【作者】 胡玲爱；

【导师】 胡健；

【作者基本信息】 中国医科大学 ， 内科学， 2010， 博士

【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。更多还原

【Abstract】 IntroductionApoptosis 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.更多还原