梓醇对H_2O_2诱导HUVECs和H9c2细胞凋亡的影响及其机制的研究
摘要
前言
凋亡在众多的病理生理过程中起着重要的作用。细胞生长和凋亡之间的平衡决定着许多病理生理过程,如高血压,动脉粥样硬化,血管疾病的发生发展。在动脉粥样硬化形成过程中过多的活性氧的产生加速了动脉粥样硬化,最终导致了心血管疾病的发生。
H2O2是体内氧化代谢的产物,也是一种活性氧,它不仅能直接氧化细胞膜上的脂质及蛋白,而且能自由穿过细胞膜,导致脂质过氧化反应和细胞内DNA的损伤。H2O2还可以通过降低细胞内的抗氧化系统诱导细胞凋亡。进一步的研究表明H2O2刺激线粒体释放细胞色素C (Cytochrome C, Cyt-C),引起细胞结构,功能,代谢的改变,进而引起血管疾病的发生。
梓醇(catalpol)是一种环烯醚萜葡萄糖苷类化合物,在神经细胞及其他细胞系的研究中发现它具有抗凋亡的作用。体外实验发现梓醇能抑制H2O2诱导PC12细胞凋亡,激活细胞内信号转导通路促进分化。梓醇对心血管氧化应激的作用还未有报道,但有研究表明梓醇能抑制脑缺血再灌注动物模型中的氧化应激,在外周组织如肾脏中,梓醇也具有保护作用。因此,我们很有理由去研究梓醇是否也能对抗心血管疾病中的氧化应激损伤。
Akt信号途径是细胞重要的促增殖、迁移及存活信号途径。多种生长因子均可通过PI3K途径激活Akt/PKB。完全活化的Akt通过磷酸化和钝化其下游底物,经由多种途径而促进细胞的存活。例如胞质内Bad能被Akt磷酸化,磷酸化后的Bad不能转位进入线粒体,抑制Cyt-C释放入胞质,减少caspase-3活化,进而抑制细胞凋亡。根据功能可将Bcl-2基因家族分为两类：一类是抗凋亡的,如Bcl-2、Bcl-XL；一类是促凋亡的,如Bax、Bad等。越来越多的研究发现,位于线粒体外膜的Bcl-2家族蛋白,能通过改变线粒体细胞膜的通透性,诱导细胞凋亡。
本实验观察了在离体细胞给予梓醇治疗是否可以减轻H2O2损伤导致内皮及心肌细胞凋亡以及分析了梓醇抗血管内皮及心肌细胞凋亡的信号转导机制,尤其是梓醇的这种保护作用是否是部分通过激活传导通路PI3K/Akt,调节Bcl-2和Bax的表达而实现的。
材料与方法
(1)人脐静脉内皮细胞,H9c2细胞培养,梓醇和／或H2O2共同孵育,收集细胞及细胞培养液；分别加入PI3K的特异性抑制剂wortmannin及LY294002进行干预,收集细胞及细胞培养液,用于指标检测。
(2)采用MTT法检测梓醇对HUVECs及H9c2细胞存活率的影响。
(3)应用LDH, SOD, MDA检测试剂盒检测细胞LDH, SOD, MDA的活性。
(4)采用激光共聚焦法检测细胞内活性氧(ROS)的浓度。
(5)应用Annexin V-FITC、TUNEL和hoechst 33258试剂盒检测细胞的凋亡。
(6)采用western blotting法检测HUVECs及H9c2细胞Akt、p-Akt、p-Bad、Bcl-2和Bax蛋白的表达。
(7)采用real time RT-PCR法检测内皮细胞Akt、Bad、Bcl-2及Bax mRNA的表达。
(8)采用免疫组化法检测心肌细胞p-Akt、Bcl-2和Bax蛋白的表达。
(9)各实验组数据均用x±s表示,利用spss11.5统计软件分析,组间差异比较采用单因素方差分析。P<0.05为有统计学差异。
结果
(1) Catalpol终浓度为0.1～10μg/ml时可促进细胞存活,呈剂量依赖性。但catalpol终浓度高至1000μg/ml时则出现增殖抑制效应。
(2) 10μg/ml catalpol处理0h、24h、48h、72h,较对照组细胞存活率明显升高。Catalpol作用72h时,HUVECs存活率达峰值；梓醇作用48h时,H9c2存活率达峰值。
(3)不同浓度0.1μg/ml、1μg/ml、10μg/ml catalpol预处理可以减轻H2O2诱导的细胞损伤,随着catalpol浓度的增加,相应的MTT检测的OD值也随之增加。
(4)H2O2组在H2O2作用后内皮及心肌细胞受到损伤,LDH较C组明显增高,损伤后MDA亦较C组明显增高,SOD较C组明显降低(均P<0.05)。Catalpol (0.1～10μg/ml)预处理培养细胞,在H202损伤过程中,均较H202组细胞损伤小,LDH和MDA含量明显减少,而SOD则较H2O2组明显增加。
(5)经H2O2处理24h后,细胞内荧光强度明显增强,用不同的浓度catalpol (0.1～10μg/ml)预处理24h后,与H2O2组相比细胞内荧光强度明显下降为,呈浓度依赖性。
(6)内皮细胞C组可见少许的TUNEL阳性细胞。与C组相比,细胞在H2O2(100μmol/L)中培养24h明显增加TUNEL阳性细胞数,然而,当用catalpol (0.1～10μg/ml)预处理24h后加入H2O2(100μmol／L)作用24h,凋亡率明显下降,且随着catalpol剂量增加凋亡率降低。
(7)正常的心肌细胞核呈圆形或椭圆形,染色均匀,呈正常的蓝色,而凋亡细胞的细胞核有明显的染色质边聚呈致密浓染,或呈碎块状致密浓染,变形亮度增强,颜色发白。Hoechst 33258结果显示：C组细胞少见白色亮点,H2O2组细胞见大量凋亡细胞。经不同浓度catalpol处理后,白色亮点呈逐渐减少趋势,凋亡细胞明显减低,呈剂量相关性。
(8) Annexin-FITC/PI检测内皮细胞凋亡率显示C组细胞的存活率为78.7±1.2%；而H2O2(100μmol／L)组细胞的凋亡率显著升高,凋亡率为35.9±0.6%,说明H2O2诱导了内皮细胞的凋亡。用不同浓度的catalpol (0.1、1、10μg/ml)预处理各组细胞后再施以H2O2作用,细胞的凋亡率出现不同程度的下降,而且随着catalpol浓度的升高,各组细胞的凋亡率也随之依次下降,细胞凋亡率分别降低至27.6±0.6%,22.6±0.8%和19.1±0.4%。
H202组心肌细胞凋亡率明显高于C组(P<0.01);与H2O2组比较,catalpol1、catalpol2、catalpol3组细胞凋亡率明显降低(P<0.01),且随着catalpol剂量增加凋亡率降低。
(9)Western blotting结果显示：H2O2作用24h后,Akt在各组中都有表达,差异无统计学意义(P>0.05), p-Akt, p-Bad, Bcl-2蛋白表达量较C组明显减少,Bax蛋白表达较C组明显增加,差异具有显著性(p<0.01)。catalpol 0.1,1, 10μg/ml可以剂量依赖性地增加p-Akt, p-Bad, Bcl-2蛋白表达,降低Bax蛋白的表达,与H2O2组比较差均有显著性(P＜0.05, P<0.01)。Wortmannin和LY294002预处理可拮抗catalpol部分保护作用。
免疫组化结果显示,在光镜下,心肌细胞阳性表达的p-Akt, Bcl-2, Bax蛋白主要积聚在胞浆中,呈棕色反应,各组蛋白表达强度不一致。积分光密度值检测结果显示,与C组比较,H2O2组细胞p-Akt, Bcl-2蛋白表达减弱(P<0.05,P<0.01),Bax蛋白表达显著增强(P<0.01)；与H2O2组比较,catalpol1、catalpol2、catalpol3组细胞的p-Akt, Bcl-2蛋白表达增高(P<0.01),Bax蛋白表达显著减弱(P<0.01)。Wortmannin和LY294002可拮抗catalpol部分保护作用。
(10) Real time RT-PCR结果显示：H2O2作用24h后,Akt, Bad, Bcl-2 mRNA表达量较C组明显减少,Bax mRNA表达较C组明显增加。Catalpol 0.1,1,10μg/ml可以剂量依赖性地增加.Akt, Bad, Bcl-2 mRNA表达,降低Bax mRNA的表达,与H2O2组比较差均有显著性(P＜0.05, P＜0.01)。Wortmannin和LY294002预处理可拮抗catalpol部分保护作用。
结论
(1) Catalpol对HUVECs、H9c2细胞存活率的影响具有时间和剂量依赖性。
(2) Catalpol通过降低MDA,增加SOD减轻H2O2诱导的细胞损伤。
(3) Catalpol可能通过清除细胞内过多的ROS来参与H2O2诱导细胞损伤的保护。
(4) PI3K/Akt-Bad途径可能为catalpol抗H2O2诱导细胞凋亡的重要信号转导机制。
(5) Catalpol还可能通过直接调节凋亡相关基因Bcl-2、Bax的表达而抑制细胞凋亡。
Introduction
Apoptosis plays a fundamental role in the development of numerous pathophysiological states. The balance between cell growth and apoptosis is likely to determine whether pathological phenomena such as hypertension, atherosclerosis and vascular disease occur. The excess generation of reactive oxygen species in the vascular system during the atherogenic process has been reported to enhance atherosclerotic lesion formation, resulting in the development of cardiovascular disease.
Hydrogen peroxide (H2O2), one of the most common reactive oxygen species, can easily penetrate the plasma membrane and cause lipid peroxidation and DNA damage in cells. H2O2 induces apoptosis by disrupting the cell's natural antioxidant defense system. It has been shown that H2O2 can stimulate cytochrome c release from the mitochondria, resulting in the loss of endothelial integrity and subsequent vascular disease.
Catalpol, a major chemical constituent of Rehmannia glutinosa Libosch, is an iridoid glucoside which exerts well known anti-apoptotic effects in neuronal cells and other cell lines. Findings from in vitro experiments have revealed that catalpol is able to activate the intracellular signal transduction pathway inducing neuronal differentiation and attenuate H2O2-induced apoptosis in PC 12 cells. The effects of catalpol on cardial oxidative stress have not been described. However a protective effect against oxidative stress damage of neurons in global and focal cerebral has been reported. Catalpol has also been shown to protect against oxidative stress in peripheral tissues such as the kidneys. Therefore, it seems reasonable to investigate whether catalpol is able to protect the cardiovascular from oxidative stress injury.
Akt is a critical component of the intracellular signaling pathway involved in regulating cell survival and apoptosis. Various growth and survival factors can activate this protein kinase. It has been reported that Akt can phosphorylate and inactivate Bad, thus inhibiting cell death. Among the Bcl-2 family, several members such as Bcl-2 and Bcl-xL induce cell survival, while other members such as Bad and Bax promote cell death. Further to this, it has been shown that members of the Bcl-2 family, which are located on the mitochondrial membrane, can alter mitochondrial membrane permeability and trigger apoptosis.
In the present study, we examined the effect of catalpol on H2O2-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and H9c2 cells. We also examined potential mechanisms underlying catalpol-associated protection, including reactive oxygen species scavenging, alterations in the phosphatidylinositol 3-kinase (PI3K)/Akt-Bad signaling pathway and changes in Bcl-2 and Bax expression.
Materials and methods
(1) We cultured HUVECs and H9c2 cells, and examined the effect of catalpol and H2O2 on cells. We also examined the effect of wortmannin and LY294002 on H2O2 incubated cells.
(2) The cell viablity of HUVECs and H9c2 was determined by MTT analysis.
(3) The concentration of malondialdehyde (MDA), lactate dehydrogenase (LDH) as well as the activity of superoxide dismutase (SOD) were were all determined by using commercially available kits.
(4) The level of intracellular reactive oxygen species was quantified by 2', 7'-dichlorofluorescein diacetate assay.
(5) Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and hoechst 33258 assay.
(6) Expression of Akt, p-Akt, p-Bad, Bcl-2 and Bax activity was quantified by western blotting analysis.
(7) Expression of Akt, Bad, Bcl-2 and Bax mRNA was determined by real-time semiquantitative reverse transcription-polymerase chain reaction method.
(8) Expression of p-Akt, Bcl-2 and Bax activity was detected by immunohistochemistry.
(9) All data are presented as mean±standard deviation (S.D.). Differences between mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA). The results were considered to be statistically significant when P＜0.05.
Results
(1) Incubation of HUVECs and H9c2 cells with different concentrations of catalpol (0.1,1,10μg/ml) for 48 h increased the viability of cells in a dose-dependent manner. The protective effect of catalpol was almost inhibited and cell viability reduced compared with the control group at a concentration of 1000μg/ml.
(2) Increased rates of cell survival were apparent 24 h after treatment. The magnitude of cell survival peaked at 72 h in HUVEC and at 48h in H9c2.
(3) Pre-incubation of cells with different concentrations of catalpol (0.1,1 or 10μg/ml) before H2O2 exposure increased viability in a dose-dependent manner.
(4) Compared with the control, treatment of cells with 100μM of H2O2 for 24 h caused significantly less activities of SOD. However, catalpol (0.1,1 or 10μg/ml) pretreatment significantly attenuated the changes of SOD activities in a dose dependent fashion, compared to the H2O2 group. In addition, cells treated with 100μM of H2O2 for 24 h caused more MDA and LDH levels, while pre-incubation of cells with catalpol (0.1,1 or 10μg/ml) markedly attenuated the increases compared to the H2O2 group.
(5) As expected, DCF fluorescence in cells exposed to H2O2 was strikingly increased compared to fluorescence in control group. Pre-treatment with catalpol (0.1, 1 or 10μg/ml) significantly inhibited DCF fluorescence caused by H2O2 exposure in a dose-dependent fashion.
(6) Few TUNEL-positive nuclei were evident in the control group. Exposure of cells to 100μM H2O2 for 24 h, however, resulted in a marked increase in the number of such nuclei. Pre-treatment with catalpol reduced the K2O2-induced increase in the number of cells with TUNEL-positive nuclei.
(7) In the control group were shown as round-shaped nuclei with homogenous fluorescence intensity. However, a marked increase of apoptotic cells which contained heterogeneous intensity, chromatin condensation, and fragmentation appeared after 24 h treatment with 100μM H2O2. Pre-treatment of catalpol completely protected cells from morphological changes by H2O2.
(8) Flow cytometry in HUVECs revealed that 78.7±1.2% of cells in the control group were viable. In contrast,35.9±0.6% of cells treated with 100μM H2O2 for 24 h were in early apoptosis or late apoptosis/necrosis. Pre-treatment of cells with catalpol (0.1,1 or 10μg/ml) for 24 h reduced the percentage of apoptotic cells associated with H2O2 exposure from 35.9±0.6% to 27.6±0.6%,22.6±0.8% and 19.1±0.4%, respectively.
Exposure of H9c2 cells to 100μM H2O2 for 24 h resulted in an increase in cellular apoptosis as revealed by flow cytometry. Pre-treatment of cells with catalpol (0.1,1 or 10μg/ml) for 24 h prior to H2O2 reduced the percentage of apoptotic cells from 14.30±0.41% to 8.97±0.36%,7.81±0.06% and 6.38±0.43%, respectively, in a concentration dependent manner.
(9) The protein phosphorylated Akt, Bad, Bcl-2 and Bax were analyzed by western blotting and immunohistochemistry. Catalpol (0.1,1 or 10μg/ml) induced Akt and Bad protein phosphorylation, increased Bcl-2, decreased Bax in a dose-dependent manner Both wortmannin and LY294002 markedly inhibited catalpol-induced Akt and Bad phosphorylation, increased Bax, decreased Bcl-2.
(10) We investigated the mRNA expression of Akt, Bad, Bcl-2 and Bax. Akt, Bad, Bcl-2 mRNA levels were significantly increased in catalpol (0.1,1 or 10μg/ml) pre-treated groups, compared to the control group. While H2O2 exposure significantly reduced Akt, Bad, Bcl-2 levels compared to control. Catalpol (0.1,1 or 10μg/ml) pre-treatment down-regulated Bax levels in cells exposed to H2O2. This effect was partially inhibited by concurrent wortmannin or LY294002 treatment.
Conclusions
(1) Pre-incubation of cells with 0.1,1 or 10μg/ml of catalpol resulted in a time-and dose-dependent manner variation of cell viability.
(2) Catalpol may protect cells from H2O2-induced injury by strengthened the changes of SOD activities and attenuated the levels of MDA.
(3) Scavenging of reactive oxygen species is involved in the mechanisms underlying the protective effect of catalpol against H2O2-induced injury.
(4) Catalpol may protect cells from H2O2-induced apoptosis at least part by activating the PI3K/Akt-Bad signaling pathway.
(5) Bcl-2 and Bax are involved in mediating the anti-apoptotic effects associated with catalpol treatment in HUVECs and H9c2 cells exposed to H2O2.
凋亡在众多的病理生理过程中起着重要的作用。细胞生长和凋亡之间的平衡决定着许多病理生理过程,如高血压,动脉粥样硬化,血管疾病的发生发展。在动脉粥样硬化形成过程中过多的活性氧的产生加速了动脉粥样硬化,最终导致了心血管疾病的发生。
H2O2是体内氧化代谢的产物,也是一种活性氧,它不仅能直接氧化细胞膜上的脂质及蛋白,而且能自由穿过细胞膜,导致脂质过氧化反应和细胞内DNA的损伤。H2O2还可以通过降低细胞内的抗氧化系统诱导细胞凋亡。进一步的研究表明H2O2刺激线粒体释放细胞色素C (Cytochrome C, Cyt-C),引起细胞结构,功能,代谢的改变,进而引起血管疾病的发生。
梓醇(catalpol)是一种环烯醚萜葡萄糖苷类化合物,在神经细胞及其他细胞系的研究中发现它具有抗凋亡的作用。体外实验发现梓醇能抑制H2O2诱导PC12细胞凋亡,激活细胞内信号转导通路促进分化。梓醇对心血管氧化应激的作用还未有报道,但有研究表明梓醇能抑制脑缺血再灌注动物模型中的氧化应激,在外周组织如肾脏中,梓醇也具有保护作用。因此,我们很有理由去研究梓醇是否也能对抗心血管疾病中的氧化应激损伤。
Akt信号途径是细胞重要的促增殖、迁移及存活信号途径。多种生长因子均可通过PI3K途径激活Akt/PKB。完全活化的Akt通过磷酸化和钝化其下游底物,经由多种途径而促进细胞的存活。例如胞质内Bad能被Akt磷酸化,磷酸化后的Bad不能转位进入线粒体,抑制Cyt-C释放入胞质,减少caspase-3活化,进而抑制细胞凋亡。根据功能可将Bcl-2基因家族分为两类：一类是抗凋亡的,如Bcl-2、Bcl-XL；一类是促凋亡的,如Bax、Bad等。越来越多的研究发现,位于线粒体外膜的Bcl-2家族蛋白,能通过改变线粒体细胞膜的通透性,诱导细胞凋亡。
本实验观察了在离体细胞给予梓醇治疗是否可以减轻H2O2损伤导致内皮及心肌细胞凋亡以及分析了梓醇抗血管内皮及心肌细胞凋亡的信号转导机制,尤其是梓醇的这种保护作用是否是部分通过激活传导通路PI3K/Akt,调节Bcl-2和Bax的表达而实现的。
材料与方法
(1)人脐静脉内皮细胞,H9c2细胞培养,梓醇和／或H2O2共同孵育,收集细胞及细胞培养液；分别加入PI3K的特异性抑制剂wortmannin及LY294002进行干预,收集细胞及细胞培养液,用于指标检测。
(2)采用MTT法检测梓醇对HUVECs及H9c2细胞存活率的影响。
(3)应用LDH, SOD, MDA检测试剂盒检测细胞LDH, SOD, MDA的活性。
(4)采用激光共聚焦法检测细胞内活性氧(ROS)的浓度。
(5)应用Annexin V-FITC、TUNEL和hoechst 33258试剂盒检测细胞的凋亡。
(6)采用western blotting法检测HUVECs及H9c2细胞Akt、p-Akt、p-Bad、Bcl-2和Bax蛋白的表达。
(7)采用real time RT-PCR法检测内皮细胞Akt、Bad、Bcl-2及Bax mRNA的表达。
(8)采用免疫组化法检测心肌细胞p-Akt、Bcl-2和Bax蛋白的表达。
(9)各实验组数据均用x±s表示,利用spss11.5统计软件分析,组间差异比较采用单因素方差分析。P<0.05为有统计学差异。
结果
(1) Catalpol终浓度为0.1～10μg/ml时可促进细胞存活,呈剂量依赖性。但catalpol终浓度高至1000μg/ml时则出现增殖抑制效应。
(2) 10μg/ml catalpol处理0h、24h、48h、72h,较对照组细胞存活率明显升高。Catalpol作用72h时,HUVECs存活率达峰值；梓醇作用48h时,H9c2存活率达峰值。
(3)不同浓度0.1μg/ml、1μg/ml、10μg/ml catalpol预处理可以减轻H2O2诱导的细胞损伤,随着catalpol浓度的增加,相应的MTT检测的OD值也随之增加。
(4)H2O2组在H2O2作用后内皮及心肌细胞受到损伤,LDH较C组明显增高,损伤后MDA亦较C组明显增高,SOD较C组明显降低(均P<0.05)。Catalpol (0.1～10μg/ml)预处理培养细胞,在H202损伤过程中,均较H202组细胞损伤小,LDH和MDA含量明显减少,而SOD则较H2O2组明显增加。
(5)经H2O2处理24h后,细胞内荧光强度明显增强,用不同的浓度catalpol (0.1～10μg/ml)预处理24h后,与H2O2组相比细胞内荧光强度明显下降为,呈浓度依赖性。
(6)内皮细胞C组可见少许的TUNEL阳性细胞。与C组相比,细胞在H2O2(100μmol/L)中培养24h明显增加TUNEL阳性细胞数,然而,当用catalpol (0.1～10μg/ml)预处理24h后加入H2O2(100μmol／L)作用24h,凋亡率明显下降,且随着catalpol剂量增加凋亡率降低。
(7)正常的心肌细胞核呈圆形或椭圆形,染色均匀,呈正常的蓝色,而凋亡细胞的细胞核有明显的染色质边聚呈致密浓染,或呈碎块状致密浓染,变形亮度增强,颜色发白。Hoechst 33258结果显示：C组细胞少见白色亮点,H2O2组细胞见大量凋亡细胞。经不同浓度catalpol处理后,白色亮点呈逐渐减少趋势,凋亡细胞明显减低,呈剂量相关性。
(8) Annexin-FITC/PI检测内皮细胞凋亡率显示C组细胞的存活率为78.7±1.2%；而H2O2(100μmol／L)组细胞的凋亡率显著升高,凋亡率为35.9±0.6%,说明H2O2诱导了内皮细胞的凋亡。用不同浓度的catalpol (0.1、1、10μg/ml)预处理各组细胞后再施以H2O2作用,细胞的凋亡率出现不同程度的下降,而且随着catalpol浓度的升高,各组细胞的凋亡率也随之依次下降,细胞凋亡率分别降低至27.6±0.6%,22.6±0.8%和19.1±0.4%。
H202组心肌细胞凋亡率明显高于C组(P<0.01);与H2O2组比较,catalpol1、catalpol2、catalpol3组细胞凋亡率明显降低(P<0.01),且随着catalpol剂量增加凋亡率降低。
(9)Western blotting结果显示：H2O2作用24h后,Akt在各组中都有表达,差异无统计学意义(P>0.05), p-Akt, p-Bad, Bcl-2蛋白表达量较C组明显减少,Bax蛋白表达较C组明显增加,差异具有显著性(p<0.01)。catalpol 0.1,1, 10μg/ml可以剂量依赖性地增加p-Akt, p-Bad, Bcl-2蛋白表达,降低Bax蛋白的表达,与H2O2组比较差均有显著性(P＜0.05, P<0.01)。Wortmannin和LY294002预处理可拮抗catalpol部分保护作用。
免疫组化结果显示,在光镜下,心肌细胞阳性表达的p-Akt, Bcl-2, Bax蛋白主要积聚在胞浆中,呈棕色反应,各组蛋白表达强度不一致。积分光密度值检测结果显示,与C组比较,H2O2组细胞p-Akt, Bcl-2蛋白表达减弱(P<0.05,P<0.01),Bax蛋白表达显著增强(P<0.01)；与H2O2组比较,catalpol1、catalpol2、catalpol3组细胞的p-Akt, Bcl-2蛋白表达增高(P<0.01),Bax蛋白表达显著减弱(P<0.01)。Wortmannin和LY294002可拮抗catalpol部分保护作用。
(10) Real time RT-PCR结果显示：H2O2作用24h后,Akt, Bad, Bcl-2 mRNA表达量较C组明显减少,Bax mRNA表达较C组明显增加。Catalpol 0.1,1,10μg/ml可以剂量依赖性地增加.Akt, Bad, Bcl-2 mRNA表达,降低Bax mRNA的表达,与H2O2组比较差均有显著性(P＜0.05, P＜0.01)。Wortmannin和LY294002预处理可拮抗catalpol部分保护作用。
结论
(1) Catalpol对HUVECs、H9c2细胞存活率的影响具有时间和剂量依赖性。
(2) Catalpol通过降低MDA,增加SOD减轻H2O2诱导的细胞损伤。
(3) Catalpol可能通过清除细胞内过多的ROS来参与H2O2诱导细胞损伤的保护。
(4) PI3K/Akt-Bad途径可能为catalpol抗H2O2诱导细胞凋亡的重要信号转导机制。
(5) Catalpol还可能通过直接调节凋亡相关基因Bcl-2、Bax的表达而抑制细胞凋亡。
Introduction
Apoptosis plays a fundamental role in the development of numerous pathophysiological states. The balance between cell growth and apoptosis is likely to determine whether pathological phenomena such as hypertension, atherosclerosis and vascular disease occur. The excess generation of reactive oxygen species in the vascular system during the atherogenic process has been reported to enhance atherosclerotic lesion formation, resulting in the development of cardiovascular disease.
Hydrogen peroxide (H2O2), one of the most common reactive oxygen species, can easily penetrate the plasma membrane and cause lipid peroxidation and DNA damage in cells. H2O2 induces apoptosis by disrupting the cell's natural antioxidant defense system. It has been shown that H2O2 can stimulate cytochrome c release from the mitochondria, resulting in the loss of endothelial integrity and subsequent vascular disease.
Catalpol, a major chemical constituent of Rehmannia glutinosa Libosch, is an iridoid glucoside which exerts well known anti-apoptotic effects in neuronal cells and other cell lines. Findings from in vitro experiments have revealed that catalpol is able to activate the intracellular signal transduction pathway inducing neuronal differentiation and attenuate H2O2-induced apoptosis in PC 12 cells. The effects of catalpol on cardial oxidative stress have not been described. However a protective effect against oxidative stress damage of neurons in global and focal cerebral has been reported. Catalpol has also been shown to protect against oxidative stress in peripheral tissues such as the kidneys. Therefore, it seems reasonable to investigate whether catalpol is able to protect the cardiovascular from oxidative stress injury.
Akt is a critical component of the intracellular signaling pathway involved in regulating cell survival and apoptosis. Various growth and survival factors can activate this protein kinase. It has been reported that Akt can phosphorylate and inactivate Bad, thus inhibiting cell death. Among the Bcl-2 family, several members such as Bcl-2 and Bcl-xL induce cell survival, while other members such as Bad and Bax promote cell death. Further to this, it has been shown that members of the Bcl-2 family, which are located on the mitochondrial membrane, can alter mitochondrial membrane permeability and trigger apoptosis.
In the present study, we examined the effect of catalpol on H2O2-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and H9c2 cells. We also examined potential mechanisms underlying catalpol-associated protection, including reactive oxygen species scavenging, alterations in the phosphatidylinositol 3-kinase (PI3K)/Akt-Bad signaling pathway and changes in Bcl-2 and Bax expression.
Materials and methods
(1) We cultured HUVECs and H9c2 cells, and examined the effect of catalpol and H2O2 on cells. We also examined the effect of wortmannin and LY294002 on H2O2 incubated cells.
(2) The cell viablity of HUVECs and H9c2 was determined by MTT analysis.
(3) The concentration of malondialdehyde (MDA), lactate dehydrogenase (LDH) as well as the activity of superoxide dismutase (SOD) were were all determined by using commercially available kits.
(4) The level of intracellular reactive oxygen species was quantified by 2', 7'-dichlorofluorescein diacetate assay.
(5) Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and hoechst 33258 assay.
(6) Expression of Akt, p-Akt, p-Bad, Bcl-2 and Bax activity was quantified by western blotting analysis.
(7) Expression of Akt, Bad, Bcl-2 and Bax mRNA was determined by real-time semiquantitative reverse transcription-polymerase chain reaction method.
(8) Expression of p-Akt, Bcl-2 and Bax activity was detected by immunohistochemistry.
(9) All data are presented as mean±standard deviation (S.D.). Differences between mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA). The results were considered to be statistically significant when P＜0.05.
Results
(1) Incubation of HUVECs and H9c2 cells with different concentrations of catalpol (0.1,1,10μg/ml) for 48 h increased the viability of cells in a dose-dependent manner. The protective effect of catalpol was almost inhibited and cell viability reduced compared with the control group at a concentration of 1000μg/ml.
(2) Increased rates of cell survival were apparent 24 h after treatment. The magnitude of cell survival peaked at 72 h in HUVEC and at 48h in H9c2.
(3) Pre-incubation of cells with different concentrations of catalpol (0.1,1 or 10μg/ml) before H2O2 exposure increased viability in a dose-dependent manner.
(4) Compared with the control, treatment of cells with 100μM of H2O2 for 24 h caused significantly less activities of SOD. However, catalpol (0.1,1 or 10μg/ml) pretreatment significantly attenuated the changes of SOD activities in a dose dependent fashion, compared to the H2O2 group. In addition, cells treated with 100μM of H2O2 for 24 h caused more MDA and LDH levels, while pre-incubation of cells with catalpol (0.1,1 or 10μg/ml) markedly attenuated the increases compared to the H2O2 group.
(5) As expected, DCF fluorescence in cells exposed to H2O2 was strikingly increased compared to fluorescence in control group. Pre-treatment with catalpol (0.1, 1 or 10μg/ml) significantly inhibited DCF fluorescence caused by H2O2 exposure in a dose-dependent fashion.
(6) Few TUNEL-positive nuclei were evident in the control group. Exposure of cells to 100μM H2O2 for 24 h, however, resulted in a marked increase in the number of such nuclei. Pre-treatment with catalpol reduced the K2O2-induced increase in the number of cells with TUNEL-positive nuclei.
(7) In the control group were shown as round-shaped nuclei with homogenous fluorescence intensity. However, a marked increase of apoptotic cells which contained heterogeneous intensity, chromatin condensation, and fragmentation appeared after 24 h treatment with 100μM H2O2. Pre-treatment of catalpol completely protected cells from morphological changes by H2O2.
(8) Flow cytometry in HUVECs revealed that 78.7±1.2% of cells in the control group were viable. In contrast,35.9±0.6% of cells treated with 100μM H2O2 for 24 h were in early apoptosis or late apoptosis/necrosis. Pre-treatment of cells with catalpol (0.1,1 or 10μg/ml) for 24 h reduced the percentage of apoptotic cells associated with H2O2 exposure from 35.9±0.6% to 27.6±0.6%,22.6±0.8% and 19.1±0.4%, respectively.
Exposure of H9c2 cells to 100μM H2O2 for 24 h resulted in an increase in cellular apoptosis as revealed by flow cytometry. Pre-treatment of cells with catalpol (0.1,1 or 10μg/ml) for 24 h prior to H2O2 reduced the percentage of apoptotic cells from 14.30±0.41% to 8.97±0.36%,7.81±0.06% and 6.38±0.43%, respectively, in a concentration dependent manner.
(9) The protein phosphorylated Akt, Bad, Bcl-2 and Bax were analyzed by western blotting and immunohistochemistry. Catalpol (0.1,1 or 10μg/ml) induced Akt and Bad protein phosphorylation, increased Bcl-2, decreased Bax in a dose-dependent manner Both wortmannin and LY294002 markedly inhibited catalpol-induced Akt and Bad phosphorylation, increased Bax, decreased Bcl-2.
(10) We investigated the mRNA expression of Akt, Bad, Bcl-2 and Bax. Akt, Bad, Bcl-2 mRNA levels were significantly increased in catalpol (0.1,1 or 10μg/ml) pre-treated groups, compared to the control group. While H2O2 exposure significantly reduced Akt, Bad, Bcl-2 levels compared to control. Catalpol (0.1,1 or 10μg/ml) pre-treatment down-regulated Bax levels in cells exposed to H2O2. This effect was partially inhibited by concurrent wortmannin or LY294002 treatment.
Conclusions
(1) Pre-incubation of cells with 0.1,1 or 10μg/ml of catalpol resulted in a time-and dose-dependent manner variation of cell viability.
(2) Catalpol may protect cells from H2O2-induced injury by strengthened the changes of SOD activities and attenuated the levels of MDA.
(3) Scavenging of reactive oxygen species is involved in the mechanisms underlying the protective effect of catalpol against H2O2-induced injury.
(4) Catalpol may protect cells from H2O2-induced apoptosis at least part by activating the PI3K/Akt-Bad signaling pathway.
(5) Bcl-2 and Bax are involved in mediating the anti-apoptotic effects associated with catalpol treatment in HUVECs and H9c2 cells exposed to H2O2.
引文
1 Shigematsu S, Arita M. Anoxia depresses sodium-calcium exchange currents in guinea-pig ventricular myocytes. J Mol Cell Cardiol.1999; 31:895-906.
2 Wattanapitayakul SK, Bauer JA. Oxidative pathways in cardiovascular disease:roles, mechanisms, and therapeutic implications. Pharmacol Ther. 2001; 89:187-206.
3 Hinata M, Matsuoka I, Iwamoto T, et al. Mechanism of Na+/Ca2+ Exchanger Activation by Hydrogen Peroxide in Guinea-Pig Ventricular Myocytes. J Pharmacol Sci.2007; 103:283-292.
4 Hamilton KL. Antioxidants and cardioprotection. Med SciSports Exerc.2007; 39:1544-1553.
5 Nie R, Xia R, Zhong X, et al. Salvia miltiorrhiza treatment during early reperfusion reduced postischemic myocardial injury in the rat. Can J Physiol Pharmacol.2007; 85:1012-1019.
6 Ikizler M, Erkasap N, Dernek S, et al. Dietary polyphenol quercetin protects rat hearts during reperfusion:enhanced antioxidant capacity with chronic treatment. Anadolu Kardiyol Derg. 2007; 7:404-410.
7 王亮,戴振华,孙伟,等.缬沙坦对乳鼠心肌细胞过氧化损伤的保护作用.南京医科大学学报.2007；27：948-951.
8 郑延松,李源,张珊红.用低浓度过氧化氢建立心肌细胞氧化损伤模型.第四军医大学学报.2001：22：1849-1851.
9 于震,周红艳,王军.地黄药理作用研究进展.中医研究.2001；14：43-45.
10 李献平,刘敏,刘世昌,等.四大怀药延缓衰老作用的研究.中西医结合杂志.1991；11：486-488.
11 袁媛.怀地黄补血作用的实验研究.中国中药杂志.1992；17：366-369.
12 陈力真,冯杏婉,周金黄,等.地黄多糖b的免疫抑瘤作用及其机理.中国药理学与毒理学杂志.1993；7：153-156.
13 陈力真,冯杏婉,周金黄.地黄多糖b对正常及5180荷瘤小鼠T淋巴细胞功能的影响.中国药理学与毒理学杂志.1994；8：125-127.
14 刘副军,程军平,赵修南,等.地黄多糖对正常小鼠造血干细胞、祖细胞及外周血象的影响.中药药理与临床.1996；2：12-14.
15 久保道德.不同炮制方法与地黄成分的变化及血液流变学改善作用的关系.药学杂志(日).1996：116：155-168.
16 张汝学,顾国明,张永祥,等.地黄低聚糖对实验性糖尿病与高血糖大鼠糖代谢的调节作用.中药药理与临床.1996;1：14-17.
17 曹凯,刘永平,李素婷,等.熟地、菊花、山药、牛膝等四大怀药对小鼠脑线粒体单胺氧化酶活力的影响.中国老年学杂志.1998;18：102-103.
18 苗明三.(怀)地黄多糖对衰老模型小鼠免疫器宫的影响.河南中医.1999；19：30-33.
19 Kim HM, An CS, Jung KY, et al. Rehmannia glutinosa inhibits tumor necrosis factora and interleukin-1 secretion from mouse astrocytes. Pharmacol Res.1999; 40:171-176.
20 Zhang R, Zhou J, Jia Z, et al. Hypoglycemic effect of Rehmannia glutinosa oligosaccharide in hyperglycemic and alloxan-induced diabetic rats and its mechanism. J Ethnopharmacol.2004; 90:39-43.
21 曾艳,贾正平,张汝学,等.地黄寡糖在2型糖尿病大鼠模型上的降血糖作用及机制.中国药理学通报.2006；22：40-42.
22 李更生,于震,王慧森.地黄化学成分与药理研究进展.国外医学中药分册.2004；26：74-78.
23 Jiang B, Liu JH, Bao YM, et al. Catalpol inhibits apoptosis in hydrogen peroxide induced PC 12 cells by preventing cytochrome c release and inactivating of caspase cascade. Toxicon. 2004; 43:53-59.
24 Li DQ, Bao YM, Li Y, et al. Catalpol modulates the expressions of Bcl-2 and Bax and attenuates apoptosis in gerbils after ischemic injury. Brain Res.2006; 1115:179-185.
25 Li DQ, Duan YL, Bao YM, et al. Neuroprotection of catalpol in transient global ischemia in gerbils. Neurosci Res.2004; 50:169-177.
26 Li DQ, Li Y, Liu Y, et al. Catalpol prevents the loss of CA1 hippocampal neurons and reduces working errors in gerbils after ischemia-reperfusion injury. Toxicon.2005; 46:845-851.
27 lsmailoglu UB, Saracoglu I, Harput US, et al. Efects of phenylpropanoid and iridoid glycosides on free radical-induced impairment of endothelium-dependent relaxation in rat aortic rings. J Ethnopharmacol.2002; 79:193-197.
28 OrtizdeUrbina AV, Martin ML, et al. In vitro antispasmodic activity of peracetylated penstemonoside, aucubin and catalpol. Planta Med.1994; 60:512-515.
29 司徒镇强,吴军正.四唑盐比色法(MTT法).细胞培养.西安：世界图书出版公司西安公司.2001;56-57.
30 GrayJ. Measurement of lipid oxidation:a review. J Am oil Chem Soc.1978; 55:539-545.
31 Niwa K, Inanami O, Yamamori T, et al. Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide. Antioxid Redox Signal.2003; 5: 713-722.
32 任德成,杜冠华.活性氧对蛋白激酶和基因表达调节作用的研究进展.功国药理学通舰.2003；19：489-492.
33 Qin HT, Chun W, Feng XY, et al. MAPK pathway mediates the protective effects of onychin on oxidative stress-induecd apoptosis in ECV304 endothelial cells. Life Sciences.2004; 76: 487-497.
34 Stemien-Oterp A, Karsan A, Cornejo CJ, et al. Mechanisma of hypoxia-induced endothelial cell death. J Biol Chem.1999; 274:8039-8051.
35 Dinuneler S, Haendeler J, Galle J, et al. Oxidized low-density lipoprotein induces apoptosis of human ECs by activation of CPP32-like proteases. Circulation.1997; 95:1760-1763.
36 Laude K, Thuillez C, Richard V. Coronary endothelial dysfunction after ischemia and reperfusion:a new therapeautic target? Brazzz J Med Res.2001; 34:1-7.
37 Bombeli T, Schwartz BR, Harlan JM. Endothelial cells undergoing apoptosis become proadhesive for nonactivated platelets. Blood.1999; 93:3831-3838.
38 Bombeli T, Karsan A, Tait JF, et al. Apoptotic vascular endothelial cells become procoagulant. Blood.1997; 89:2429-2442.
39 Vogt CJ, Schmid-Schonbein GM. Microvascular endothelial cell death and rarefaction in the glucocorticoid-induced hypertensive rat. Microcirculation.2001; 8:129-139.
40 Zou MH, Shi C, Cohen RA. High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H(2) receptor-mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells. Diabetes.2002; 51:198-203.
41 Holleyman C, Larson D, Hunter K. Stimulation of ischemia reperfusion in endothelial cell culture increase apoptosis. J Extra Corpor Technol.2001; 33:175-180.
42 徐慧,郑杨,佟倩.卡托普利晚期预处理对人内皮细胞缺氧复氧损伤的保护作用机制.中国动脉硬化杂志.2004;3：291-295.
43 Zhang J, Tan Z, Tran ND. Chemical hypoxia-ischemia induces apoptosis in cerebromicrovascular endothelial cell. Brain.Res.2000; 877:134-140.
44 Hong N, Browning J, Howard T, et al. Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and transforming growth factorbeta. Endothelium.1999; 7: 175-180.
45 Brutsaert DL. Cardiac endothelial myocardial signaling:its role in cardiac growth, contractkle performance.and rhythmicty. Physiological Reviews.2003,83:59-115.
46 Saraste A, Puikki K, Kallajoki M, et al. Apoptosis in human acute myocardial infarction. Circulation.1997; 95:320-323.
47 Jiang BH, Zheng JZ, Aoki M. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci USA.2000; 97:1749-1753.
48 Vanthaesebroeck B, Alessi DR, The PI3K-PDK1 connection:more than just a road to PKB. Biochem J.2000; 346:561-576.
49 Long X, Boluyt MO, Hipolito ML, et al. Crow MT.P53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invest.1997; 99:2635-2643.
50 Kirshenbaum LA, de Moissac D. The bcl-2 gene product prevents programmed cell death of ventricular myocytes. Circulation.1997; 96:1580-1585.
51 Jacobson MD, Burme JF, King MP, et al. Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature.1993; 361:365-369.
52 Shawm C, Huang JQ, Rezaiefar P, et al. Co-localization of the cysteine caspase-3 with apoptotic myoctyes after in vivo myocardial ischemia and reperfusion in the rat. J Mol Cell Cardiol.1998; 30:733-742.
53 Nunez G, Benedict MA, Hu Y, et al. Caspases:the proteases of the apoptotic pathway. Oneogene.1998; 17:3237-3245.
54 Brennere, Cadiou H, Vieria HL. Bel-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oneogene.2000; 19:329-336.
55 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods.2001; 25:402-408.
56 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc.2008; 3:1101-1108.
57 Savill J. Apoptosis:a mechanism for regulation of the cell complement of inflamed glomeruli. kidney Int.1992; 41:607-612.
58 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragment. Cell Biol.1992; 119:493-501.
59 彭黎明.六种细胞凋亡检测方法的比较.中华病理学杂志.1999;28：55-57.
60 邹伟,李兆育,李春雷,等.蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用.生理科学进展.2000；31：120-124.
61 Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem.1998; 67: 481-507.
62 Jones PF, Jakubowicz T, Pitossi FJ, et al. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci USA. 1991; 88:41714175.
63 Bellacosa A, Testa JR, Staal SP, et al. Aretroviral oncogene, Akt, encoding a serine-threonine kinase containing an SH2-like region. Science.1991; 254:274-277.
64 Coffer PJ, Woodgett JR. Molecular cloning and characterization of a novel putative protein-Serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem.1991; 201:475-481.
65 Datta K, Bellacosa A, Chan TO, et al. Akt is a grant a direct target of the phosphatidylinositol 3-kinase. Activation by growth factors, v-src and v-Ha-Ras in Sf9 and mammalian cells. J Biol Chem.1996; 271:30835-30839.
66 Franke TF, Hornik CP, segev L, et al. PI3K/Akt and apoptosis:size matters. Oncogene.2003; 22:8983-8998.
67 Fresno Vara JA, Casado E, Castro J, et al. PI3K/Akt signaling pathway and cancer. Cancer Treat Rev.2004; 30:193-204.
68 Jiang X, Wang X. Cytochrome C-mediated apoptosis. Annu Rev Biochem.2004; 73:87-106.
69 Downwrd J. PI3-kinase, Akt and cell survival. Sem in Cell Dev Biol.2004; 15:177-182.
70 Diehl JA, Cheng M, Roussel MF, et al. Glycogen synthase kinase-3 beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev.1998; 12:3499-3511.
71 Nusse R. Wnts and Hedgehogs:lipid-modified proteins and similarities in signalind mechanisms at the cell surface. Development.2003; 130:5297-5305.
72 Zong WX, EdelsteinL C, Chen C, et al. The prosurvival Bcl-2 homolog Bfl-1/Al is a direct transcriptional target of NF-KB that blocks TNF alpha induced apoptosis. Genes Dev.1999; 13: 382-387.
73 Strasser A, Harris AW, Jacks T, et al. DNA damage can induce apoptosis in proliferating lymphoid cells via p53 independent mechanisms inhibitable by Bcl-2. Cell.1994; 79:329-39.
74 Yin XM, Oltvai ZN, Korsmeyer S J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature.1994; 369:321-323.
75 Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell.1993; 74:609-619.
76 Yang E, Zha J, Jockel J, et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell.1995; 80:285-291.
77 Harris MH, Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membre permeability. Cell Death Dir.2000; 71:1182-1191.
78 Cosulich SC, Savory PJ, Clarke PR. Bcl-2 regulates amplification of caspase activation by cytochrome C. Curr Biol.1999; 9:147-150.
79 Bossy-Wetzel E, Green DR. Caspases induce cytochrome C release from mitochondria by activating cytosolic factors. J Biol Chem.1999; 274:17484-17490.
80 Sutton VR, Dvais JE, Cancilla M, et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B mediated caspase activation. J Exp Med.2000; 192: 1403-1414.
81 Stoka V, Turk B, Schendel SL, et al. Lysosomal portease path-ways to apoptosis. Clevagae of bid, not Pro-caspases, is the most likely way. J Biol Chem.2001,276:3149-3157.
82 Chan PH. Reactive oxygen radicals in singaling and damage in the ischemic barin. J Cereb. Blood Flow Metba.2001; 21:2-14.
1 Gottlieb RA, Burleson KO, Kloner RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest.1994; 94:1621-1628.
2 姚震,焦解歌,冯建章.心肌缺血再灌注损伤与细胞凋亡关系的实验研究.海南医学院学报.2000：6：129-133.
3 穆军升.NO与心血管疾病细胞凋亡.国外医学生理病理科学与临床分册.2001；21：389-391.
4 Chakrabarti S, Hoque AN, Karrnazyn M. A rapid ischemia induced apoptosis in isolated rat hearts and its attenuation by the sodium hydrogen exchang inhibitor HOE 642 (cariporide). J Mol Cell Cardiol.1997; 29:3169-3174.
5 马桂兰.细胞凋亡与冠心病.医学综述.2000;6：491492.
6 Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest.1996; 74:86-107.
7 Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res.1996; 79:949-956.
8 Gottlieb RA, Burleson KO, Khmer RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. Clin Invest.1994; 94:1621-1628.
9 Zhao ZQ, Nakamura M, Wang NP, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion. Apoptosis.2001; 6:279-290.
10 Ling H, Lou Y. Total flavones from Elshohzia blanda reduce infarct size during acute myocardial ischemia by inhibiting myocardial apoptosis in rats. Ethno pharmacol.2005; 101: 169-175.
11 赵卫红,寿好长,阎福岭,等.细胞凋亡的生物学特征.郑州：河南医科大学出版社.1997；7-16.
12 Ferrer L, Friguls B, Dalof E, et al. Caspase-dependent and caspase-in-dependent signalling of apoptosis in the penumbrao fllowing middlecerebral artery occlusion in the adult rat. Neuorpathol Appl Neurobiol.2003; 29:472-481.
13 Okamura T, Miura T, Takemura G. Efect of caspase inhibitors on myocardial in farct size and myocyte DNA fragmentation in the ischemia repefusedr at heart. Cardiovasc Res.2000; 45: 642-650.
14 Bialik S, Cryns VL, Drineie A. The mitochondrial apoptotie pathway is activated by serum and glucose deprivation in cardiacmyocytes. Circ Res.1999; 85:403-415.
15 Saikmuar P, Dong Z, Mikhailov V. Apopotsis:definition mechanismand relevence to disese. Am J Med.1999; 107:489-506.
16 Harris MH, Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membre permeability. Cell Death Dir.2000; 71:1182-1191.
17 Cosulich SC, Savory PJ. Clarke PR. Bcl-2 regulates amplification of caspase activation by cytochrome C. Curr Biol.1999; 9:147-150.
18 Bossy-Wetzel E, Green DR. Caspases induce cytochrome C release from mitochondria by activating cytosolic factors. J Biol Chem.1999; 274:1740-1749.
19 Sutton VR, Dvais JE, Cancilla M, et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B mediated caspase activation. J Exp Med.2000; 192: 1403-1414.
20 Stoka V, Turk B, Schendel SL, et al. Lysosomal portease path-ways to apoptosis:Clevagae of bid, not procaspases, is the most likely way. J Biol Chem.2001; 276:3149-3157.
21 Madeddu F, Naska S, Bozzi Y. BDNF down-regulates the caspase-3 pathway in injured geniculo-cortical neurones. Neuroreport.2004; 15:2045-2049.
22 Zhang WR, Kitagawa H, Hayashi T, et al. Topical applification of neurotrophin-3 attenuates isehemie brain injuy after transient middle cerebarl artey occlusion in rats. Brain Res.1999; 842:211-214.
23 Katoh S, MitsuiY, Kitnai K, et al. The rescuing effect of nevre growth factor is the result of up-regulation of bcl-2 in hyperoxia-induced apoptosis of a subclone of pheochomocytoma cells PC12h. Neurosci Lett.1997; 232:71-74.
24 Kiprianova I, Freimna TM, Desiderato S, et al. Brain-derived neurotrophic factor prevents neuronal death and glial activation after global isehemia in the rat. J Neurosci Res.1999; 56: 21-27.
25 Cao W, Carney JM, Duchon A, et al. Oxygen free radical involvement ischemia and reperfusion injury to brain. Neurosci. Lett.1988; 88:233-238.
26 Facchinetti F, Dawson VL, Dwason TM. Free radical as mediators of neuronal injury. Cell Mol Neurobiol.1998; 18:667-682.
27 Chan PH. Reactive oxygen radicals in singaling and damage in the ischemic barin. J Cereb. Blood Flow Metba.2001; 21:2-14.
28 Fujio Y, Nguyen T, Wencker D. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation.2000; 101:660-667.
29 Yodli R, Tomms F. Effect of multiple phosphorylation events on the trailscrip on factor FKHR, FKHRL1 and AFX.Biochem.Soc. Frmm.2002; 30:391-397.
30 Kajstura J, Fiordaliso F, Andreoli AM, et al. IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotension Ⅱ-mediated oxide stress. Diabetes.2001; 50: 1414-1424.
31 Matsui T, Tao J, del Monte F, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation.2001; 104:330-335.
32 Kovacs P, Bak Ⅰ, Szemdrei L, et al. Non-specific caspase inhibition reduces infarct size and improve post-ischemic recovery in isolated ischemial reperfusion rat hearts. Naunyn Schmiedebergs Arch Pharmocal.2001; 364:501-507.
33 Anwar A, Zahid AA, Schiidegger KJ, et al. Tumor necrosis factor-alpha results IGF-1 and IGFBP-3 expression in vascular smooth musle. Circulation.2002; 105:1220-1225.
34 Levine AI. P53:the cellular gatekeeper for growth and division. Cell.1997; 88:323-331.
35 Vousden KH. Activation of the p53 tumor suppressor protein. J Biochim Biophys Acta.2002; 1602:47-59.
36 Bargonetti J, Manfredi J. Multiple roles of the tumor suppressor p53. J Curr Opin Oncol.2002; 14:86-91.
37 Vousden KH, Lu X. Live or let die:the cells response to p53. Nat Rev Cancer.2002; 2: 594-604.
38 Regula KM, Kirshenbaum LA. p53 activates the mitochondrial death pathway and apoptosis of ventricular myocytes independent of de novo gene transcription. J Mol Cell Cardiol.2001; 33: 1435-1445.
39 Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. J Mol Cell.2003; 11:577-590.
40 Zhao ZQ, Velez DA, Wang NP, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion. Apoptosis.2001; 6:279-290.
41 Maed K, Hata R, Gillardon F, et al. Aggravation of brain injuru atfer transient focal ischemia in P53-deficient mice. J Mol Brain Res.2001; 889:54-61.
42 Mc Ghana L, Hakim AM, Robertson GS. Hippcampal Myc and p53 expression following transient global isehemia. Brain Res Mol Brain Res.1998; 56:133-145.
43 Maulik N, Sasaki H, Addya S. et al. Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors. FEBS Lett.2000; 485:7-12.
44 Culmsee C, Zhu X, Yu QS, et al. A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid beta-peptide. J Neurochem.2001; 77: 220-228.
45 Tomasevic G, Shamloo M, Israeli D, et al. Activation of p53 and its target genes p21(WAF1/Cipl) and PAG608/Wig-1 in ischemic preconditioning. Mol Brain Res.1999; 70: 304-313.
46 Long x, Bolhyt MO, Hipolito M1, et al. r63 and the hypocia-induced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invesr.1997; 99:2635-2643.
47 谢红光.受体与细胞信号转导.见：刘正湘主编.实用心血管受体学,北京：科学出版社,2001；28-32.
48 赵正航,臧伟进,于晓红.腺苷对模拟缺血再灌注豚鼠心室肌细胞动作电位的影响及其离子机制.中国药理学通报.2003;19：274-278.
49 刘睿编译.调节细胞凋亡的信号传递机制.国外医学免疫学分册.1995;18：270-272.
50 Arenzana-Seisdedos F, Turpin P, Rodriguez M, el al. Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci.1997; 110:369-378.
51 Li C, Browder W, Kao RI. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol.1997; 276:543-552.
52 陈冬梅,汪海.血管内皮细胞功能与心血管疾病相关因子研究进展.中国药理学通报.2003：19：361-365.
53 Sawa Y, Morishita R, Suzaki K, et al. A novel strategy for myocardial protection using in vivo transfection of cis element decoyagainst NF-kB binding site. Circulation.1997; 96:280-285.
54 Squadrito F, Ahavilla D, Squadrito G, el al. Tacrolimus limits polymorphonuclear leucocyte accumulation and protects against myocardial ischaemia-reperfusion injury. J Mol Cell Cardiol. 2000; 32:429-440.
55 唐旭东,姜建春,姜大春.三七总皂甘对心肌缺血／再灌注中中性粒细胞核因子-κB活化及其粘附的影响.中国药理学通报.2002；18：556-560.
56 Maulik N, Galang N. Differential regulation of Bel-2, AP-1 and NF-kappaB on cardiomyocyte apoptosls during myccardial ischemic stress adaptation. FEBS Lett.1999; 443:331-336.
57 Kirahenbaum LA. Bcl-2 intersects the NF-kappaB signaling pathway and suppresses apoptosis in ventricular myocytes. Clin invest Med.2000; 22:322-330.
58 Ashkenazi A, Dixit VM. Death receptor:signaling and modulation. Seience.1998; 281: 1305-1308.
59 Yuna J, Yankner BA. Apoptosis in the nevrous system. Nature.2000; 407:802-809.
60 Hatano E, Bradham CA, Stark A, et al. The mitochondrial pemreabiliy transition augments Fas-induced apoptosis in mouse heaptoyctes. J Biol Chem.2000; 275:11814-11823.
61 Baud V, Karin M. Signal transduction by tumor necorsis and its relatives. Trends Cell Biol. 2001; 11:372-377.
62 Jiang X, Wang X. Cytochrome c pormotes caspase-9 activation inducing nucleo-tide binding to Apaf-1. J Biol Chem.2000; 275:31199-31203.
63 Wang X. The expanding role of mitochondria in apoptosis. Genes Dev.2001; 15:2922-2933.
64 Nakgawa T, Zhu H, Morishima N, et al. Caspase-12 mediates endoplasmic reticulum specific apoptosis and cytotoxicity by amyloid-beta. Nature.2000; 403:98-103.
65 Michell RH. Evolution of the diverse biological roles of inositols. Biochemical Society Symposium.2007; 74:223-246.
66 DiPaolo G, DeCamilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature. 2006; 443:651-657.
67 Berridge MJ. Unlocking the secrets of cell signaling. Annual Review of Physiology.2005; 67: 1-21.
68 Vanhaesebroeck B, Leevers SJ, Ahmadi K, et al. Synthesis and function of 3-phosphorylated inositol lipids. Annual Review of Biochemistry.2001; 70:535-602.
69 Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem.1998; 67: 481-507.
70 Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kina-ses as regulators of growth and metabolism. Nature Reviews. Genetics.2006; 7:606-619.
71 Maffucci T, Brancaccio A, Piccolo E, et al. Insulin induces phosphate-dylinositol-3-phosphate formation through TC10 activation. EMBO Journal.2003; 22:4178-4189.
72 Lindmo K, Stenmark H. Regulation of membrane traffic by phosphoinositide 3-kinases. J Cell Sci.2006; 119:605-614.
73 Hirsch E, Lembo G, Montrucchio G, et al. Signaling through PI3Kgamma:a common platform for leukocyte, platelet and cardiovascular stress sensing. Thromb Haemost.2006; 95:29-35.
74 Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci.2001; 114: 1439-1445.
75 Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection:more than just a road to PKB Biochem J.2000; 346:561-576.
76 Vanhaesebroeck B, Leevers SJ, Ahmadi K, et al. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem.2001; 70:535-602.
77 McMuUen JR, Jennings GL. Diferences between pathological and physiological cardiac hypertrophy:novel therapeutic strategies to treat heart failure. Clin Exp Pharmacol Physiol. 2007; 34:255-262.
78 Shiojima I, Walsh K. Regulation of cardiac growth and coronary angiogenesis by the Akt/ PKB signaling pathway. Genes Dev.2006; 20:3347-3365
79 Muslin AJ, DeBosch B. Role of Akt in cardiac growth and metabolism. Nova rtis Found Symp. 2006; 274:118-126.
80 DeBosch B, Treskov I, Lupu TS, et al. Aktl is required for physiological cardiac growth. Circulation.2006; 113:2097-2104.
81 McMulhn JR, Shioi T, Zhang L, et al. Deletion of ribosomal S6 kinases does not attenuate pathological, physiolocal or insulin-like growth factor 1 receptor-phosphoinositide 3-kinase-induced cardiac hypertrophy. Mol Cell Bid.2004; 24:6231-6240.
82 Armstrong SC. Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res.2004; 61:427-436.
83 Chiari PC, Bienengraeber MW, Pagel PS, et al. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction:evidence for anesthetic-induced postcond-itioning in rabbits. Anesthesiology. 2005; 102:102-109.
84 Yellon DM, Downey JM. Preconditioning the myocardium:from cellular physiology to clinical cardiology. Physiol Rev.2003; 83:1113-1151.
85 Burgering BM, Medema RH. Decisions on life and death:FOXO Forkhead transcription factors are in command when PKB/Akt is off duty. J Leukoc Biol.2003; 73:689-701.
86 Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell.1999; 96:857-868.
87 Rena G, Guo S, Cichy SC, et al. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem 1999; 274:17179-17183.
88 Cantley LC. The phosphoinositide 3-kinases pathway. Science.2002; 296:1655-1657.
89 Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell.1997; 91:231-241.
90 Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-Kinase/Akt cell survival pathway. J Biol Chem.1998; 273:19929-19932.
91 Khoynezhad A, Jalali Z, Tortolani AJ. Apoptosis:pathophysiology and therapeutic implications for the cardiac surgeon. Ann Thorac Surg.2004; 78:1109-1118
92 Moolman JA, Hartley S, Van Wyk J. et al. Inhibition of myocardial apoptosis by ischaemic and beta-adrenergic preconditioning is dependent on p38 MAPK. Cardi ovasc Drugs Ther. 2006; 20:13-25.
93 Tissier R, Waintraub X, Couvreur N. Pharmacological postconditioning with the phytoestrogen genistein. J Mol Cell Cardiol.2007; 42:79-87.
94 Burger D, Xiang F, Hammoud L, et al. Role of Heme Oxygenase-1 in the cardioprotective effects of erythropoietin during myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol.2009; 296:H84-93.
95 Gao JP, Chua CC, Chen ZY. Resistin, an adipocytokine, offers protection against acute myocardial infarction. J Mol Cell Cardiol.2007; 43:601-609.
96 Ghibu S, Lauzier B, Delemasure S, et al. Antioxidant properties of alpha-lipoic acid:effects on red blood membrane permeability and adaptation of isolated rat heart to reversible ischemia. Mol Cell Biochem.2009; 320:141-148.
97 Blokhin IO, Vlasov TD, Galagudza MM, et al. Role of sodium-calcium exchanger in the myocardial protection against ischemia-reperfusion injury. Ross Fiziol Zh Im I M Sechenova. 2008; 94:284-292.
98 Zhang JH, Chen ZW, Wu Z. Late protedtive effect of pharmacological precongitioning with total flavones of rhododendra against myocardial ischemia-reperfusion injury. Can J Physiol Pharmacol.2008; 86:131-138.
99 Fuglesteg BN, Suleman N, Tiron C, et al. Signal transducer and activator of transcription 3 is involved in the cardioprotective signaling pathway activated bu insulin therapy at reperfusion. Basic Res Cardiol.2008; 103:444-453.
100 Lu X, Hamilton JA, Shen J, et al. Role of tumor necrosis factor-alpha in myocardial dysfunction and apoptosis during hindlimb ischemia and reperfusion. Crit Care Med.2006; 34: 484-491.
101 Das A, Xi L, Kukreja RC. Phosphodiestprase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis, Essential role of nitric oxide signaling. J Bioi Chem. 2005；280：12944-12955.
102 陈锐群,卢存寿,张丽丽,等.地黄的研究.阴健和郭力弓主编.中药现代研究与临床应用.北京：学苑出版社.1994；272-279.
103 都恒青,郭湘云,赵曦,等.地黄的研究.楼之岑和秦波主编.常用中药材品种整理和质量研究.北方编第二册.北京：北京医科大学出版社.1995；839-841.
104 Isao K, Masayuki Y. Chemical constituents of rehmanniae radix, cornifructus, dioscoreae rhizoma. Modern Eastern Medicine.1986; 7:55-62.
105 中华人民共和国药典2000年版一部.北京：化学工业出版社.2000；94.
106 Jiang B, Liu JH, Bao YM, et al. Catalpol inhibits apoptosis in hydrogen peroxide induced PC 12 cells by preventing cytochrome c release and inactivating of caspase cascade. Toxicon. 2004; 43:53-59.
107 Li DQ, Bao YM, Li Y, et al. Catalpol modulates the expressions of Bcl-2 and Bax and attenuates apoptosis in gerbils after ischemic injury. Brain Res.2006; 1115:179-185.
108 Li DQ, Duan YL, Bao YM, et al. Neuroprotection of catalpol in transient global ischemia in gerbils. Neurosci Res.2004; 50:169-177.
109 Li DQ, Li Y, Liu Y, et al. Catalpol prevents the loss of CA1 hippocampal neurons and reduces working errors in gerbils after ischemia-reperfusion injury. Toxicon.2005; 46:845-851.
110 Hu LA, Sun YK, Hu J. Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax. Eur. J. Pharmacol.2010; 628:155-163.
2 Wattanapitayakul SK, Bauer JA. Oxidative pathways in cardiovascular disease:roles, mechanisms, and therapeutic implications. Pharmacol Ther. 2001; 89:187-206.
3 Hinata M, Matsuoka I, Iwamoto T, et al. Mechanism of Na+/Ca2+ Exchanger Activation by Hydrogen Peroxide in Guinea-Pig Ventricular Myocytes. J Pharmacol Sci.2007; 103:283-292.
4 Hamilton KL. Antioxidants and cardioprotection. Med SciSports Exerc.2007; 39:1544-1553.
5 Nie R, Xia R, Zhong X, et al. Salvia miltiorrhiza treatment during early reperfusion reduced postischemic myocardial injury in the rat. Can J Physiol Pharmacol.2007; 85:1012-1019.
6 Ikizler M, Erkasap N, Dernek S, et al. Dietary polyphenol quercetin protects rat hearts during reperfusion:enhanced antioxidant capacity with chronic treatment. Anadolu Kardiyol Derg. 2007; 7:404-410.
7 王亮,戴振华,孙伟,等.缬沙坦对乳鼠心肌细胞过氧化损伤的保护作用.南京医科大学学报.2007；27：948-951.
8 郑延松,李源,张珊红.用低浓度过氧化氢建立心肌细胞氧化损伤模型.第四军医大学学报.2001：22：1849-1851.
9 于震,周红艳,王军.地黄药理作用研究进展.中医研究.2001；14：43-45.
10 李献平,刘敏,刘世昌,等.四大怀药延缓衰老作用的研究.中西医结合杂志.1991；11：486-488.
11 袁媛.怀地黄补血作用的实验研究.中国中药杂志.1992；17：366-369.
12 陈力真,冯杏婉,周金黄,等.地黄多糖b的免疫抑瘤作用及其机理.中国药理学与毒理学杂志.1993；7：153-156.
13 陈力真,冯杏婉,周金黄.地黄多糖b对正常及5180荷瘤小鼠T淋巴细胞功能的影响.中国药理学与毒理学杂志.1994；8：125-127.
14 刘副军,程军平,赵修南,等.地黄多糖对正常小鼠造血干细胞、祖细胞及外周血象的影响.中药药理与临床.1996；2：12-14.
15 久保道德.不同炮制方法与地黄成分的变化及血液流变学改善作用的关系.药学杂志(日).1996：116：155-168.
16 张汝学,顾国明,张永祥,等.地黄低聚糖对实验性糖尿病与高血糖大鼠糖代谢的调节作用.中药药理与临床.1996;1：14-17.
17 曹凯,刘永平,李素婷,等.熟地、菊花、山药、牛膝等四大怀药对小鼠脑线粒体单胺氧化酶活力的影响.中国老年学杂志.1998;18：102-103.
18 苗明三.(怀)地黄多糖对衰老模型小鼠免疫器宫的影响.河南中医.1999；19：30-33.
19 Kim HM, An CS, Jung KY, et al. Rehmannia glutinosa inhibits tumor necrosis factora and interleukin-1 secretion from mouse astrocytes. Pharmacol Res.1999; 40:171-176.
20 Zhang R, Zhou J, Jia Z, et al. Hypoglycemic effect of Rehmannia glutinosa oligosaccharide in hyperglycemic and alloxan-induced diabetic rats and its mechanism. J Ethnopharmacol.2004; 90:39-43.
21 曾艳,贾正平,张汝学,等.地黄寡糖在2型糖尿病大鼠模型上的降血糖作用及机制.中国药理学通报.2006；22：40-42.
22 李更生,于震,王慧森.地黄化学成分与药理研究进展.国外医学中药分册.2004；26：74-78.
23 Jiang B, Liu JH, Bao YM, et al. Catalpol inhibits apoptosis in hydrogen peroxide induced PC 12 cells by preventing cytochrome c release and inactivating of caspase cascade. Toxicon. 2004; 43:53-59.
24 Li DQ, Bao YM, Li Y, et al. Catalpol modulates the expressions of Bcl-2 and Bax and attenuates apoptosis in gerbils after ischemic injury. Brain Res.2006; 1115:179-185.
25 Li DQ, Duan YL, Bao YM, et al. Neuroprotection of catalpol in transient global ischemia in gerbils. Neurosci Res.2004; 50:169-177.
26 Li DQ, Li Y, Liu Y, et al. Catalpol prevents the loss of CA1 hippocampal neurons and reduces working errors in gerbils after ischemia-reperfusion injury. Toxicon.2005; 46:845-851.
27 lsmailoglu UB, Saracoglu I, Harput US, et al. Efects of phenylpropanoid and iridoid glycosides on free radical-induced impairment of endothelium-dependent relaxation in rat aortic rings. J Ethnopharmacol.2002; 79:193-197.
28 OrtizdeUrbina AV, Martin ML, et al. In vitro antispasmodic activity of peracetylated penstemonoside, aucubin and catalpol. Planta Med.1994; 60:512-515.
29 司徒镇强,吴军正.四唑盐比色法(MTT法).细胞培养.西安：世界图书出版公司西安公司.2001;56-57.
30 GrayJ. Measurement of lipid oxidation:a review. J Am oil Chem Soc.1978; 55:539-545.
31 Niwa K, Inanami O, Yamamori T, et al. Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide. Antioxid Redox Signal.2003; 5: 713-722.
32 任德成,杜冠华.活性氧对蛋白激酶和基因表达调节作用的研究进展.功国药理学通舰.2003；19：489-492.
33 Qin HT, Chun W, Feng XY, et al. MAPK pathway mediates the protective effects of onychin on oxidative stress-induecd apoptosis in ECV304 endothelial cells. Life Sciences.2004; 76: 487-497.
34 Stemien-Oterp A, Karsan A, Cornejo CJ, et al. Mechanisma of hypoxia-induced endothelial cell death. J Biol Chem.1999; 274:8039-8051.
35 Dinuneler S, Haendeler J, Galle J, et al. Oxidized low-density lipoprotein induces apoptosis of human ECs by activation of CPP32-like proteases. Circulation.1997; 95:1760-1763.
36 Laude K, Thuillez C, Richard V. Coronary endothelial dysfunction after ischemia and reperfusion:a new therapeautic target? Brazzz J Med Res.2001; 34:1-7.
37 Bombeli T, Schwartz BR, Harlan JM. Endothelial cells undergoing apoptosis become proadhesive for nonactivated platelets. Blood.1999; 93:3831-3838.
38 Bombeli T, Karsan A, Tait JF, et al. Apoptotic vascular endothelial cells become procoagulant. Blood.1997; 89:2429-2442.
39 Vogt CJ, Schmid-Schonbein GM. Microvascular endothelial cell death and rarefaction in the glucocorticoid-induced hypertensive rat. Microcirculation.2001; 8:129-139.
40 Zou MH, Shi C, Cohen RA. High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H(2) receptor-mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells. Diabetes.2002; 51:198-203.
41 Holleyman C, Larson D, Hunter K. Stimulation of ischemia reperfusion in endothelial cell culture increase apoptosis. J Extra Corpor Technol.2001; 33:175-180.
42 徐慧,郑杨,佟倩.卡托普利晚期预处理对人内皮细胞缺氧复氧损伤的保护作用机制.中国动脉硬化杂志.2004;3：291-295.
43 Zhang J, Tan Z, Tran ND. Chemical hypoxia-ischemia induces apoptosis in cerebromicrovascular endothelial cell. Brain.Res.2000; 877:134-140.
44 Hong N, Browning J, Howard T, et al. Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and transforming growth factorbeta. Endothelium.1999; 7: 175-180.
45 Brutsaert DL. Cardiac endothelial myocardial signaling:its role in cardiac growth, contractkle performance.and rhythmicty. Physiological Reviews.2003,83:59-115.
46 Saraste A, Puikki K, Kallajoki M, et al. Apoptosis in human acute myocardial infarction. Circulation.1997; 95:320-323.
47 Jiang BH, Zheng JZ, Aoki M. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci USA.2000; 97:1749-1753.
48 Vanthaesebroeck B, Alessi DR, The PI3K-PDK1 connection:more than just a road to PKB. Biochem J.2000; 346:561-576.
49 Long X, Boluyt MO, Hipolito ML, et al. Crow MT.P53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invest.1997; 99:2635-2643.
50 Kirshenbaum LA, de Moissac D. The bcl-2 gene product prevents programmed cell death of ventricular myocytes. Circulation.1997; 96:1580-1585.
51 Jacobson MD, Burme JF, King MP, et al. Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature.1993; 361:365-369.
52 Shawm C, Huang JQ, Rezaiefar P, et al. Co-localization of the cysteine caspase-3 with apoptotic myoctyes after in vivo myocardial ischemia and reperfusion in the rat. J Mol Cell Cardiol.1998; 30:733-742.
53 Nunez G, Benedict MA, Hu Y, et al. Caspases:the proteases of the apoptotic pathway. Oneogene.1998; 17:3237-3245.
54 Brennere, Cadiou H, Vieria HL. Bel-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oneogene.2000; 19:329-336.
55 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods.2001; 25:402-408.
56 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc.2008; 3:1101-1108.
57 Savill J. Apoptosis:a mechanism for regulation of the cell complement of inflamed glomeruli. kidney Int.1992; 41:607-612.
58 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragment. Cell Biol.1992; 119:493-501.
59 彭黎明.六种细胞凋亡检测方法的比较.中华病理学杂志.1999;28：55-57.
60 邹伟,李兆育,李春雷,等.蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用.生理科学进展.2000；31：120-124.
61 Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem.1998; 67: 481-507.
62 Jones PF, Jakubowicz T, Pitossi FJ, et al. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci USA. 1991; 88:41714175.
63 Bellacosa A, Testa JR, Staal SP, et al. Aretroviral oncogene, Akt, encoding a serine-threonine kinase containing an SH2-like region. Science.1991; 254:274-277.
64 Coffer PJ, Woodgett JR. Molecular cloning and characterization of a novel putative protein-Serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem.1991; 201:475-481.
65 Datta K, Bellacosa A, Chan TO, et al. Akt is a grant a direct target of the phosphatidylinositol 3-kinase. Activation by growth factors, v-src and v-Ha-Ras in Sf9 and mammalian cells. J Biol Chem.1996; 271:30835-30839.
66 Franke TF, Hornik CP, segev L, et al. PI3K/Akt and apoptosis:size matters. Oncogene.2003; 22:8983-8998.
67 Fresno Vara JA, Casado E, Castro J, et al. PI3K/Akt signaling pathway and cancer. Cancer Treat Rev.2004; 30:193-204.
68 Jiang X, Wang X. Cytochrome C-mediated apoptosis. Annu Rev Biochem.2004; 73:87-106.
69 Downwrd J. PI3-kinase, Akt and cell survival. Sem in Cell Dev Biol.2004; 15:177-182.
70 Diehl JA, Cheng M, Roussel MF, et al. Glycogen synthase kinase-3 beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev.1998; 12:3499-3511.
71 Nusse R. Wnts and Hedgehogs:lipid-modified proteins and similarities in signalind mechanisms at the cell surface. Development.2003; 130:5297-5305.
72 Zong WX, EdelsteinL C, Chen C, et al. The prosurvival Bcl-2 homolog Bfl-1/Al is a direct transcriptional target of NF-KB that blocks TNF alpha induced apoptosis. Genes Dev.1999; 13: 382-387.
73 Strasser A, Harris AW, Jacks T, et al. DNA damage can induce apoptosis in proliferating lymphoid cells via p53 independent mechanisms inhibitable by Bcl-2. Cell.1994; 79:329-39.
74 Yin XM, Oltvai ZN, Korsmeyer S J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature.1994; 369:321-323.
75 Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell.1993; 74:609-619.
76 Yang E, Zha J, Jockel J, et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell.1995; 80:285-291.
77 Harris MH, Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membre permeability. Cell Death Dir.2000; 71:1182-1191.
78 Cosulich SC, Savory PJ, Clarke PR. Bcl-2 regulates amplification of caspase activation by cytochrome C. Curr Biol.1999; 9:147-150.
79 Bossy-Wetzel E, Green DR. Caspases induce cytochrome C release from mitochondria by activating cytosolic factors. J Biol Chem.1999; 274:17484-17490.
80 Sutton VR, Dvais JE, Cancilla M, et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B mediated caspase activation. J Exp Med.2000; 192: 1403-1414.
81 Stoka V, Turk B, Schendel SL, et al. Lysosomal portease path-ways to apoptosis. Clevagae of bid, not Pro-caspases, is the most likely way. J Biol Chem.2001,276:3149-3157.
82 Chan PH. Reactive oxygen radicals in singaling and damage in the ischemic barin. J Cereb. Blood Flow Metba.2001; 21:2-14.
1 Gottlieb RA, Burleson KO, Kloner RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest.1994; 94:1621-1628.
2 姚震,焦解歌,冯建章.心肌缺血再灌注损伤与细胞凋亡关系的实验研究.海南医学院学报.2000：6：129-133.
3 穆军升.NO与心血管疾病细胞凋亡.国外医学生理病理科学与临床分册.2001；21：389-391.
4 Chakrabarti S, Hoque AN, Karrnazyn M. A rapid ischemia induced apoptosis in isolated rat hearts and its attenuation by the sodium hydrogen exchang inhibitor HOE 642 (cariporide). J Mol Cell Cardiol.1997; 29:3169-3174.
5 马桂兰.细胞凋亡与冠心病.医学综述.2000;6：491492.
6 Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest.1996; 74:86-107.
7 Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res.1996; 79:949-956.
8 Gottlieb RA, Burleson KO, Khmer RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. Clin Invest.1994; 94:1621-1628.
9 Zhao ZQ, Nakamura M, Wang NP, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion. Apoptosis.2001; 6:279-290.
10 Ling H, Lou Y. Total flavones from Elshohzia blanda reduce infarct size during acute myocardial ischemia by inhibiting myocardial apoptosis in rats. Ethno pharmacol.2005; 101: 169-175.
11 赵卫红,寿好长,阎福岭,等.细胞凋亡的生物学特征.郑州：河南医科大学出版社.1997；7-16.
12 Ferrer L, Friguls B, Dalof E, et al. Caspase-dependent and caspase-in-dependent signalling of apoptosis in the penumbrao fllowing middlecerebral artery occlusion in the adult rat. Neuorpathol Appl Neurobiol.2003; 29:472-481.
13 Okamura T, Miura T, Takemura G. Efect of caspase inhibitors on myocardial in farct size and myocyte DNA fragmentation in the ischemia repefusedr at heart. Cardiovasc Res.2000; 45: 642-650.
14 Bialik S, Cryns VL, Drineie A. The mitochondrial apoptotie pathway is activated by serum and glucose deprivation in cardiacmyocytes. Circ Res.1999; 85:403-415.
15 Saikmuar P, Dong Z, Mikhailov V. Apopotsis:definition mechanismand relevence to disese. Am J Med.1999; 107:489-506.
16 Harris MH, Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membre permeability. Cell Death Dir.2000; 71:1182-1191.
17 Cosulich SC, Savory PJ. Clarke PR. Bcl-2 regulates amplification of caspase activation by cytochrome C. Curr Biol.1999; 9:147-150.
18 Bossy-Wetzel E, Green DR. Caspases induce cytochrome C release from mitochondria by activating cytosolic factors. J Biol Chem.1999; 274:1740-1749.
19 Sutton VR, Dvais JE, Cancilla M, et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B mediated caspase activation. J Exp Med.2000; 192: 1403-1414.
20 Stoka V, Turk B, Schendel SL, et al. Lysosomal portease path-ways to apoptosis:Clevagae of bid, not procaspases, is the most likely way. J Biol Chem.2001; 276:3149-3157.
21 Madeddu F, Naska S, Bozzi Y. BDNF down-regulates the caspase-3 pathway in injured geniculo-cortical neurones. Neuroreport.2004; 15:2045-2049.
22 Zhang WR, Kitagawa H, Hayashi T, et al. Topical applification of neurotrophin-3 attenuates isehemie brain injuy after transient middle cerebarl artey occlusion in rats. Brain Res.1999; 842:211-214.
23 Katoh S, MitsuiY, Kitnai K, et al. The rescuing effect of nevre growth factor is the result of up-regulation of bcl-2 in hyperoxia-induced apoptosis of a subclone of pheochomocytoma cells PC12h. Neurosci Lett.1997; 232:71-74.
24 Kiprianova I, Freimna TM, Desiderato S, et al. Brain-derived neurotrophic factor prevents neuronal death and glial activation after global isehemia in the rat. J Neurosci Res.1999; 56: 21-27.
25 Cao W, Carney JM, Duchon A, et al. Oxygen free radical involvement ischemia and reperfusion injury to brain. Neurosci. Lett.1988; 88:233-238.
26 Facchinetti F, Dawson VL, Dwason TM. Free radical as mediators of neuronal injury. Cell Mol Neurobiol.1998; 18:667-682.
27 Chan PH. Reactive oxygen radicals in singaling and damage in the ischemic barin. J Cereb. Blood Flow Metba.2001; 21:2-14.
28 Fujio Y, Nguyen T, Wencker D. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation.2000; 101:660-667.
29 Yodli R, Tomms F. Effect of multiple phosphorylation events on the trailscrip on factor FKHR, FKHRL1 and AFX.Biochem.Soc. Frmm.2002; 30:391-397.
30 Kajstura J, Fiordaliso F, Andreoli AM, et al. IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotension Ⅱ-mediated oxide stress. Diabetes.2001; 50: 1414-1424.
31 Matsui T, Tao J, del Monte F, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation.2001; 104:330-335.
32 Kovacs P, Bak Ⅰ, Szemdrei L, et al. Non-specific caspase inhibition reduces infarct size and improve post-ischemic recovery in isolated ischemial reperfusion rat hearts. Naunyn Schmiedebergs Arch Pharmocal.2001; 364:501-507.
33 Anwar A, Zahid AA, Schiidegger KJ, et al. Tumor necrosis factor-alpha results IGF-1 and IGFBP-3 expression in vascular smooth musle. Circulation.2002; 105:1220-1225.
34 Levine AI. P53:the cellular gatekeeper for growth and division. Cell.1997; 88:323-331.
35 Vousden KH. Activation of the p53 tumor suppressor protein. J Biochim Biophys Acta.2002; 1602:47-59.
36 Bargonetti J, Manfredi J. Multiple roles of the tumor suppressor p53. J Curr Opin Oncol.2002; 14:86-91.
37 Vousden KH, Lu X. Live or let die:the cells response to p53. Nat Rev Cancer.2002; 2: 594-604.
38 Regula KM, Kirshenbaum LA. p53 activates the mitochondrial death pathway and apoptosis of ventricular myocytes independent of de novo gene transcription. J Mol Cell Cardiol.2001; 33: 1435-1445.
39 Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. J Mol Cell.2003; 11:577-590.
40 Zhao ZQ, Velez DA, Wang NP, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion. Apoptosis.2001; 6:279-290.
41 Maed K, Hata R, Gillardon F, et al. Aggravation of brain injuru atfer transient focal ischemia in P53-deficient mice. J Mol Brain Res.2001; 889:54-61.
42 Mc Ghana L, Hakim AM, Robertson GS. Hippcampal Myc and p53 expression following transient global isehemia. Brain Res Mol Brain Res.1998; 56:133-145.
43 Maulik N, Sasaki H, Addya S. et al. Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors. FEBS Lett.2000; 485:7-12.
44 Culmsee C, Zhu X, Yu QS, et al. A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid beta-peptide. J Neurochem.2001; 77: 220-228.
45 Tomasevic G, Shamloo M, Israeli D, et al. Activation of p53 and its target genes p21(WAF1/Cipl) and PAG608/Wig-1 in ischemic preconditioning. Mol Brain Res.1999; 70: 304-313.
46 Long x, Bolhyt MO, Hipolito M1, et al. r63 and the hypocia-induced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invesr.1997; 99:2635-2643.
47 谢红光.受体与细胞信号转导.见：刘正湘主编.实用心血管受体学,北京：科学出版社,2001；28-32.
48 赵正航,臧伟进,于晓红.腺苷对模拟缺血再灌注豚鼠心室肌细胞动作电位的影响及其离子机制.中国药理学通报.2003;19：274-278.
49 刘睿编译.调节细胞凋亡的信号传递机制.国外医学免疫学分册.1995;18：270-272.
50 Arenzana-Seisdedos F, Turpin P, Rodriguez M, el al. Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci.1997; 110:369-378.
51 Li C, Browder W, Kao RI. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol.1997; 276:543-552.
52 陈冬梅,汪海.血管内皮细胞功能与心血管疾病相关因子研究进展.中国药理学通报.2003：19：361-365.
53 Sawa Y, Morishita R, Suzaki K, et al. A novel strategy for myocardial protection using in vivo transfection of cis element decoyagainst NF-kB binding site. Circulation.1997; 96:280-285.
54 Squadrito F, Ahavilla D, Squadrito G, el al. Tacrolimus limits polymorphonuclear leucocyte accumulation and protects against myocardial ischaemia-reperfusion injury. J Mol Cell Cardiol. 2000; 32:429-440.
55 唐旭东,姜建春,姜大春.三七总皂甘对心肌缺血／再灌注中中性粒细胞核因子-κB活化及其粘附的影响.中国药理学通报.2002；18：556-560.
56 Maulik N, Galang N. Differential regulation of Bel-2, AP-1 and NF-kappaB on cardiomyocyte apoptosls during myccardial ischemic stress adaptation. FEBS Lett.1999; 443:331-336.
57 Kirahenbaum LA. Bcl-2 intersects the NF-kappaB signaling pathway and suppresses apoptosis in ventricular myocytes. Clin invest Med.2000; 22:322-330.
58 Ashkenazi A, Dixit VM. Death receptor:signaling and modulation. Seience.1998; 281: 1305-1308.
59 Yuna J, Yankner BA. Apoptosis in the nevrous system. Nature.2000; 407:802-809.
60 Hatano E, Bradham CA, Stark A, et al. The mitochondrial pemreabiliy transition augments Fas-induced apoptosis in mouse heaptoyctes. J Biol Chem.2000; 275:11814-11823.
61 Baud V, Karin M. Signal transduction by tumor necorsis and its relatives. Trends Cell Biol. 2001; 11:372-377.
62 Jiang X, Wang X. Cytochrome c pormotes caspase-9 activation inducing nucleo-tide binding to Apaf-1. J Biol Chem.2000; 275:31199-31203.
63 Wang X. The expanding role of mitochondria in apoptosis. Genes Dev.2001; 15:2922-2933.
64 Nakgawa T, Zhu H, Morishima N, et al. Caspase-12 mediates endoplasmic reticulum specific apoptosis and cytotoxicity by amyloid-beta. Nature.2000; 403:98-103.
65 Michell RH. Evolution of the diverse biological roles of inositols. Biochemical Society Symposium.2007; 74:223-246.
66 DiPaolo G, DeCamilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature. 2006; 443:651-657.
67 Berridge MJ. Unlocking the secrets of cell signaling. Annual Review of Physiology.2005; 67: 1-21.
68 Vanhaesebroeck B, Leevers SJ, Ahmadi K, et al. Synthesis and function of 3-phosphorylated inositol lipids. Annual Review of Biochemistry.2001; 70:535-602.
69 Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem.1998; 67: 481-507.
70 Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kina-ses as regulators of growth and metabolism. Nature Reviews. Genetics.2006; 7:606-619.
71 Maffucci T, Brancaccio A, Piccolo E, et al. Insulin induces phosphate-dylinositol-3-phosphate formation through TC10 activation. EMBO Journal.2003; 22:4178-4189.
72 Lindmo K, Stenmark H. Regulation of membrane traffic by phosphoinositide 3-kinases. J Cell Sci.2006; 119:605-614.
73 Hirsch E, Lembo G, Montrucchio G, et al. Signaling through PI3Kgamma:a common platform for leukocyte, platelet and cardiovascular stress sensing. Thromb Haemost.2006; 95:29-35.
74 Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci.2001; 114: 1439-1445.
75 Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection:more than just a road to PKB Biochem J.2000; 346:561-576.
76 Vanhaesebroeck B, Leevers SJ, Ahmadi K, et al. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem.2001; 70:535-602.
77 McMuUen JR, Jennings GL. Diferences between pathological and physiological cardiac hypertrophy:novel therapeutic strategies to treat heart failure. Clin Exp Pharmacol Physiol. 2007; 34:255-262.
78 Shiojima I, Walsh K. Regulation of cardiac growth and coronary angiogenesis by the Akt/ PKB signaling pathway. Genes Dev.2006; 20:3347-3365
79 Muslin AJ, DeBosch B. Role of Akt in cardiac growth and metabolism. Nova rtis Found Symp. 2006; 274:118-126.
80 DeBosch B, Treskov I, Lupu TS, et al. Aktl is required for physiological cardiac growth. Circulation.2006; 113:2097-2104.
81 McMulhn JR, Shioi T, Zhang L, et al. Deletion of ribosomal S6 kinases does not attenuate pathological, physiolocal or insulin-like growth factor 1 receptor-phosphoinositide 3-kinase-induced cardiac hypertrophy. Mol Cell Bid.2004; 24:6231-6240.
82 Armstrong SC. Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res.2004; 61:427-436.
83 Chiari PC, Bienengraeber MW, Pagel PS, et al. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction:evidence for anesthetic-induced postcond-itioning in rabbits. Anesthesiology. 2005; 102:102-109.
84 Yellon DM, Downey JM. Preconditioning the myocardium:from cellular physiology to clinical cardiology. Physiol Rev.2003; 83:1113-1151.
85 Burgering BM, Medema RH. Decisions on life and death:FOXO Forkhead transcription factors are in command when PKB/Akt is off duty. J Leukoc Biol.2003; 73:689-701.
86 Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell.1999; 96:857-868.
87 Rena G, Guo S, Cichy SC, et al. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem 1999; 274:17179-17183.
88 Cantley LC. The phosphoinositide 3-kinases pathway. Science.2002; 296:1655-1657.
89 Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell.1997; 91:231-241.
90 Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-Kinase/Akt cell survival pathway. J Biol Chem.1998; 273:19929-19932.
91 Khoynezhad A, Jalali Z, Tortolani AJ. Apoptosis:pathophysiology and therapeutic implications for the cardiac surgeon. Ann Thorac Surg.2004; 78:1109-1118
92 Moolman JA, Hartley S, Van Wyk J. et al. Inhibition of myocardial apoptosis by ischaemic and beta-adrenergic preconditioning is dependent on p38 MAPK. Cardi ovasc Drugs Ther. 2006; 20:13-25.
93 Tissier R, Waintraub X, Couvreur N. Pharmacological postconditioning with the phytoestrogen genistein. J Mol Cell Cardiol.2007; 42:79-87.
94 Burger D, Xiang F, Hammoud L, et al. Role of Heme Oxygenase-1 in the cardioprotective effects of erythropoietin during myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol.2009; 296:H84-93.
95 Gao JP, Chua CC, Chen ZY. Resistin, an adipocytokine, offers protection against acute myocardial infarction. J Mol Cell Cardiol.2007; 43:601-609.
96 Ghibu S, Lauzier B, Delemasure S, et al. Antioxidant properties of alpha-lipoic acid:effects on red blood membrane permeability and adaptation of isolated rat heart to reversible ischemia. Mol Cell Biochem.2009; 320:141-148.
97 Blokhin IO, Vlasov TD, Galagudza MM, et al. Role of sodium-calcium exchanger in the myocardial protection against ischemia-reperfusion injury. Ross Fiziol Zh Im I M Sechenova. 2008; 94:284-292.
98 Zhang JH, Chen ZW, Wu Z. Late protedtive effect of pharmacological precongitioning with total flavones of rhododendra against myocardial ischemia-reperfusion injury. Can J Physiol Pharmacol.2008; 86:131-138.
99 Fuglesteg BN, Suleman N, Tiron C, et al. Signal transducer and activator of transcription 3 is involved in the cardioprotective signaling pathway activated bu insulin therapy at reperfusion. Basic Res Cardiol.2008; 103:444-453.
100 Lu X, Hamilton JA, Shen J, et al. Role of tumor necrosis factor-alpha in myocardial dysfunction and apoptosis during hindlimb ischemia and reperfusion. Crit Care Med.2006; 34: 484-491.
101 Das A, Xi L, Kukreja RC. Phosphodiestprase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis, Essential role of nitric oxide signaling. J Bioi Chem. 2005；280：12944-12955.
102 陈锐群,卢存寿,张丽丽,等.地黄的研究.阴健和郭力弓主编.中药现代研究与临床应用.北京：学苑出版社.1994；272-279.
103 都恒青,郭湘云,赵曦,等.地黄的研究.楼之岑和秦波主编.常用中药材品种整理和质量研究.北方编第二册.北京：北京医科大学出版社.1995；839-841.
104 Isao K, Masayuki Y. Chemical constituents of rehmanniae radix, cornifructus, dioscoreae rhizoma. Modern Eastern Medicine.1986; 7:55-62.
105 中华人民共和国药典2000年版一部.北京：化学工业出版社.2000；94.
106 Jiang B, Liu JH, Bao YM, et al. Catalpol inhibits apoptosis in hydrogen peroxide induced PC 12 cells by preventing cytochrome c release and inactivating of caspase cascade. Toxicon. 2004; 43:53-59.
107 Li DQ, Bao YM, Li Y, et al. Catalpol modulates the expressions of Bcl-2 and Bax and attenuates apoptosis in gerbils after ischemic injury. Brain Res.2006; 1115:179-185.
108 Li DQ, Duan YL, Bao YM, et al. Neuroprotection of catalpol in transient global ischemia in gerbils. Neurosci Res.2004; 50:169-177.
109 Li DQ, Li Y, Liu Y, et al. Catalpol prevents the loss of CA1 hippocampal neurons and reduces working errors in gerbils after ischemia-reperfusion injury. Toxicon.2005; 46:845-851.
110 Hu LA, Sun YK, Hu J. Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax. Eur. J. Pharmacol.2010; 628:155-163.