达水平显著增加(P<0.05);与H/R组、H/R+pCMV6组比较,H/R+pCMV6-SERP1组H9c2心肌细胞中存活率、Bac-l蛋白表达水平显著升高,细胞凋亡率、Bax、Caspase-3蛋白表达水平显著降低(P<0.05).结论 SERP1过表达可能通过上调Bacl-2蛋白,下调Bax、Caspase-3蛋白表达,对缺氧/复氧诱导的H9c2心肌细胞凋亡有一定保护作用.     

香青兰及其含药血清对H9c2心肌细胞三种缺氧/复氧损伤模型的影响

目的:比较香青兰醇提物及其含药血清对H9c2心肌细胞3种缺氧/复氧损伤模型的影响,考察香青兰对心肌缺血/再灌注损伤的保护作用。方法:体外培养H9c2心肌细胞,以Na2S2O4,N2和厌氧袋为缺氧环境分别建立缺氧/复氧损伤模型,以T-SOD,MDA,LDH为指标,考察香青兰醇提物(1,10,100μg.mL-1)及其含药血清对H9c2心肌细胞缺氧/复氧损伤的影响。结果:与模型组比较,香青兰醇提物及其含药血清能明显抑制缺氧/复氧损伤H9c2心肌细胞LDH释放和MDA含量,升高T-SOD活力(P<0.05或0.01)。结论:香青兰醇提物及其含药血清对H9c2心肌细胞缺氧/复氧损伤具有保护作用,提示香青兰具有保护心肌缺血/再灌注损伤的作用。     

Baicalin protects H9c2 cardiomyocytes against hypoxia/reoxygenation‐induced apoptosis and oxidative stress through activation of mitochondrial aldehyde dehydrogenase 2

Baicalin, a flavonoid glycoside separated from Scutellaria baicalensis, has cardioprotection against ischaemia/reperfusion (I/R) injury. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is considered as an endogenous protective mechanism against I/R injury depending on its anti‐oxidant and anti‐apoptotic characteristics. The present study demonstrates whether ALDH2 contributes to the cardioprotection of baicalin against hypoxia/reoxygenation (H/R)‐inudced H9c2 cardiomyocytes injury. Our results observed that H/R treatment resulted in a significant decrease in cells viability and obvious increases in caspase‐3 activity and apoptosis rate in H9c2 cells, while these alterations were evidently reversed by baicalin pretreatment. Simultaneously, baicalin mitigated H/R‐induced the decreases in the levels of ALDH2 mRNA and protein as well as the activity of ALDH2 in H9c2 cells. However, we found that daidzin, an ALDH2 antagonist, remarkably attenuated baicalin‐elicited inhibitory action on H/R‐induced the downregulation of cells viability and Bcl‐2 protein expression, and the upregulations of caspase‐3 activity, apoptosis rate, cytochrome c and Bax proteins expressions in H9c2 cells. In addition, baicalin reversed H/R‐induced oxidative stress as evidenced by the downregulation of malondialdehyde (MAD) and 4‐hydroxy aldehydes (4‐HNE) levels, the inhibition of endogenous reactive oxygen species (ROS) generation, and the downregulation of superoxide dismutase (SOD) activity induced by H/R treatment, while these effects were also blocked by daidzin. Furthermore, we found that Alda‐1, an ALDH2 agonist, also abolished H/R‐induced cytotoxicity, apoptosis, and oxidative stress, indicating that ALDH2 mediated H/R‐induced H9c2 cell injury. Overall, these results suggested that baicalin prevents H/R‐induced apoptosis and oxidative stress through enhancing ALDH activity and expression in H9c2 cardiomyocytes.     

NDUFA4L2 protects against ischaemia/reperfusion‐induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4‐like 2 (NDUFA4L2) on myocardial ischaemia‐reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia‐reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT‐PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl‐2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down‐regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R‐induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl‐2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down‐regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury.     

pFLAG-AMPKα2真核表达质粒构建及对心肌缺氧/复氧损伤的作用

目的：构建大鼠pFLAG-AMPKα2真核表达质粒,分析AMPKα2过表达对心肌细胞缺氧/复氧损伤的影响。方法：提取H9c2心肌样细胞的mRNA,反转录为cDNA。PCR扩增AMPKα2基因的cDNA全长,并将其克隆至pFLAG-CMV-4构建pFLAG-AMPKα2重组质粒。转染pFLAG-AMPKα2至H9c2细胞,West-ern Blot测定AMPKα2的蛋白表达情况,建立缺氧/复氧（A/R）损伤模型,MTT法检测心肌细胞的存活率,生物自动分析仪检测LDH活性,流式细胞术检测各实验组心肌细胞凋亡程度、细胞内ROS的变化,试剂盒检测细胞内抗氧化酶（SOD、GSH-Px）活性。结果：AMPKα2全长基因序列克隆至真核表达载体pFLAG-CMV-4,酶切鉴定片段大小为1700bp,Western blot检测转染pFLAG-AMPKα2后,AMPKα2蛋白在H9c2细胞中高表达。H9c2细胞遭受A/R损伤,心肌细胞存活率下降,LDH活性增加,细胞凋亡加重,ROS生成增加,SOD及GSH-Px的酶活性下降,pFLAG-AMPKα2重组质粒的导入可明显逆转、改善上述各项指标,从而对抗A/R损伤。结论：构建成功的pFlAG-AMPKα2重组质粒能对抗A/R所致的心肌细胞损伤,其机制与改善心肌细胞的氧化应激作用有关。     

吡那地尔、美托洛尔、谷氨酰胺、胰岛素单独及联合使用对H9c2心肌细胞缺氧/复氧损伤的保护作用及机制

目的研究吡那地尔(pinacidil,Pin)、美托洛尔(metoprolol,Met)、谷氨酰胺(glutamine,Glu)、胰岛素(insulin,Ins)四药联用对缺氧/复氧(hypoxia/reoxygenation,H/R)所致H9c2心肌细胞损伤的保护作用并探讨其作用机制。方法将培养的H9c2心肌细胞随机分为7组,即①正常对照(Con)组;②H/R组;③Pin组;④Met组;⑤Glu组;⑥Ins组;⑦四药联用(PMGI)组。测定各组H9c2心肌细胞的存活率;收集培养液测定乳酸脱氢酶(lactate dehydrogenase,LDH)活性,超氧化物歧化酶(superoxide dismutase,SOD)活性;Western印迹法检测保护性蛋白热休克蛋白70(heat shock protein 70,HSP70)的表达情况。结果对于H/R损伤的H9c2心肌细胞,四药联用组与药物单用组或H/R组相比能明显提高细胞存活率,保护细胞膜,减少LDH渗漏;增加抗氧化能力,提高SOD含量;增加HSP70的表达。结论联合用药组与药物单用组相比保护作用增强。     

艾司洛尔对缺氧复氧诱导的心肌细胞AMPK/GSK-3β通路及凋亡的影响

目的 探讨艾司洛尔对缺氧复氧诱导的心肌细胞凋亡的影响,并初步研究其作用机制.方法 体外培养大鼠心肌细胞(H9c2),并建立缺氧/复氧(I/R)损伤模型,随机分为对照组、H/R组和艾司洛尔低、中、高剂量(0.2、5.0、25.0μmol/L)组;噻唑蓝(MTT)法检测各组H9c2细胞存活率变化情况;膜联蛋白V-异硫氰酸荧...     

钴原卟啉对H9c2心肌细胞缺氧/复氧损伤的保护作用

目的研究钴原卟啉(COPP)预处理在H9c2心肌细胞缺氧/复氧损伤中的作用及分子机制。方法建立H9C2心肌细胞缺氧/复氧模型。在H9c2心肌细胞缺氧/复氧前用COPP预处理,并用锌原卟啉(Znpp),全反视黄酸(ATAR)分别抑制HO-1及Nrf2-ARE。检测细胞上清液中LDH、CK的水平变化;用RT-PCR法分析HO-1mRNA表达水平;Westernblot分析HO-1、Nrf2的蛋白表达水平。结果与缺氧/复氧组比,COPP预处理组中LDH和CK水平均明显降低,而HO-1mRNA水平,蛋白水平及细胞核的Nrf2蛋白水平均明显增加;Znpp的加入阻断了COPP预处理对心肌细胞的保护作用;ATAR的加入抑制了Nrf2在细胞核的聚集,进而抑制了COPP对HO-1的诱导。结论COPP预处理诱导H9c2心肌细胞HO-1过表达,具有抗心肌细胞缺氧/复氧损伤的保护作用;其机制与Nrf2-ARE信号通路相关。