三七皂苷R1及Pim-2在H_2O_2诱导的心肌细胞损伤中的作用机制研究
|
摘要
一、目的：
近年来,国内外大量研究文献都表明氧化应激是许多病理因素造成心血管损伤的共同机制,在许多心血管疾病的病理过程中都有过量氧自由基产生,同时抗氧自由基防御机制却受到抑制,如动脉粥样硬化(AS)、高血压病、缺血性心脏病、高脂血症等。氧化应激损伤心肌细胞的转归与氧化应激的程度相关,在一定应激强度范围内,氧化应激对心肌细胞造成的损伤是可逆的,而超过一定限度,则对心肌细胞造成不可逆的损伤,主要包括心肌细胞的凋亡和坏死。
原癌基因Pim(Proviral integration site of murine)家族是哺乳动物细胞中的丝/苏氨酸激酶,属于钙调蛋白依赖性的蛋白激酶,包括Pim-1、Pim-2和Pim-3。大量的实验研究已证实,Pim激酶的上调与细胞凋亡的抑制存在显著的相关性。Pim-2持续表达能够促进细胞长期抵抗各种凋亡信号的刺激。研究证实Pim-1在Akt通路中保护心肌细胞损伤,而在Pim-1缺失小鼠中Pim-2的表达增高了4.6倍；Pim-1缺失小鼠在心梗后7天,Pim-2的表达增高2.75倍。但Pim-2在心肌细胞中的表达及作用尚缺乏研究,有必要探讨Pim-2在心肌细胞损伤中的作用及机制。
三七是具有多种心血管活性的中药,临床广泛应用于心血管疾病的防治。现代化学和药理学研究发现三七总皂苷(Panax notoginseng saponin, PNS)是三七的主要活性成分,含有多种单体皂苷主要有：人参皂苷Rb1,人参皂苷Rg1,三七皂苷R1。本课题以三七皂苷R1为研究对象,旨在了解三七皂苷R1对心肌细胞损伤的保护作用,并探讨其参与调节的细胞信号通路,凋亡相关蛋白,并了解其对Pim-2的调控作用。
二、方法：
1.乳鼠心肌细胞的原代培养外科无菌条件下取出乳鼠心脏,用胰酶40C过夜,终止消化,再用胶原酶消化液逐步消化法分离单个心肌细胞,接种至培养瓶中,采取差速贴壁分离法以去除心肌成纤维细胞,得到纯度较高的心肌细胞用于实验。
2.H202致心肌细胞氧化应激模型建立心肌细胞正常培养2-3天后,选取细胞密度在80%-95%以上的,自律性搏动120～140次/min的心肌细胞用于实验,选取不同浓度与时间点的H2O2干预心肌细胞,选择合适的浓度及干预时间用于后续实验。
3.三七皂苷R1预处理心肌细胞心肌细胞培养的第2-3天,细胞生长接近80%融合时,心肌细胞预先用不同浓度三七皂苷R1培养24h,进行三七皂苷R1毒性测试,选取合适的高、中、低剂量组进行相关后续实验。
4.MTT比色法测定心肌细胞活力将心肌细胞接种于九十六孔板,按实验分组给予相应的处理因素后,按MTT法具体操作步骤进行操作,在酶标仪490nm处测其OD值并计算各组心肌细胞存活率。
5.流式细胞术检测心肌细胞凋亡率心肌细胞按照实验分组处理后,用不含EDTA的胰酶消化收集,以Annexin V-FITC/Propidium Iodide进行染色,室温、避光、反应5～15min,上流式细胞仪(激发波长488nm,发射波长530mm)进行检测。
6.心肌细胞LDH、MDA、SOD的检测将心肌细胞接种于六孔培养板中,按不同实验目的进行分组处理,处理完毕后,分别按照LDH、MDA、SOD检测试剂盒说明书进行操作,在酶标仪420nm处测量每组OD值并计算各组细胞内LDH、MDA、SOD的含量。实验重复三次以上。
7.实时定量PCR检测心肌细胞内Pim-2mRNA的表达心肌细胞按照2×107/孔的浓度接种至六孔板中,取正常培养2-3天后状态最佳的心肌细胞用于实验。实验分组依次为：正常对照组、H2O20.5、1、2、3、6h五个时间组,50μMH2O2干预心肌细胞。用Trizol提取心肌细胞总RNA,在紫外分光光度计上测定OD260/OD280,鉴定提取的RNA纯度和浓度。然后,反转录合成cDNA,根据Gene Bank中Pim-2、GAPDH的基因序列资料,借助于计算机软件PrimerPremier5.0设计引物,上机进行目的基因的荧光定量检测。
8. SiRNA沉默心肌细胞Pim-2基因调合适的细胞浓度分别接种于六孔板或九十六孔板中,正常培养2-3天左右,当贴璧细胞达到80%-90%融合时,取状态良好的细胞进行实验。siPORTTM NeoFXTM转染试剂与DMEM培养基混匀室温孵育15min, siRNA稀释液等体积混匀,孵育15min。然后将混合液加入培养板混匀。转染心肌细胞48-72h后收集细胞,再进行Western blot实验和流式细胞术检测心肌细胞凋亡率。
9. Western blot法检测心肌细胞蛋白的表达心肌细胞按实验分组处理完成后,以Western及IP细胞裂解液提取心肌细胞蛋白,BCA法进行蛋白定量,进行蛋白印迹实验。以Actin为内参,将处理好的蛋白质样品进行SDS-PAGE分离,电泳结束后将蛋白质转移到PVDF膜上,封闭后,孵育一抗和二抗,ECL显色,KODAK Image Station2000MM成像系统采集图像,获得图像用图像处理软件Image Tool3.0测定并分析条带灰度值以检测目的蛋白的表达水平。
10.统计分析实验数据采用SPSS13.0统计软件进行分析。计量资料数据以x±s表示。对数据进行正态性检验和方差齐性检验。若符合正态分布和方差齐性,则采用方差分析。若不符合正态分布和方差齐性,则采用秩和检验。方差齐性时两两比较采用LSD法,方差不齐时两两比较采用Tamhane's T2法。多组计数资料的比较(各组AV评分)采用完全随机设计资料的Kruskal-Wallis检验方法。各实验组不同缺血及再灌注时间点血流动力学指标采用重复测量方差分析。检验显著性水准a=0.05。
三、结果：
1.三七皂苷R1对H2O2诱导的心肌细胞损伤的作用
1.1H2O2对心肌细胞损伤的浓度效应关系MTT比色法检测不同浓度H2O2干预心肌细胞3h后,细胞活力显著降低,光镜下可看到细胞搏动减弱甚至停止,细胞密集区域出现大片细胞脱落。与正常对照组比较,H202干预的各组细胞活力均有所下降,经统计分析各组差异均有统计学意义(F=734.372,P<0.001),且随着H202浓度的增加各组细胞活力出现递减趋势。
1.2不同浓度三七皂苷R1对心肌细胞活力的影响不同浓度三七皂苷R1预处理心肌细胞24h,比较各浓度三七皂苷R1作用后细胞活力与正常对照组的差异。经统计分析,三七皂苷R1浓度为0.1×10-6mol/L、1×10mol/L、10×10-6mol/L时细胞活力与正常对照组比较差异无统计学意义(P均>0.05),而浓度为50×10-6mol/L、100×10-6mol/L时,作用心肌细胞24h后细胞活力较正常对照组显著降低(P<0.001)。
1.3三七皂苷R1预处理对H2O2干预的心肌细胞活力的影响三七皂苷R1低、中、高浓度(0.1×10-6mol/L、1×10-6mol/L、10×10-6mol/L)预处理心肌细胞24h,然后以H2O2(50pmol/L)作用细胞3h,同时设立正常对照组、模型组,比较各不同浓度三七皂苷R1对H2O2诱导的心肌细胞活力的影响。经统计分析表明,各组间细胞活力差异有统计学意义((F=519.758,P<0.001)。三七皂苷R1预处理后可呈浓度依赖性减轻H2O2处理后心肌细胞活力的下降。
1.4三七皂苷R1预处理对H2O2干预的心肌细胞凋亡率的影响流式细胞术检测各组心肌细胞凋亡率,比较各不同浓度三七皂苷R1对H2O2诱导的心肌细胞凋亡率的影响。经三七皂苷R1预处理后,各组细胞凋亡与模型组相比有所减少,尤其是高浓度组的凋亡情况显著改善。经统计分析,各组差异均有统计学意义(P<0.0001)。
1.5三七皂苷R1预处理对H2O2干预的心肌细胞LDH、MDA、SOD的影响试剂盒检测各组心肌细胞LDH、MDA、SOD值,结果与正常对照组比较,模型组心肌细胞SOD活性显著降低,而LDH活性、MDA含量则显著增加(P<0.001)；三七皂苷R1可呈剂量依赖性增加SOD活性,降低LDH活性、MDA含量,各组间比较差异具有统计学意义(P<0.001)。
2三七皂苷R1对H2O2诱导的心肌细胞凋亡信号通路、凋亡相关蛋白表达的影响
2.1三七皂苷R1预处理对H202干预的心肌细胞MAPK信号通路的影响
2.1.1Western Blot检测各组细胞中P-ERK1/2.总ERK1/2蛋白表达的变化,分析目的条带的光密度值(IOD)。与正常组相比,模型组(H2O2) P-ERK1/2表达显著提高,三七皂苷R1低、中、高剂量组与模型组相比P-ERK1/2表达显著降低,其中高剂量组表达降低最为显著。各组间P-ERK1/2表达存在显著差异(P<0.01)。
2.1.2Western Blot检测各组细胞中P-JNK、总JNK蛋白表达的变化,分析目的条带的光密度值(IOD)。与正常组相比,模型组(H2O2) P-JNK表达显著提高(P<0.001),三七皂苷R1低、中、高剂量组与模型组相比表达无显著差异(P>0.05)。
2.1.3Western Blot检测各组细胞中P-P38、总P38蛋白表达的变化,分析目的条带的光密度值(IOD)。与正常组相比,模型组(H2O2) P-P38表达显著提高(P<0.001),三七皂苷R1低、中、高剂量组与模型组相比P-P38表达无统计学意义(P>0.05)。
2.2三七皂苷R1预处理心肌细胞凋亡蛋白Bax/Bcl-2表达的变化用Western Blot检测各组细胞中Box、Bcl-2蛋白表达的变化,分析目的条带的光密度值(IOD),正常组Bax、Bcl-2有一定量的表达,与正常组相比,模型组Bax表达显著增高,三七皂苷R1低、中、高剂量组与模型组相比表达逐渐降低(P<0.001)；与正常组相比,模型组Bcl-2表达量降低,三七皂苷R1低、中、高剂量组与模型组相比表达逐渐增高(P<0.001)。与正常组相比,模型组Bax/Bcl-2值显著增高,三七皂苷R1低、中、高剂量组与模型组相比表达逐渐降低(P<0.001)。
3Pim-2在H202诱导的心肌细胞损伤中的表达及作用
3.1H202显著上调心肌细胞内Pim-2mRNA的表达用浓度为50μmol/L的H202干预心肌细胞0.5、1、2、3、6h,实验设正常对照组。应用实时荧光定量PCR的方法检测各组心肌细胞内Pim-2mRNA的表达时发现,H202干预后的心肌细胞Pim-2mRNA的表达有所增加,各组差异有统计学意义(P<0.001)。其中H2O20.5、1h组与正常对照组相比,差异无统计学意义(P=0.056)；其余各浓度组与正常对照组相比,差异均有统计学意义(P<0.001),尤其是H2023h组Pim-2mRNA的水平增加最显著；H2O26h组心肌细胞Pim-2mRNA表达水平有所降低,但仍高于正常对照组。
3.2H202显著上调心肌细胞内Pim-2蛋白的表达应用Western blot法检测H202干预不同时间后心肌细胞内Pim-2的蛋白水平发现,H202干预后的心肌细胞Pim-2蛋白表达增高,其增高的趋势与实时荧光定量PCR检测出的Pim-2mRNA增高趋势一致,各组差异有统计学意义(P<0.001)。
3.3Pim-2siRNA干扰对细胞凋亡的影响实验分为正常组,H202组,siRNA组(H2O2+siRNA),阴性对照组(H2O2+NC)。结果显示模型组与正常组相比,细胞凋亡率显著增加,siRNA干扰组细胞凋亡率进一步增加(P<0.001),阴性对照组与模型组相比无显著差异(P>0.05),与正常组相比显著增加(P<0.001)。
3.4Pim-2siRNA干扰对细胞LDH、MDA、SOD的影响心肌细胞经Pim-2siRNA干扰后,心肌细胞SOD活性较模型组显著降低,而LDH、MDA含量较模型组显著增高,各指标组间差异具有统计学意义(P<.001),阴性对照组与模型组相比差异无统计学意义(P>0.05)。
4Pim-2蛋白表达调控
4.1三七皂苷R1上调Pim-2蛋白的表达通过Western Blot技术检测各组心肌细胞Pim-2蛋白表达,结果显示模型组(H202)与正常组相比Pim-2表达显著增高,三七皂苷R1各剂量组Pim-2表达进一步增高,其中三七皂苷R1高剂量组表达增高最为显著,各组间差异有统计学意义(P<0.01)。
4.2Pim-2siRNA干扰对三七皂苷R1预处理心肌细胞凋亡率的影响实验分为正常组、模型组(H202)、三七皂苷R1组(H2O2+NG R110μM)、siRNA转染组(H2O2+NG R110μM+siRNA)、siRNA阴性对照组。流式细胞术检测各组细胞凋亡率,结果模型组与正常组相比凋亡率显著增高(P<0.001),三七皂苷R1组与模型组相比显著下降,siRNA组、siRNA阴性对照组与三七皂苷R1组相比无显著差异(P>0.05)。
4.3Pim-2siRNA干扰对心肌细胞Bax/Bcl-2表达的影响实验分为正常组,H202组,siRNA组(H2O2+siRNA),阴性对照组。用WB检测各组细胞中Bax、 Bcl-2蛋白表达的变化,分析目的条带的光密度值(IOD),与正常组相比,模型组Bax表达显著增高,siRNA组与模型组相比表达进一步增高(P<0.01),阴性对照组与模型组相比Bax表达无显著差异。与正常组相比,模型组Bcl-2表达量降低,siRNA组与模型组相比表达进一步降低(P<0.0I),阴性对照组与模型组相比无显著差异。模型组与正常组相比,Bax/Bcl-2值显著增高,siRNA组进一步增高(P<0.01),阴性对照组无显著差异。
四、结论：
1、H202能抑制心肌细胞活力,促进心肌细胞凋亡的发生,导致氧化平衡体系的破坏。三七皂苷R1可以抑制H202诱导的心肌细胞损伤,增强心肌细胞活力,保护心肌细胞凋亡,增强细胞抗氧活酶活力。
2、H202促进心肌细胞ERK、JNK、P38的磷酸化,三七皂苷R1下调H202干预的心肌细胞中ERK的磷酸化,而对JNK、P38的磷酸化无显著调控。三七皂苷R1下调H2O2干预的心肌细胞中Bax蛋白的表达,上调Bcl-2蛋白的表达。表明三七皂苷R1抗心肌细胞凋亡与调节ERK信号通路、凋亡相关蛋白有关。
3、H202能诱导心肌细胞中Pim-2激酶的表达,Pim-2siRNA干扰可有效沉默Pim-2基因,Pim-2蛋白表达对心肌细胞损伤其保护作用。
4、三七皂苷R1上调H202干预的心肌细胞中Pim-2的表达,但siRNA干扰Pim-2后,三七皂苷R1仍能抗心肌细胞凋亡,表明三七皂苷R1抗心肌细胞凋亡并非唯一通过Pim-2调控。Pim-2能调节H2O2干预的心肌细胞中凋亡相关蛋白Bcl-2的表达。
Objective:
In recent years, a large number of study both at home and abroad have shown that oxidative stress related apoptosis is the common mechanism of cardiovascular damage caused by many different pathological factors. There are excessive oxygen free radicals generated in many pathological processes of cardiovascular diseases, such as atherosclerosis (AS), hypertension, ischemic heart disease, hyperlipidemia and so on, while the anti-oxidative defensive mechanism in these processes are inhibited. The damage of myocardial cells cause by oxidative stress is relevant to its intensity. It's reversible in a certain stress intensity range. But the damage can become irreversible, which mainly includes myocyte apoptosis and necrosis, if beyond the certain range.
The proto-oncogene Pim (Proviral integration site of murine) family in mammalian cells is serine/threonine kinase belongings to the calmodulin-dependent protein kinase, including Pim-1, Pim-2and Pim-3. Many experimental studies have confirmed the existence of a significant correlation between the upregulation of Pim kinase and inhibition of apoptosis. Pim-2expression continued to promote long-term cell resistance to various apoptosis signal stimulation. It's confirmed that Pim-1protects myocardial injury via Akt pathway, the expression of Pim-2in Pim-1deficient mice increased4.6times;7days after myocardial infarction of Pim-1-deficient mice, Pim-2expression increased2.75times. But Pim-2expression and function in myocardial cells is still unknown, it is necessary to explore it's function and mechanism of myocardial injury.
Radix Notoginseng is a medicine with a variety of cardiovascular activity, it's widely used in clinical in the prevention and treatment of cardiovascular disease. With the advance of modern chemistry and pharmacology, Panax notoginseng saponins (Panax notoginseng saponin, PNS) is identified to be the main active ingredient of the Radix Notoginseng, contains a variety of monomer saponin, such as ginsenoside Rbl, ginsenoside Rgl, the notoginsenoside R1. Focusing on notoginsenoside R1in this study, our purpose is to understand the protective effect of notoginsenoside R1in myocardial injury, and to investigate the cell signaling pathways and proteins involved in apoptosis regulation, and Pim-2effect in notoginsenoside R1protection.
Method:
1. Primary culture of neonatal rat cardiomyocytes
Neonatal rats' hearts were removed in surgical sterile conditions, digested in trypsin in4℃overnight, and then gradually digested the heart tissue with collagenase into isolated cells. Cells were inoculated into a culture flask. Differential adhesion separation method was used to remove cardiac fibroblasts in order to obtain pure myocardial cells in this study.
2. Model of H2O2-induced oxidative stress in myocardial cells
Normal myocardial cells were cultured for2-3days, proliferated to reached the density of80%-95%. Cells that pulsed at the rate120～140times per minute were selected for the use of experimental myocardial cells. Different time points and concentrations were tested for H2O2intervention. Proper concentration of H2O2and time point to intervene was used in follow-up experiment.
3. Notoginsenoside R1myocardial preconditioning
Cultured myocardial cells first2-3days, cell growth close to80%confluence, myocardial cells pretreated with different concentrations notoginsenoside R1cultured24h, toxicity tests conducted Notoginsenoside R1, select the appropriate high, medium and low dose groups related follow-up experiments.
4. Assessment of cell viability (MTT assay)
Cardiomyocytes were seeded in96-well culture plates at a density of1×104cells/well, were incubated at37℃for48h, and then pretreated with different drugs depend on experimental groups. After that, the cells were washed with phosphate-buffered saline (PBS) three times and MTT dye was added to each well for the last4h of treatment. The reaction stopped with the addition of dimethyl sulfoxide (DMSO) and the optical density was determined at490nm on a multi-well plate reader. All groups were assayed in six wells and the mean for each group was calculated.
5. Analysis of cardiomyocyte apoptosis with flow cytometry
Myocardial cells according to the experimental groups were digested by EDTA-free trypsin, stained with to Annexin V-FITC/Propidium Iodide, incubated at room temperature and protected from light for5～15min, analysed by flow cytometry (excitation wavelength488nm, emission wave length530nm).
6. Detection the level of intracellular LDH、MDA、SOD in myocardial cells
Myocardial cells were seeded in six wells, grouped by different experimental treatment. Analysises were done in accordance with the instruction of LDH, MDA, SOD detection kit after treatment, each set of OD values measured at420nm in a microplate reader and the contents of LDH, MDA, SOD in every sample was calculated according to these values. Each experiment was repeated three times.
7. Real-time quantitative PCR analysis of myocardial cells Pim-2mRNA expression.
Cardiomyocytes were seeded to6-well plates2×107/well, cultured for2-3days. Cells were collected in optimal condition for the following experiment. Samples were divided as following groups:normal groups, control groups,0.5,1,2,3,6hours H2O2treated group,50μM H2O2was used for myocardial cells. The total RNA of cardiomyocytes was extracted with Trizol, purity and concentration of the extracted RNA was measured on a UV spectrophotometer. Then cDNA was synthesized by reverse transcription, and fluorescence quanititavie dectection of the target gene was performed afterwards. The primer of Pim-2gene was designed according to Gene Bank sequence imformation, by means of Primerpremier5.0software.
7. SiRNA silencing myocardial cells'Pim-2expression.
Cells were cultured in six wells and ninty-six wells for2or3days to reach80%～90%confluent, cells of optimal condition were used for further expriments. SiPORTTM NeoFXTM was incubated with DMEM and SiRNA diluent in the same volume for15min at room temperature. The mixture was added into the culture plates for transfection for next48～72h.After that cells were collected for Western blot analysis and flow cytometry analysis of cardiomyocyte apoptosis rates.
8. Western blot analysis of the protein expression of myocardial cells. Myocardial cells were solubilized after complication of the treatment by using the Western or IP cell lysates, protein quantitation was performed by using BCA method before the extrats went for immunoblotting analysis. Total proteins were separated on a SDS-PAGE gel, and transferred to a PVDF membrane after electrophoresis, incubated with primary and secondary antibody in turn after the membrane was blocked, signal was developed by using ECL substrate, acquired image by KODAK Image Station2000MM system, analysis the image with Image Tool3.0, the gray values of the lanes were measured and quantization as the expression of target proteins. Actin was used as loading control in this assay.
10. Statistical analysis:The data was analyzed by SPSS13.0. measurement data was presented as the means±SEM. Test of normality and homogeneity of variance was done in each group. If datas accorded with normality and homogeneity of variance, statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Tamhane's T2. Multiple enumeration data was analyzed by Kruskal-Wallis. Hemodynamics parameters in different time points was analyzed by repetitive measurement and analysis of variance. Level of significance a=0.05
Result:
1. Notoginsenoside Rl roles in H2O2-induced myocardial injury
1.1The concentration-response relationship of myocardial cells injury to H2O2. MTT assay shows that after treating the cardiomyocytes with different concentrations of H2O23hours, viability of cells were evidently reduced. Cells pulse became slower or even stop when observed through the microscope, there are large pieces of cells shedding in the area that cells was relative crowded. Compared to the normal control,cell viability after H2O2intervention at various concentration were all reduced, the statistical analysis of the difference between each group were statistically significant (F=734.372,P<0.001), and cell viability were more apparently reduced with higher concentration of H2O2.
1.2The different concentrations of notoginsenoside R1on myocardial cell viability. Myocardial cells were preconditioned with different concentrations of notoginsenoside for24hours. Comparisons of cell viability were made between groups treated with different concentrations of notoginsenoside R1and the normal control group. Statistical analysis showed that there are no statistically difference of cell viablility between groups treated with0.1×10-6mol/L,1×10-6mol/L,10x10-6mol/L notoginsenoside R1and groups of normal control (P>0.05), while the concentration of100×10-6mol/L groups' viability were significantly lower than controls after treating myocardial cells for24hours (P<0.01).
1.3Pretreatment of Notoginsenoside R1on myocardial cell viability in H2O2injury models. Myocardial cells were pretreated with low, medium and high concentrations (0.1×10-6mol/L,1×10-6mol/L,10×10-6mol/L) of notoginsenoside for24hours, then H2O2(SOμmol/L) was added for3hours, normal control group and model group were set up at the same time. Comparisons of myocardial cells viability were made between groups of different concentrations notoginsenoside R1in H2O2injury models. The statistical analysis showed that the cell viability difference between the groups was statistically significant ((F=519.758, P<0.001). Notoginsenoside R1pretreatment reduced the decline of myocardial cell viability in a dose-dependent manner after H2O2treatment.
1.4Pretreatment of Notoginsenoside R1reduced the apoptosis rate of myocardial cell in the H2O2injury model. The apoptotic rates detected by flow cytometry were used to compare the effects of notoginsenoside R1of multiple concentrations in H2O2-induced models. After pretreatment by notoginsenoside R1, the apoptotic rates of cells were lower campared to the model group, especially in high concentration groups, with significantly improvement of apoptotic rates, After statistical analysis, differences of each groups were statistically significant (P <0.0001).
1.5Activities of LDH, MDA and SOD in notoginsenoside R1pretreated myocardial cells of H2O2intervention. Compared with the normal control group, SOD activity of model groups were significantly reduced, while the activity of LDH, MDA was significantly increased (P<0.001). Notoginsenoside R1lowered SOD activity in a dose-dependent manner, while increased LDH and MDA activity. The difference among the groups was statistically significant (P<0.001).
2Notoginsenoside R1effects on H2O2-induced myocardial apoptosis signaling pathways and apoptosis-related protein expression
2.1Notoginsenoside R1effects on H2O2intervention myocardial cells MAPK signaling pathway
2.1.1Western Blot detection of protein expression of p-ERK1/2, total ERK1/2in each group and analysis of the target bands by optical density (IOD). Compared with normal group, model group (H2O2) p-ERK1/2expressions were significantly increased. P-ERK1/2expression in groups of low, medium and high dose of notoginsenoside R1pretreatment were significantly reduced compared to the modele groups, especially in of the high-dose groups. There are significant differences of p-ERK1/2expression among the groups (P<0.01).
2.1.2Western Blot detection of protein expression of P-JNK, total JNK in each group and analysis of the target bands by optical density (IOD). Compared with the normal group, model group (H2O2) P-JNK expression was significantly increased (P <0.001). There are no significant difference between groups of multiple concentrations of notoginsenoside Rl pretreatment and model groups (P>0.05).
2.1.3Western Blot detection of protein expression of P-P38, P38in each group and analysis of the target bands by optical density (IOD). Compared with normal group, model group (H2O2) P-P38expression was significantly increased (P<0.001), notoginsenoside R1low. There are no significant difference between groups of multiple concentrations of notoginsenoside R1pretreatment and model groups (P>0.05).
2.2Changes of expression of Bax/Bcl-2apoptotic proteins of cardiomyocytes after pretreatment of notoginsenoside R1. Bax/Bcl-2expression was detected by Western Blotting analysis in each group; analyses of the target band optical density (IOD) were performed aferward. Normal groups have a certain amount of Bax, Bcl-2expression, Bax expression of model groups were significantly higher than the normal groups, notoginsenoside R1groups have a decreased Bax expression compared to model groups (P<0.01); Compared with the normal group, model groups have reduced Bcl-2expression. Notoginsenoside R1groups expression of BCL-2were gradually increased compared to model groups (P<0.01). The Bax/Bcl-2ratio of model groups was significantly higher to normal groups, notoginsenoside R1groups have reduced Bax/Bcl-2compared to model groups (P<0.01).
3Pim-2expressions and its role in H2O2-induced myocardial injury
3.1H2O2intervention significantly upregulated Pim-2mRNA expression in myocardial cells.50mM/L H2O2was addd to myocardial cells cultures for0.5,1,2,3,6h, the normal control group was setted up at the same time. Pim-2mRNA expression analysed by real-time quantitative PCR. It's showed that Pim-2mRNA expression increased in groups after the intervention of H2O2.There are statistically significant between groups (P<0.001). Comparsions between each groups were statistically analysed too.Which0.5h and1h groups showed no significant difference compared to the control group (P>0.05); the remaining groups were significant different between each others (P<0.0001), especially in3h, Pim-2mRNA level increased to the most obvious level;6h groups showed reduced Pim-2mRNA expression, but the level was still higher than the normal control group.
3.2H2O2intervention significantly increased Pim-2protein expression in myocardiocytes. Pim-2protein expressions were increased after H2O2intervention, indicated by Westernblotting analysis of Pim-2protein expression at different time points. The growth of Pim-2expressions were correlation with the growth of mRNA expression analysed by real-time fluorescence quantitative PCR. The difference between each groups were statistically significant (P<0.01).
3.3Pim-2siRNA interference on apoptosis of myocardial cells. Samples of myocardial cells cultures were randomly divided into normal group, H2O2group, siRNA group (H2O2+siRNA), and negative control group (H2O2+NC). The results show model group compared with the normal group, the apoptosis rate of model group was increased significantly compared to the normal control group. The siRNA interference further increased the apoptosis rate (P<0.001), negative control group was significant different compared to the normal group (P<0.001), but no statistical difference when compared to the model group (P>0.05).
3.4Impact of Pim-2siRNA interference on activity of LDH, MDA and SOD. SOD activity after Pim-2siRNA interference was significantly reduced compared with the model group, while LDH, MDA content compared with the model group was significantly increased, the differences between groups has statistical significance (P <0.001). Indexes of negative control group were not statistically significant (P>0.05) when compared with the model group.
4Pim-2protein expression and regulation
4.1Notoginsenoside R1increased Pim-2protein expression. Pim-2protein expression of myocardial cells was analysed by Western Blotting, the results showPim-2expression of the model group (H2O2) was significantly higher than the normal group, notoginsenoside R1of different concentration further increased the Pim-2expression, the most Pim-2expressoin was observed in the high dose notoginsenosdie R1group. Difference among the groups was statistically significant (P<0.01).
4.2Pim-2siRNA interference on notoginsenoside Rl pre-treatment H2O2injured cardiac myocyte. Samples of myocardial cells were divided into normal group, model group (H2O2), notoginsenoside R1group (H2O2+NG Rl10μM), siRNA transfection group (H2O2+NG R1lOμM+siRNA), siRNA negative control group. Apoptosis rates were analysed by flow cytometry, the result showed that the model group was significantly higher compared with the normal group (P<0.001), apoptotic rates in oginsenoside R1group were decreased compared with the model group. There are no significant difference between siRNA group, siRNA negative control group and notoginsenoside R1group (P>0.05).
4.3Pim-2siRNA interference on Bax/Bcl-2protein expressons. Samples of myocardial cells were divided into normal group, H2O2group, siRNA group (H2O2+siRNA), and negative control group. Western blotting was used to detect the expression in Bax, Bcl-2protein, the target bands were analysed with optical density (IOD). The expression of Bax in model group was significantly higher than normal control group. SiRNA group showed further increase compared with model group expression (P<0.01), there is no significant difference on Bax expression between negative control group and model group. Bcl-2expression of model group was decreased when compared to normal group, SiRNA group showed further decrease compared with model group expression (P<0.01), there is no significant difference on BCL-2expression between negative control group and model group. Bax/Bcl-2ratio was significantly increased when comparison was made through the model group and normal group (P<0.01), there was further increase in SiRNA group, but no significant difference between negative control group and normal group..
Conclusion:
1, H2O2can inhibit cardiac activity, and promote apoptosis of cardiac myocyte, resulting in the destruction of the oxidative balance system. Notoginsenoside R1can inhibit H2O2-induced myocardial injury, enhance myocardial cell viability, cell antioxidant activity, and protect cardiac myocyte from apoptosis.
2. H2O2enhanced myocardial cells phosphorylation of ERK, JNK, P38proteins. Notoginsenoside R1reduced phosphorylation of JNK after H2O2intervention of myocardial cells, but did not have significant regulation on ERK and P38phosphrylation. Bax expression decreased, while Bcl-2expression increased simultanteously in H2O2intervention after pretreatment of notoginsenoside R1. It's suggested that Notoginsenoside R1protect cardiomyocyte from apoptosis through it's regulation of the ERK signaling pathway and apoptosis-related proteins.
3. H2O2can induce expression of Pim-2kinase in myocardial cells. Pim-2siRNA interference can effectively silencens Pim-2gene, and blocked its protective effect on myocardial injury.
4. Pim-2expression in the myocardial cells increased by Notoginsenoside R1in H2O2intervention. Notoginsenoside R1can still prevent cardiomyocyte apoptosis dispite of SiRNA interference of Pim-2, indicating that functions of notoginsenoside R1against myocardial cell apoptosis is not only regulated by Pim-2, but also through variety of apoptosis-related proteins such as Bax and Bcl-2.
近年来,国内外大量研究文献都表明氧化应激是许多病理因素造成心血管损伤的共同机制,在许多心血管疾病的病理过程中都有过量氧自由基产生,同时抗氧自由基防御机制却受到抑制,如动脉粥样硬化(AS)、高血压病、缺血性心脏病、高脂血症等。氧化应激损伤心肌细胞的转归与氧化应激的程度相关,在一定应激强度范围内,氧化应激对心肌细胞造成的损伤是可逆的,而超过一定限度,则对心肌细胞造成不可逆的损伤,主要包括心肌细胞的凋亡和坏死。
原癌基因Pim(Proviral integration site of murine)家族是哺乳动物细胞中的丝/苏氨酸激酶,属于钙调蛋白依赖性的蛋白激酶,包括Pim-1、Pim-2和Pim-3。大量的实验研究已证实,Pim激酶的上调与细胞凋亡的抑制存在显著的相关性。Pim-2持续表达能够促进细胞长期抵抗各种凋亡信号的刺激。研究证实Pim-1在Akt通路中保护心肌细胞损伤,而在Pim-1缺失小鼠中Pim-2的表达增高了4.6倍；Pim-1缺失小鼠在心梗后7天,Pim-2的表达增高2.75倍。但Pim-2在心肌细胞中的表达及作用尚缺乏研究,有必要探讨Pim-2在心肌细胞损伤中的作用及机制。
三七是具有多种心血管活性的中药,临床广泛应用于心血管疾病的防治。现代化学和药理学研究发现三七总皂苷(Panax notoginseng saponin, PNS)是三七的主要活性成分,含有多种单体皂苷主要有：人参皂苷Rb1,人参皂苷Rg1,三七皂苷R1。本课题以三七皂苷R1为研究对象,旨在了解三七皂苷R1对心肌细胞损伤的保护作用,并探讨其参与调节的细胞信号通路,凋亡相关蛋白,并了解其对Pim-2的调控作用。
二、方法：
1.乳鼠心肌细胞的原代培养外科无菌条件下取出乳鼠心脏,用胰酶40C过夜,终止消化,再用胶原酶消化液逐步消化法分离单个心肌细胞,接种至培养瓶中,采取差速贴壁分离法以去除心肌成纤维细胞,得到纯度较高的心肌细胞用于实验。
2.H202致心肌细胞氧化应激模型建立心肌细胞正常培养2-3天后,选取细胞密度在80%-95%以上的,自律性搏动120～140次/min的心肌细胞用于实验,选取不同浓度与时间点的H2O2干预心肌细胞,选择合适的浓度及干预时间用于后续实验。
3.三七皂苷R1预处理心肌细胞心肌细胞培养的第2-3天,细胞生长接近80%融合时,心肌细胞预先用不同浓度三七皂苷R1培养24h,进行三七皂苷R1毒性测试,选取合适的高、中、低剂量组进行相关后续实验。
4.MTT比色法测定心肌细胞活力将心肌细胞接种于九十六孔板,按实验分组给予相应的处理因素后,按MTT法具体操作步骤进行操作,在酶标仪490nm处测其OD值并计算各组心肌细胞存活率。
5.流式细胞术检测心肌细胞凋亡率心肌细胞按照实验分组处理后,用不含EDTA的胰酶消化收集,以Annexin V-FITC/Propidium Iodide进行染色,室温、避光、反应5～15min,上流式细胞仪(激发波长488nm,发射波长530mm)进行检测。
6.心肌细胞LDH、MDA、SOD的检测将心肌细胞接种于六孔培养板中,按不同实验目的进行分组处理,处理完毕后,分别按照LDH、MDA、SOD检测试剂盒说明书进行操作,在酶标仪420nm处测量每组OD值并计算各组细胞内LDH、MDA、SOD的含量。实验重复三次以上。
7.实时定量PCR检测心肌细胞内Pim-2mRNA的表达心肌细胞按照2×107/孔的浓度接种至六孔板中,取正常培养2-3天后状态最佳的心肌细胞用于实验。实验分组依次为：正常对照组、H2O20.5、1、2、3、6h五个时间组,50μMH2O2干预心肌细胞。用Trizol提取心肌细胞总RNA,在紫外分光光度计上测定OD260/OD280,鉴定提取的RNA纯度和浓度。然后,反转录合成cDNA,根据Gene Bank中Pim-2、GAPDH的基因序列资料,借助于计算机软件PrimerPremier5.0设计引物,上机进行目的基因的荧光定量检测。
8. SiRNA沉默心肌细胞Pim-2基因调合适的细胞浓度分别接种于六孔板或九十六孔板中,正常培养2-3天左右,当贴璧细胞达到80%-90%融合时,取状态良好的细胞进行实验。siPORTTM NeoFXTM转染试剂与DMEM培养基混匀室温孵育15min, siRNA稀释液等体积混匀,孵育15min。然后将混合液加入培养板混匀。转染心肌细胞48-72h后收集细胞,再进行Western blot实验和流式细胞术检测心肌细胞凋亡率。
9. Western blot法检测心肌细胞蛋白的表达心肌细胞按实验分组处理完成后,以Western及IP细胞裂解液提取心肌细胞蛋白,BCA法进行蛋白定量,进行蛋白印迹实验。以Actin为内参,将处理好的蛋白质样品进行SDS-PAGE分离,电泳结束后将蛋白质转移到PVDF膜上,封闭后,孵育一抗和二抗,ECL显色,KODAK Image Station2000MM成像系统采集图像,获得图像用图像处理软件Image Tool3.0测定并分析条带灰度值以检测目的蛋白的表达水平。
10.统计分析实验数据采用SPSS13.0统计软件进行分析。计量资料数据以x±s表示。对数据进行正态性检验和方差齐性检验。若符合正态分布和方差齐性,则采用方差分析。若不符合正态分布和方差齐性,则采用秩和检验。方差齐性时两两比较采用LSD法,方差不齐时两两比较采用Tamhane's T2法。多组计数资料的比较(各组AV评分)采用完全随机设计资料的Kruskal-Wallis检验方法。各实验组不同缺血及再灌注时间点血流动力学指标采用重复测量方差分析。检验显著性水准a=0.05。
三、结果：
1.三七皂苷R1对H2O2诱导的心肌细胞损伤的作用
1.1H2O2对心肌细胞损伤的浓度效应关系MTT比色法检测不同浓度H2O2干预心肌细胞3h后,细胞活力显著降低,光镜下可看到细胞搏动减弱甚至停止,细胞密集区域出现大片细胞脱落。与正常对照组比较,H202干预的各组细胞活力均有所下降,经统计分析各组差异均有统计学意义(F=734.372,P<0.001),且随着H202浓度的增加各组细胞活力出现递减趋势。
1.2不同浓度三七皂苷R1对心肌细胞活力的影响不同浓度三七皂苷R1预处理心肌细胞24h,比较各浓度三七皂苷R1作用后细胞活力与正常对照组的差异。经统计分析,三七皂苷R1浓度为0.1×10-6mol/L、1×10mol/L、10×10-6mol/L时细胞活力与正常对照组比较差异无统计学意义(P均>0.05),而浓度为50×10-6mol/L、100×10-6mol/L时,作用心肌细胞24h后细胞活力较正常对照组显著降低(P<0.001)。
1.3三七皂苷R1预处理对H2O2干预的心肌细胞活力的影响三七皂苷R1低、中、高浓度(0.1×10-6mol/L、1×10-6mol/L、10×10-6mol/L)预处理心肌细胞24h,然后以H2O2(50pmol/L)作用细胞3h,同时设立正常对照组、模型组,比较各不同浓度三七皂苷R1对H2O2诱导的心肌细胞活力的影响。经统计分析表明,各组间细胞活力差异有统计学意义((F=519.758,P<0.001)。三七皂苷R1预处理后可呈浓度依赖性减轻H2O2处理后心肌细胞活力的下降。
1.4三七皂苷R1预处理对H2O2干预的心肌细胞凋亡率的影响流式细胞术检测各组心肌细胞凋亡率,比较各不同浓度三七皂苷R1对H2O2诱导的心肌细胞凋亡率的影响。经三七皂苷R1预处理后,各组细胞凋亡与模型组相比有所减少,尤其是高浓度组的凋亡情况显著改善。经统计分析,各组差异均有统计学意义(P<0.0001)。
1.5三七皂苷R1预处理对H2O2干预的心肌细胞LDH、MDA、SOD的影响试剂盒检测各组心肌细胞LDH、MDA、SOD值,结果与正常对照组比较,模型组心肌细胞SOD活性显著降低,而LDH活性、MDA含量则显著增加(P<0.001)；三七皂苷R1可呈剂量依赖性增加SOD活性,降低LDH活性、MDA含量,各组间比较差异具有统计学意义(P<0.001)。
2三七皂苷R1对H2O2诱导的心肌细胞凋亡信号通路、凋亡相关蛋白表达的影响
2.1三七皂苷R1预处理对H202干预的心肌细胞MAPK信号通路的影响
2.1.1Western Blot检测各组细胞中P-ERK1/2.总ERK1/2蛋白表达的变化,分析目的条带的光密度值(IOD)。与正常组相比,模型组(H2O2) P-ERK1/2表达显著提高,三七皂苷R1低、中、高剂量组与模型组相比P-ERK1/2表达显著降低,其中高剂量组表达降低最为显著。各组间P-ERK1/2表达存在显著差异(P<0.01)。
2.1.2Western Blot检测各组细胞中P-JNK、总JNK蛋白表达的变化,分析目的条带的光密度值(IOD)。与正常组相比,模型组(H2O2) P-JNK表达显著提高(P<0.001),三七皂苷R1低、中、高剂量组与模型组相比表达无显著差异(P>0.05)。
2.1.3Western Blot检测各组细胞中P-P38、总P38蛋白表达的变化,分析目的条带的光密度值(IOD)。与正常组相比,模型组(H2O2) P-P38表达显著提高(P<0.001),三七皂苷R1低、中、高剂量组与模型组相比P-P38表达无统计学意义(P>0.05)。
2.2三七皂苷R1预处理心肌细胞凋亡蛋白Bax/Bcl-2表达的变化用Western Blot检测各组细胞中Box、Bcl-2蛋白表达的变化,分析目的条带的光密度值(IOD),正常组Bax、Bcl-2有一定量的表达,与正常组相比,模型组Bax表达显著增高,三七皂苷R1低、中、高剂量组与模型组相比表达逐渐降低(P<0.001)；与正常组相比,模型组Bcl-2表达量降低,三七皂苷R1低、中、高剂量组与模型组相比表达逐渐增高(P<0.001)。与正常组相比,模型组Bax/Bcl-2值显著增高,三七皂苷R1低、中、高剂量组与模型组相比表达逐渐降低(P<0.001)。
3Pim-2在H202诱导的心肌细胞损伤中的表达及作用
3.1H202显著上调心肌细胞内Pim-2mRNA的表达用浓度为50μmol/L的H202干预心肌细胞0.5、1、2、3、6h,实验设正常对照组。应用实时荧光定量PCR的方法检测各组心肌细胞内Pim-2mRNA的表达时发现,H202干预后的心肌细胞Pim-2mRNA的表达有所增加,各组差异有统计学意义(P<0.001)。其中H2O20.5、1h组与正常对照组相比,差异无统计学意义(P=0.056)；其余各浓度组与正常对照组相比,差异均有统计学意义(P<0.001),尤其是H2023h组Pim-2mRNA的水平增加最显著；H2O26h组心肌细胞Pim-2mRNA表达水平有所降低,但仍高于正常对照组。
3.2H202显著上调心肌细胞内Pim-2蛋白的表达应用Western blot法检测H202干预不同时间后心肌细胞内Pim-2的蛋白水平发现,H202干预后的心肌细胞Pim-2蛋白表达增高,其增高的趋势与实时荧光定量PCR检测出的Pim-2mRNA增高趋势一致,各组差异有统计学意义(P<0.001)。
3.3Pim-2siRNA干扰对细胞凋亡的影响实验分为正常组,H202组,siRNA组(H2O2+siRNA),阴性对照组(H2O2+NC)。结果显示模型组与正常组相比,细胞凋亡率显著增加,siRNA干扰组细胞凋亡率进一步增加(P<0.001),阴性对照组与模型组相比无显著差异(P>0.05),与正常组相比显著增加(P<0.001)。
3.4Pim-2siRNA干扰对细胞LDH、MDA、SOD的影响心肌细胞经Pim-2siRNA干扰后,心肌细胞SOD活性较模型组显著降低,而LDH、MDA含量较模型组显著增高,各指标组间差异具有统计学意义(P<.001),阴性对照组与模型组相比差异无统计学意义(P>0.05)。
4Pim-2蛋白表达调控
4.1三七皂苷R1上调Pim-2蛋白的表达通过Western Blot技术检测各组心肌细胞Pim-2蛋白表达,结果显示模型组(H202)与正常组相比Pim-2表达显著增高,三七皂苷R1各剂量组Pim-2表达进一步增高,其中三七皂苷R1高剂量组表达增高最为显著,各组间差异有统计学意义(P<0.01)。
4.2Pim-2siRNA干扰对三七皂苷R1预处理心肌细胞凋亡率的影响实验分为正常组、模型组(H202)、三七皂苷R1组(H2O2+NG R110μM)、siRNA转染组(H2O2+NG R110μM+siRNA)、siRNA阴性对照组。流式细胞术检测各组细胞凋亡率,结果模型组与正常组相比凋亡率显著增高(P<0.001),三七皂苷R1组与模型组相比显著下降,siRNA组、siRNA阴性对照组与三七皂苷R1组相比无显著差异(P>0.05)。
4.3Pim-2siRNA干扰对心肌细胞Bax/Bcl-2表达的影响实验分为正常组,H202组,siRNA组(H2O2+siRNA),阴性对照组。用WB检测各组细胞中Bax、 Bcl-2蛋白表达的变化,分析目的条带的光密度值(IOD),与正常组相比,模型组Bax表达显著增高,siRNA组与模型组相比表达进一步增高(P<0.01),阴性对照组与模型组相比Bax表达无显著差异。与正常组相比,模型组Bcl-2表达量降低,siRNA组与模型组相比表达进一步降低(P<0.0I),阴性对照组与模型组相比无显著差异。模型组与正常组相比,Bax/Bcl-2值显著增高,siRNA组进一步增高(P<0.01),阴性对照组无显著差异。
四、结论：
1、H202能抑制心肌细胞活力,促进心肌细胞凋亡的发生,导致氧化平衡体系的破坏。三七皂苷R1可以抑制H202诱导的心肌细胞损伤,增强心肌细胞活力,保护心肌细胞凋亡,增强细胞抗氧活酶活力。
2、H202促进心肌细胞ERK、JNK、P38的磷酸化,三七皂苷R1下调H202干预的心肌细胞中ERK的磷酸化,而对JNK、P38的磷酸化无显著调控。三七皂苷R1下调H2O2干预的心肌细胞中Bax蛋白的表达,上调Bcl-2蛋白的表达。表明三七皂苷R1抗心肌细胞凋亡与调节ERK信号通路、凋亡相关蛋白有关。
3、H202能诱导心肌细胞中Pim-2激酶的表达,Pim-2siRNA干扰可有效沉默Pim-2基因,Pim-2蛋白表达对心肌细胞损伤其保护作用。
4、三七皂苷R1上调H202干预的心肌细胞中Pim-2的表达,但siRNA干扰Pim-2后,三七皂苷R1仍能抗心肌细胞凋亡,表明三七皂苷R1抗心肌细胞凋亡并非唯一通过Pim-2调控。Pim-2能调节H2O2干预的心肌细胞中凋亡相关蛋白Bcl-2的表达。
Objective:
In recent years, a large number of study both at home and abroad have shown that oxidative stress related apoptosis is the common mechanism of cardiovascular damage caused by many different pathological factors. There are excessive oxygen free radicals generated in many pathological processes of cardiovascular diseases, such as atherosclerosis (AS), hypertension, ischemic heart disease, hyperlipidemia and so on, while the anti-oxidative defensive mechanism in these processes are inhibited. The damage of myocardial cells cause by oxidative stress is relevant to its intensity. It's reversible in a certain stress intensity range. But the damage can become irreversible, which mainly includes myocyte apoptosis and necrosis, if beyond the certain range.
The proto-oncogene Pim (Proviral integration site of murine) family in mammalian cells is serine/threonine kinase belongings to the calmodulin-dependent protein kinase, including Pim-1, Pim-2and Pim-3. Many experimental studies have confirmed the existence of a significant correlation between the upregulation of Pim kinase and inhibition of apoptosis. Pim-2expression continued to promote long-term cell resistance to various apoptosis signal stimulation. It's confirmed that Pim-1protects myocardial injury via Akt pathway, the expression of Pim-2in Pim-1deficient mice increased4.6times;7days after myocardial infarction of Pim-1-deficient mice, Pim-2expression increased2.75times. But Pim-2expression and function in myocardial cells is still unknown, it is necessary to explore it's function and mechanism of myocardial injury.
Radix Notoginseng is a medicine with a variety of cardiovascular activity, it's widely used in clinical in the prevention and treatment of cardiovascular disease. With the advance of modern chemistry and pharmacology, Panax notoginseng saponins (Panax notoginseng saponin, PNS) is identified to be the main active ingredient of the Radix Notoginseng, contains a variety of monomer saponin, such as ginsenoside Rbl, ginsenoside Rgl, the notoginsenoside R1. Focusing on notoginsenoside R1in this study, our purpose is to understand the protective effect of notoginsenoside R1in myocardial injury, and to investigate the cell signaling pathways and proteins involved in apoptosis regulation, and Pim-2effect in notoginsenoside R1protection.
Method:
1. Primary culture of neonatal rat cardiomyocytes
Neonatal rats' hearts were removed in surgical sterile conditions, digested in trypsin in4℃overnight, and then gradually digested the heart tissue with collagenase into isolated cells. Cells were inoculated into a culture flask. Differential adhesion separation method was used to remove cardiac fibroblasts in order to obtain pure myocardial cells in this study.
2. Model of H2O2-induced oxidative stress in myocardial cells
Normal myocardial cells were cultured for2-3days, proliferated to reached the density of80%-95%. Cells that pulsed at the rate120～140times per minute were selected for the use of experimental myocardial cells. Different time points and concentrations were tested for H2O2intervention. Proper concentration of H2O2and time point to intervene was used in follow-up experiment.
3. Notoginsenoside R1myocardial preconditioning
Cultured myocardial cells first2-3days, cell growth close to80%confluence, myocardial cells pretreated with different concentrations notoginsenoside R1cultured24h, toxicity tests conducted Notoginsenoside R1, select the appropriate high, medium and low dose groups related follow-up experiments.
4. Assessment of cell viability (MTT assay)
Cardiomyocytes were seeded in96-well culture plates at a density of1×104cells/well, were incubated at37℃for48h, and then pretreated with different drugs depend on experimental groups. After that, the cells were washed with phosphate-buffered saline (PBS) three times and MTT dye was added to each well for the last4h of treatment. The reaction stopped with the addition of dimethyl sulfoxide (DMSO) and the optical density was determined at490nm on a multi-well plate reader. All groups were assayed in six wells and the mean for each group was calculated.
5. Analysis of cardiomyocyte apoptosis with flow cytometry
Myocardial cells according to the experimental groups were digested by EDTA-free trypsin, stained with to Annexin V-FITC/Propidium Iodide, incubated at room temperature and protected from light for5～15min, analysed by flow cytometry (excitation wavelength488nm, emission wave length530nm).
6. Detection the level of intracellular LDH、MDA、SOD in myocardial cells
Myocardial cells were seeded in six wells, grouped by different experimental treatment. Analysises were done in accordance with the instruction of LDH, MDA, SOD detection kit after treatment, each set of OD values measured at420nm in a microplate reader and the contents of LDH, MDA, SOD in every sample was calculated according to these values. Each experiment was repeated three times.
7. Real-time quantitative PCR analysis of myocardial cells Pim-2mRNA expression.
Cardiomyocytes were seeded to6-well plates2×107/well, cultured for2-3days. Cells were collected in optimal condition for the following experiment. Samples were divided as following groups:normal groups, control groups,0.5,1,2,3,6hours H2O2treated group,50μM H2O2was used for myocardial cells. The total RNA of cardiomyocytes was extracted with Trizol, purity and concentration of the extracted RNA was measured on a UV spectrophotometer. Then cDNA was synthesized by reverse transcription, and fluorescence quanititavie dectection of the target gene was performed afterwards. The primer of Pim-2gene was designed according to Gene Bank sequence imformation, by means of Primerpremier5.0software.
7. SiRNA silencing myocardial cells'Pim-2expression.
Cells were cultured in six wells and ninty-six wells for2or3days to reach80%～90%confluent, cells of optimal condition were used for further expriments. SiPORTTM NeoFXTM was incubated with DMEM and SiRNA diluent in the same volume for15min at room temperature. The mixture was added into the culture plates for transfection for next48～72h.After that cells were collected for Western blot analysis and flow cytometry analysis of cardiomyocyte apoptosis rates.
8. Western blot analysis of the protein expression of myocardial cells. Myocardial cells were solubilized after complication of the treatment by using the Western or IP cell lysates, protein quantitation was performed by using BCA method before the extrats went for immunoblotting analysis. Total proteins were separated on a SDS-PAGE gel, and transferred to a PVDF membrane after electrophoresis, incubated with primary and secondary antibody in turn after the membrane was blocked, signal was developed by using ECL substrate, acquired image by KODAK Image Station2000MM system, analysis the image with Image Tool3.0, the gray values of the lanes were measured and quantization as the expression of target proteins. Actin was used as loading control in this assay.
10. Statistical analysis:The data was analyzed by SPSS13.0. measurement data was presented as the means±SEM. Test of normality and homogeneity of variance was done in each group. If datas accorded with normality and homogeneity of variance, statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Tamhane's T2. Multiple enumeration data was analyzed by Kruskal-Wallis. Hemodynamics parameters in different time points was analyzed by repetitive measurement and analysis of variance. Level of significance a=0.05
Result:
1. Notoginsenoside Rl roles in H2O2-induced myocardial injury
1.1The concentration-response relationship of myocardial cells injury to H2O2. MTT assay shows that after treating the cardiomyocytes with different concentrations of H2O23hours, viability of cells were evidently reduced. Cells pulse became slower or even stop when observed through the microscope, there are large pieces of cells shedding in the area that cells was relative crowded. Compared to the normal control,cell viability after H2O2intervention at various concentration were all reduced, the statistical analysis of the difference between each group were statistically significant (F=734.372,P<0.001), and cell viability were more apparently reduced with higher concentration of H2O2.
1.2The different concentrations of notoginsenoside R1on myocardial cell viability. Myocardial cells were preconditioned with different concentrations of notoginsenoside for24hours. Comparisons of cell viability were made between groups treated with different concentrations of notoginsenoside R1and the normal control group. Statistical analysis showed that there are no statistically difference of cell viablility between groups treated with0.1×10-6mol/L,1×10-6mol/L,10x10-6mol/L notoginsenoside R1and groups of normal control (P>0.05), while the concentration of100×10-6mol/L groups' viability were significantly lower than controls after treating myocardial cells for24hours (P<0.01).
1.3Pretreatment of Notoginsenoside R1on myocardial cell viability in H2O2injury models. Myocardial cells were pretreated with low, medium and high concentrations (0.1×10-6mol/L,1×10-6mol/L,10×10-6mol/L) of notoginsenoside for24hours, then H2O2(SOμmol/L) was added for3hours, normal control group and model group were set up at the same time. Comparisons of myocardial cells viability were made between groups of different concentrations notoginsenoside R1in H2O2injury models. The statistical analysis showed that the cell viability difference between the groups was statistically significant ((F=519.758, P<0.001). Notoginsenoside R1pretreatment reduced the decline of myocardial cell viability in a dose-dependent manner after H2O2treatment.
1.4Pretreatment of Notoginsenoside R1reduced the apoptosis rate of myocardial cell in the H2O2injury model. The apoptotic rates detected by flow cytometry were used to compare the effects of notoginsenoside R1of multiple concentrations in H2O2-induced models. After pretreatment by notoginsenoside R1, the apoptotic rates of cells were lower campared to the model group, especially in high concentration groups, with significantly improvement of apoptotic rates, After statistical analysis, differences of each groups were statistically significant (P <0.0001).
1.5Activities of LDH, MDA and SOD in notoginsenoside R1pretreated myocardial cells of H2O2intervention. Compared with the normal control group, SOD activity of model groups were significantly reduced, while the activity of LDH, MDA was significantly increased (P<0.001). Notoginsenoside R1lowered SOD activity in a dose-dependent manner, while increased LDH and MDA activity. The difference among the groups was statistically significant (P<0.001).
2Notoginsenoside R1effects on H2O2-induced myocardial apoptosis signaling pathways and apoptosis-related protein expression
2.1Notoginsenoside R1effects on H2O2intervention myocardial cells MAPK signaling pathway
2.1.1Western Blot detection of protein expression of p-ERK1/2, total ERK1/2in each group and analysis of the target bands by optical density (IOD). Compared with normal group, model group (H2O2) p-ERK1/2expressions were significantly increased. P-ERK1/2expression in groups of low, medium and high dose of notoginsenoside R1pretreatment were significantly reduced compared to the modele groups, especially in of the high-dose groups. There are significant differences of p-ERK1/2expression among the groups (P<0.01).
2.1.2Western Blot detection of protein expression of P-JNK, total JNK in each group and analysis of the target bands by optical density (IOD). Compared with the normal group, model group (H2O2) P-JNK expression was significantly increased (P <0.001). There are no significant difference between groups of multiple concentrations of notoginsenoside Rl pretreatment and model groups (P>0.05).
2.1.3Western Blot detection of protein expression of P-P38, P38in each group and analysis of the target bands by optical density (IOD). Compared with normal group, model group (H2O2) P-P38expression was significantly increased (P<0.001), notoginsenoside R1low. There are no significant difference between groups of multiple concentrations of notoginsenoside R1pretreatment and model groups (P>0.05).
2.2Changes of expression of Bax/Bcl-2apoptotic proteins of cardiomyocytes after pretreatment of notoginsenoside R1. Bax/Bcl-2expression was detected by Western Blotting analysis in each group; analyses of the target band optical density (IOD) were performed aferward. Normal groups have a certain amount of Bax, Bcl-2expression, Bax expression of model groups were significantly higher than the normal groups, notoginsenoside R1groups have a decreased Bax expression compared to model groups (P<0.01); Compared with the normal group, model groups have reduced Bcl-2expression. Notoginsenoside R1groups expression of BCL-2were gradually increased compared to model groups (P<0.01). The Bax/Bcl-2ratio of model groups was significantly higher to normal groups, notoginsenoside R1groups have reduced Bax/Bcl-2compared to model groups (P<0.01).
3Pim-2expressions and its role in H2O2-induced myocardial injury
3.1H2O2intervention significantly upregulated Pim-2mRNA expression in myocardial cells.50mM/L H2O2was addd to myocardial cells cultures for0.5,1,2,3,6h, the normal control group was setted up at the same time. Pim-2mRNA expression analysed by real-time quantitative PCR. It's showed that Pim-2mRNA expression increased in groups after the intervention of H2O2.There are statistically significant between groups (P<0.001). Comparsions between each groups were statistically analysed too.Which0.5h and1h groups showed no significant difference compared to the control group (P>0.05); the remaining groups were significant different between each others (P<0.0001), especially in3h, Pim-2mRNA level increased to the most obvious level;6h groups showed reduced Pim-2mRNA expression, but the level was still higher than the normal control group.
3.2H2O2intervention significantly increased Pim-2protein expression in myocardiocytes. Pim-2protein expressions were increased after H2O2intervention, indicated by Westernblotting analysis of Pim-2protein expression at different time points. The growth of Pim-2expressions were correlation with the growth of mRNA expression analysed by real-time fluorescence quantitative PCR. The difference between each groups were statistically significant (P<0.01).
3.3Pim-2siRNA interference on apoptosis of myocardial cells. Samples of myocardial cells cultures were randomly divided into normal group, H2O2group, siRNA group (H2O2+siRNA), and negative control group (H2O2+NC). The results show model group compared with the normal group, the apoptosis rate of model group was increased significantly compared to the normal control group. The siRNA interference further increased the apoptosis rate (P<0.001), negative control group was significant different compared to the normal group (P<0.001), but no statistical difference when compared to the model group (P>0.05).
3.4Impact of Pim-2siRNA interference on activity of LDH, MDA and SOD. SOD activity after Pim-2siRNA interference was significantly reduced compared with the model group, while LDH, MDA content compared with the model group was significantly increased, the differences between groups has statistical significance (P <0.001). Indexes of negative control group were not statistically significant (P>0.05) when compared with the model group.
4Pim-2protein expression and regulation
4.1Notoginsenoside R1increased Pim-2protein expression. Pim-2protein expression of myocardial cells was analysed by Western Blotting, the results showPim-2expression of the model group (H2O2) was significantly higher than the normal group, notoginsenoside R1of different concentration further increased the Pim-2expression, the most Pim-2expressoin was observed in the high dose notoginsenosdie R1group. Difference among the groups was statistically significant (P<0.01).
4.2Pim-2siRNA interference on notoginsenoside Rl pre-treatment H2O2injured cardiac myocyte. Samples of myocardial cells were divided into normal group, model group (H2O2), notoginsenoside R1group (H2O2+NG Rl10μM), siRNA transfection group (H2O2+NG R1lOμM+siRNA), siRNA negative control group. Apoptosis rates were analysed by flow cytometry, the result showed that the model group was significantly higher compared with the normal group (P<0.001), apoptotic rates in oginsenoside R1group were decreased compared with the model group. There are no significant difference between siRNA group, siRNA negative control group and notoginsenoside R1group (P>0.05).
4.3Pim-2siRNA interference on Bax/Bcl-2protein expressons. Samples of myocardial cells were divided into normal group, H2O2group, siRNA group (H2O2+siRNA), and negative control group. Western blotting was used to detect the expression in Bax, Bcl-2protein, the target bands were analysed with optical density (IOD). The expression of Bax in model group was significantly higher than normal control group. SiRNA group showed further increase compared with model group expression (P<0.01), there is no significant difference on Bax expression between negative control group and model group. Bcl-2expression of model group was decreased when compared to normal group, SiRNA group showed further decrease compared with model group expression (P<0.01), there is no significant difference on BCL-2expression between negative control group and model group. Bax/Bcl-2ratio was significantly increased when comparison was made through the model group and normal group (P<0.01), there was further increase in SiRNA group, but no significant difference between negative control group and normal group..
Conclusion:
1, H2O2can inhibit cardiac activity, and promote apoptosis of cardiac myocyte, resulting in the destruction of the oxidative balance system. Notoginsenoside R1can inhibit H2O2-induced myocardial injury, enhance myocardial cell viability, cell antioxidant activity, and protect cardiac myocyte from apoptosis.
2. H2O2enhanced myocardial cells phosphorylation of ERK, JNK, P38proteins. Notoginsenoside R1reduced phosphorylation of JNK after H2O2intervention of myocardial cells, but did not have significant regulation on ERK and P38phosphrylation. Bax expression decreased, while Bcl-2expression increased simultanteously in H2O2intervention after pretreatment of notoginsenoside R1. It's suggested that Notoginsenoside R1protect cardiomyocyte from apoptosis through it's regulation of the ERK signaling pathway and apoptosis-related proteins.
3. H2O2can induce expression of Pim-2kinase in myocardial cells. Pim-2siRNA interference can effectively silencens Pim-2gene, and blocked its protective effect on myocardial injury.
4. Pim-2expression in the myocardial cells increased by Notoginsenoside R1in H2O2intervention. Notoginsenoside R1can still prevent cardiomyocyte apoptosis dispite of SiRNA interference of Pim-2, indicating that functions of notoginsenoside R1against myocardial cell apoptosis is not only regulated by Pim-2, but also through variety of apoptosis-related proteins such as Bax and Bcl-2.
引文
[1]J. L. Martind ale, N.J. H olbrook. Cellular response to oxidative stress:sig naling for suicid e and survival [J]. Cell Ph y siol,2002,192(1):1-15.
[2]徐方平,罗健东.ROS与心肌细胞凋亡[J].华北国防医药,2003,15(2)：90-93.
[3]易龙,et al.,花色苷类植物化学物抑制血管内皮细胞氧化应激损伤作用的结构一效应关系研究.第三军医大学学报DSDX,2009(21).
[4]廖德荣,et al.,血管内皮生长因子对过氧化氢诱导的内皮细胞凋亡的影响及其机制.中国动脉硬化杂志KDYZ,2006(06).
[5]Halliwell B, Gutteridge J M, Free radicals in biology and medicine [M] 12nd ed, Oxford:Clarendon Press,1989.121051
[6]Lips DJ, Bueno OF, Wilkins BJ, et al. MEK1-ERK2 signaling pathway protects myoeardium from ischemic injury in vivo[J]. Cireulation.2004。109(16): 1938-1941.
[7]Cuevas BD, Abell AN, Johnson GL. Role of mitogen activated protein kinase kinase kinases in signal integration [J]. Oncogene,2007.26(22):3159-3171.
[8]Bonni A, Brunet A, West AE, et al. Cell Survival Promoted by the Ras-MAPK Signaling Pathway by Transcription-Dependent and -Independent Mechanisms [J]. Science,1999,286(5443):1358-1362.
[9]Baines CP, Zhang J, Wang GW, et al. Mitochondrial PKC epsilonand MAPK form sign aling modules in tile murine heart:enhancedmito chondrial PKC epsilon-MAPK interactions and diferential MAPK activation in PKCepsilon-induced eardiopreteetion [J]. CireRes.2002,90(4):390-397.
[10]Casey J F, Peter S H, Kyan M C,et al. The Serin/t heronine Kinase Pim-2 is A Transcriptionally Regulated Apoptotic Inhabitor [J]. Genes Dev,2003,17: 1814-1854.
[11]Peng C, Knebel A,Morrice N A,et al. Pim Kinase Substrate Identification and Specificity [J]. J Biochem (Tokyo),2007,141 (3):353-362.
[12]Dai H,Li R,Wheeler T,et al. Pim-2 Up regulation:Biological Implications Associated with Disease Progression and Perinueral Invasion in Prostate Cancer [J]. Prostate,2005,65 (3):276-286.
[13]Muraski JR., Rota M, Misao Y, et al. Pim-1 regulates cardiomyocyte survival downstream of Akt[J]. Nat Med,2007,13(12):1467-1475.
[14]Dan L, Ming H, Bo Y, et al. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway[J]. Bio & Cell Bio,2009,41:2315-2322
[15]Jung J I, Lim SS, Choi HJ-etal.Isoliquiritigenin apoptosis by Depolarizing mitochondrial membranes in prostate cancer cells [J]. J Nutr Biochem,2006,17 (10):689-696.
[16]Kim BC, Mamura M, Choi KS, et al. Transforming growth factor betal induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells [J]. Mol Cell Biol,2002,22 (5):1369-1378.
[17]Grethe S, Coltella N, Di Renzo MF, et al. p38 MAPK downregulates phosphorrylation of Bad in doxorubicin-induced endothelial apoptosis[J]. Biochem Biophys Res Commun,2006,347 (3):781-790.
[18]Korsmeyer SJ, Wei MC, Saito M, et al. Pro2apop totic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c [J]. Cell Death Differ,2000,7 (12):1166-1173.
[19]Bachmann M, Moroy T. The serine/threonine kinase Pim-1. Int J Biochem Cell Biol,2005,37:726-730.
[20]Morishita D, Katayama R, Sekimizu K, et al. Pim kinases promote cell cycle progression by phosphorylating and down-regulating p27Kipl at the transcriptional and posttranscriptional levels[J]. Cancer Res,2008,68(13): 5076-5085.
[21]Bin Y, Marina Z, Sheldon H, et al. The Pim-2 Kinase Phpsphorylates BAD on Serine112 and Reverses BAD-induced Cell Death [J]. J Biol Chem,2003,278 (46):45358-45367.
[22]White E. The Pims and Out s of Survival Signaling:Role for the Pim-2 Protein Kinase in the Suppression of Apoptosis by Cytokines[J]. Genes Dev,2003,17: 1813-1816.
[23]Bohensky J, Shapiro I M, Leshinsky S, et al. Pim-2 is an Independent Regulator of Chondrocyte Survival and Autophagy in the Epiphyseal Growt h Plate [J]. J Cell Physiol,2007,213 (1):246-251.
[24]Casey J F, Peter S H, Kyan M C,et al. The Serin/theronine Kinase Pim-2 is A Transcriptionally Regulated Apoptotic Inhabitor [J]. Genes Dev,2003,17 1814-1854.
[25]Peng C, Knebel A,Morrice N A,et al. Pim Kinase Subst rate Identification and Specificity [J]. J Biochem (Tokyo),2007,141 (3):353-362.
[26]Dai H,Li R,Wheeler T,et al. Pim-2 Up regulation:Biological Implications Associated wit h Disease Progression and Perinueral Invasion in Prostate Cancer [J]. Prostate,2005,65 (3):276-286.
[27]Muraski JR, Rota M, Misao Y, et al. Pim-1 regulates cardiomyocyte survival downstream of Akt[J]. Nat Med,2007,13(12):1467-1475.
[28]Dan L, Ming H, Bo Y, et al. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway [J]. Bio & Cell Bio,2009,41:2315-2322
[29]李家实.中药鉴定学[M].上海：上海科学技术出版社,1998：132.
[30]Dong TX, Cui XM, Song ZH, et al. Chemical assessment ofroots of Panax notoginseng in China:regional and seasonal variations in its active constituents[J]. J Agric Food Chen,2003,51:4617-4623.
[31]周晓慧,周晓霞,杨鹤梅.三七总皂甙防治动脉粥样硬化的研究进展.承德医学院学报[J],2003,20：350-352.
[32]雷蕾.血栓通对血液流变学影响的药理学研究[J].海峡药学,2007,19：35-36.
[33]钟尚乾,张焕风,黄永禄,等.血塞通软胶囊治疗急性脑梗死及对血液流变学的影响[J].中国药物与临床,2008,8：68-69.
[34]Garcia-Villalon AL, Amezquita YM, Monge L, et al. Mechanisms of the protective effects of urocortin on coronary endothelial function during ischemia-reperfusion in rat isolated hearts[J]. Br J Pharmacol,2005,145: 490-494.
[35]Hong-Sheng Z, Sheng-Qi W. Notoginsenoside R1 inhibits TNF-α-induced fibronectin production in smooth muscle cells via the ROS/ERK pathway [J]. 2006,40(9):1664-1674.
[36]Hong-Sheng Z, Sheng-Qi W. Notoginsenoside R1 inhibits TNF-α-induced PAI-1 production in human aortic smooth muscle cells [J].2006,44(9):224-230.
[37]del Rio LA, Sandalio LM, Corpas FJ, et al.Reactive Oxygen Species and Reactive Nitrogen Species in Peroxisomes. Production, Scavenging, and Role in Cell Signaling [J].Plant Physiol,2006,141(2):330-335.
[38]Zhang WX, Wan HJ, Zeng XH, et al. Protective effect of vitamin C on rat heart mitochondria injury induced by oxygen radicals [J].HarbinYike Daxue Xuebao(J Harbin Med Univ),2001,35(2):82-84.
[39]Benard G, Bellance N, James D, et al.Mitochondrial bioenergetics and structural network organization [J]. J Cell Sci,2007,120(5):838-848.
[40]Chen ZH, Saito Y, Yoshida Y, et al. Effect of oxygen concentration on free radical-induced cytotoxicity[J]. Biosci Biotechnol Biochem.2008,72(6): 1491-1497.
[41]Sodha NR, Clements RT, Feng J, et al.The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia-reperfusion injury[J].Eur J Cardiothorac Surg,2008,33(5):906-913.
[42]Billman GE.The calcium channel antagonist, flunarizine, protects against ventricular fibrillation [J].Eur J Pharmacol,1992,212(2-3):231-5.
[43]Bognar Z, Kalai T, Palfi A, et al. A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death [J].Free Radic Biol Med,2006,41(5):835-848.
[44]Singal PK, Khaper N, Palace V, et al. The role of oxidative stress in the genesis of heart disease [J]. Cardiovasc Res,1998,40(3):426-432.
[45]Sehirli O, Tozan A, Omurtag GZ, et al.Protective effect of resveratrol against naphthalene-induced oxidative stress in mice [J]. Ecotoxicol Environ Saf,2008,71(1):301-308.
[46].Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].Br J Cancer,1972;26(4):239-257.
[47].JAmes TN. Normal and abnormal consequences of apoptosis in the human heart.From postnatal morphogenesis to paroxymal arrhythmias [J]. Circul-ation, 1994,90(1):556-573.
[48].吴青,陶宏凯,陶大昌,等.灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究[J].中国心血管病研究杂志,2004,2(11)：905.
[49].宋治远,李永华,姚青,等.缺血再灌注对在体兔窦房结细胞凋亡及凋亡相关基因表达的影响[J].中国病理生理杂志,2003,19(12)：1606-1609.
[50].Xie Z.Koyama T, Suzuki J, et al.Coronary reperfusion following is hemia: different expression of Bcl-2 and Bax proteins,and cardiomyocyte apoptosis[J].Jpn heart J,2001,42(6):759-770.
[51].McClintock DS,Santore MT,Lee VY, et al.Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death [J].Mol Cell Biol,2002;22(1):94-104.
[52].Antonsson B, Conti F, Ciavatta A, et al. Inhibition of bax channel forming activity by bcl-2 [J]. Science,1997,277 (5324):370.
[53].Naumovski L, Cleary ML. The p53-binding 53 BP2 also inferacts with bcl-2 and impedes cell cycle progression at G2/M [J].Mol Cell Biol,1996,16:3884.
[54].Saleh A, Srinivasula SM, Balkir L,et al. Negative regulation of the Apaf-lapoptosome by Hsp70[J].Nat Cell Biol,2000,2(8):476.
[55].Skulachev VP.Cytochrome C in the apoptotic and antioxidant cascades [J]. FEBS Lett,1998,423(3):275.
[56].Misao J. Hayakawa Y,Ohno M, et al. Expression of bcl-2 protein, an inhibitor of apoptosis, and Bax, an accelerator of apoptosis, in ventricular myocytes of human hearts with myocardial infarction[J]. Circulation,1996,94(7):1506-1512.
[57]Gonzalez-Gonzalez E, Lopez-Casas PP, Del Mazo J. Gene silencing by RNAi in mouse Sertoli cells[J].Reprod Biol Endocrinol.2008,6(1):29.
[58]Takei Y, Nemoto T, Mu P, et al. In vivo silencing of a molecular target by short interfering RNA electroporation:tumor vascularization correlates to delivery efficiency[J].Mol Cancer Ther.2008,7(1):211-221.
[59]Elbashir SM, Lendeckel W, Tusch T. RNA interference is mediated by 21-and 22-nucleotide RNAs[J]. Gene,2001,15:188-200
[60]McManus MT, Petersen CP, Haines BB, et al. Gene silencing using micro-RNA designed hairpins[J].RNA,2002,8:842-850.
[61]Brummelkamp T R,Bernards R,Agami R.A system for stable expression of short interfering RNAs in mammalian cells[J].Science,2002,296:550-553
[62]Sui G C,Soohoo C,Affar EIB.A DNA vector-based RNAitechnology to suppress gene expression in mammalian cells[J]. Proc Natl Acad Sci USA,2002, 99:5515-5520
[63]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.[J]. Nature.1998, 391(6669):806-811.
[64]Hammond S M, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. [J]. Nature. 2000,404(6775):293-296.
[65]Sakoda T, Kasahara N, Kedes L, et al. Calcium phosphate coprecipitation greatly enhances transduction of cardiac myocytes and vascular smooth muscle cells by lentivirus vectors.[J]. Exp Clin Cardiol.2007,12(3):133-138.
[66]Rinne A, Littwitz C, Bender K, et al. Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.[M].2009:107-123.
[67]Movassagh M, Philpott A. Cardiac differentiation in Xenopus requires the cyclin-dependent kinase inhibitor, p27Xic1[J], Cardiovasc Res.2008,79(3): 436-447.
[68]Frias M A, Rebsamen M C, Gerber-Wicht C, et al. Prostaglandin E2 activates Stat3 in neonatal rat ventricular cardiomyocytes:A role in cardiac hypertrophy.[J]. Cardiovasc Res.2007,73(1):57-65.
[69]Fluiter K, Mook O R, Baas F. The therapeutic potential of LNA-modified siRNAs:reduction of off-target effects by chemical modification of the siRNA sequence.[J]. Methods Mol Biol.2009,487:189-203.
[70]Feigner P L, Gadek T R, Holm M, et al. Lipofection:a highly efficient, lipid-mediated DNA-transfection procedure. [J]. Proc Natl Acad Sci U S A. 1987,84(21):7413-7417.
[71]Almofti M R, Harashima H, Shinohara Y, et al. Cationic liposome-mediated gene delivery:biophysical study and mechanism of internalization.[J]. Arch Biochem Biophys.2003,410(2):246-253.
[72]Nakanishi M, Noguchi A. Confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cationic cholesterol derivative.[J]. Adv Drug Deliv Rev.2001,52(3):197-207.
[73]Wrobel I, Collins D. Fusion of cationic liposomes with mammalian cells occurs after endocytosis.[J]. Biochim Biophys Acta.1995,1235(2):296-304.
[74]van der Woude I, Visser H W, ter Beest M B, et al. Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system.[J]. Biochim Biophys Acta.1995,1240(1):34-40.
[75]Gopalakrishnan B; Wolff J. siRNA and DNA transfer to cultured cells.[J]. Methods Mol Biol.2009,480:31-52.
[76]Liu D, Ren T, Gao X. Cationic transfection lipids.[J]. Curr Med Chem.2003, 10(14):1307-1315.
[77]Tseng Y C, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA.[J]. Adv Drug Deliv Rev.2009,61(9):721-731.
[78]De Rosa G, La Rotonda M I. Nano and microtechnologies for the delivery of oligonucleotides with gene silencing properties.[J]. Molecules.2009,14(8): 2801-2823.
[79]Zhang S, Zhao B, Jiang H, et al. Cationic lipids and polymers mediated vectors for delivery of siRNA.[J]. J Control Release.2007,123(1):1-10.
[80]Peng C, Knebel A,Morrice N A,et al. Pirn Kinase Substrate Identification and Specificity [J]. J Biochem (Tokyo),2007,141 (3):353-362.
[81]Dai H,Li R,Wheeler T,et al. Pim-2 Up regulation:Biological Implications Associated with Disease Progression and Perinueral Invasion in Prostate Cancer [J]. Prostate,2005,65 (3):276-286.
[82]Muraski JR., Rota M, Misao Y, et al. Pim-1 regulates cardiomyocyte survival downstream of Akt[J]. Nat Med,2007,13(12):1467-1475.
[83]Jung J I, Lim SS, Choi HJ, et al. Isoliquiritigenin induces apoptosis by Depolarizing mitochondrial membranes in prostate cancer cells [J]. J Nutr Biochem,2006,17 (10):689-696.
[84]Kim BC, Mamura M, Choi KS, et al. Transforming growth factor betal induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells [J]. Mol Cell Biol,2002,22 (5):1369-1378.
[85]Grethe S, Coltella N, Di Renzo MF, et al. p38 MAPK downregulates phosphorrylation of Bad in doxorubicin-induced endothelial apoptosis[J]. Biochem Biophys Res Commun,2006,347 (3):781-790.
[86]Korsmeyer SJ, Wei MC, Saito M, et al. Pro2apop totic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c [J]. Cell Death Differ,2000,7 (12):1166-1173.
[87]Bin Y, Marina Z, Sheldon H, et al. The Pim-2 Kinase Phpsphorylates BAD on Serinel 12 and Reverses BAD-induced Cell Deat h[J]. J Biol Chem,2003,278 (46):45358-45367.
[88]White E. The Pims and Outs of Survival Signaling:Role for the Pim-2 Protein Kinase in the Suppression of Apoptosis by Cytokines[J]. Genes Dev,2003,17: 1813-1816.
[2]徐方平,罗健东.ROS与心肌细胞凋亡[J].华北国防医药,2003,15(2)：90-93.
[3]易龙,et al.,花色苷类植物化学物抑制血管内皮细胞氧化应激损伤作用的结构一效应关系研究.第三军医大学学报DSDX,2009(21).
[4]廖德荣,et al.,血管内皮生长因子对过氧化氢诱导的内皮细胞凋亡的影响及其机制.中国动脉硬化杂志KDYZ,2006(06).
[5]Halliwell B, Gutteridge J M, Free radicals in biology and medicine [M] 12nd ed, Oxford:Clarendon Press,1989.121051
[6]Lips DJ, Bueno OF, Wilkins BJ, et al. MEK1-ERK2 signaling pathway protects myoeardium from ischemic injury in vivo[J]. Cireulation.2004。109(16): 1938-1941.
[7]Cuevas BD, Abell AN, Johnson GL. Role of mitogen activated protein kinase kinase kinases in signal integration [J]. Oncogene,2007.26(22):3159-3171.
[8]Bonni A, Brunet A, West AE, et al. Cell Survival Promoted by the Ras-MAPK Signaling Pathway by Transcription-Dependent and -Independent Mechanisms [J]. Science,1999,286(5443):1358-1362.
[9]Baines CP, Zhang J, Wang GW, et al. Mitochondrial PKC epsilonand MAPK form sign aling modules in tile murine heart:enhancedmito chondrial PKC epsilon-MAPK interactions and diferential MAPK activation in PKCepsilon-induced eardiopreteetion [J]. CireRes.2002,90(4):390-397.
[10]Casey J F, Peter S H, Kyan M C,et al. The Serin/t heronine Kinase Pim-2 is A Transcriptionally Regulated Apoptotic Inhabitor [J]. Genes Dev,2003,17: 1814-1854.
[11]Peng C, Knebel A,Morrice N A,et al. Pim Kinase Substrate Identification and Specificity [J]. J Biochem (Tokyo),2007,141 (3):353-362.
[12]Dai H,Li R,Wheeler T,et al. Pim-2 Up regulation:Biological Implications Associated with Disease Progression and Perinueral Invasion in Prostate Cancer [J]. Prostate,2005,65 (3):276-286.
[13]Muraski JR., Rota M, Misao Y, et al. Pim-1 regulates cardiomyocyte survival downstream of Akt[J]. Nat Med,2007,13(12):1467-1475.
[14]Dan L, Ming H, Bo Y, et al. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway[J]. Bio & Cell Bio,2009,41:2315-2322
[15]Jung J I, Lim SS, Choi HJ-etal.Isoliquiritigenin apoptosis by Depolarizing mitochondrial membranes in prostate cancer cells [J]. J Nutr Biochem,2006,17 (10):689-696.
[16]Kim BC, Mamura M, Choi KS, et al. Transforming growth factor betal induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells [J]. Mol Cell Biol,2002,22 (5):1369-1378.
[17]Grethe S, Coltella N, Di Renzo MF, et al. p38 MAPK downregulates phosphorrylation of Bad in doxorubicin-induced endothelial apoptosis[J]. Biochem Biophys Res Commun,2006,347 (3):781-790.
[18]Korsmeyer SJ, Wei MC, Saito M, et al. Pro2apop totic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c [J]. Cell Death Differ,2000,7 (12):1166-1173.
[19]Bachmann M, Moroy T. The serine/threonine kinase Pim-1. Int J Biochem Cell Biol,2005,37:726-730.
[20]Morishita D, Katayama R, Sekimizu K, et al. Pim kinases promote cell cycle progression by phosphorylating and down-regulating p27Kipl at the transcriptional and posttranscriptional levels[J]. Cancer Res,2008,68(13): 5076-5085.
[21]Bin Y, Marina Z, Sheldon H, et al. The Pim-2 Kinase Phpsphorylates BAD on Serine112 and Reverses BAD-induced Cell Death [J]. J Biol Chem,2003,278 (46):45358-45367.
[22]White E. The Pims and Out s of Survival Signaling:Role for the Pim-2 Protein Kinase in the Suppression of Apoptosis by Cytokines[J]. Genes Dev,2003,17: 1813-1816.
[23]Bohensky J, Shapiro I M, Leshinsky S, et al. Pim-2 is an Independent Regulator of Chondrocyte Survival and Autophagy in the Epiphyseal Growt h Plate [J]. J Cell Physiol,2007,213 (1):246-251.
[24]Casey J F, Peter S H, Kyan M C,et al. The Serin/theronine Kinase Pim-2 is A Transcriptionally Regulated Apoptotic Inhabitor [J]. Genes Dev,2003,17 1814-1854.
[25]Peng C, Knebel A,Morrice N A,et al. Pim Kinase Subst rate Identification and Specificity [J]. J Biochem (Tokyo),2007,141 (3):353-362.
[26]Dai H,Li R,Wheeler T,et al. Pim-2 Up regulation:Biological Implications Associated wit h Disease Progression and Perinueral Invasion in Prostate Cancer [J]. Prostate,2005,65 (3):276-286.
[27]Muraski JR, Rota M, Misao Y, et al. Pim-1 regulates cardiomyocyte survival downstream of Akt[J]. Nat Med,2007,13(12):1467-1475.
[28]Dan L, Ming H, Bo Y, et al. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway [J]. Bio & Cell Bio,2009,41:2315-2322
[29]李家实.中药鉴定学[M].上海：上海科学技术出版社,1998：132.
[30]Dong TX, Cui XM, Song ZH, et al. Chemical assessment ofroots of Panax notoginseng in China:regional and seasonal variations in its active constituents[J]. J Agric Food Chen,2003,51:4617-4623.
[31]周晓慧,周晓霞,杨鹤梅.三七总皂甙防治动脉粥样硬化的研究进展.承德医学院学报[J],2003,20：350-352.
[32]雷蕾.血栓通对血液流变学影响的药理学研究[J].海峡药学,2007,19：35-36.
[33]钟尚乾,张焕风,黄永禄,等.血塞通软胶囊治疗急性脑梗死及对血液流变学的影响[J].中国药物与临床,2008,8：68-69.
[34]Garcia-Villalon AL, Amezquita YM, Monge L, et al. Mechanisms of the protective effects of urocortin on coronary endothelial function during ischemia-reperfusion in rat isolated hearts[J]. Br J Pharmacol,2005,145: 490-494.
[35]Hong-Sheng Z, Sheng-Qi W. Notoginsenoside R1 inhibits TNF-α-induced fibronectin production in smooth muscle cells via the ROS/ERK pathway [J]. 2006,40(9):1664-1674.
[36]Hong-Sheng Z, Sheng-Qi W. Notoginsenoside R1 inhibits TNF-α-induced PAI-1 production in human aortic smooth muscle cells [J].2006,44(9):224-230.
[37]del Rio LA, Sandalio LM, Corpas FJ, et al.Reactive Oxygen Species and Reactive Nitrogen Species in Peroxisomes. Production, Scavenging, and Role in Cell Signaling [J].Plant Physiol,2006,141(2):330-335.
[38]Zhang WX, Wan HJ, Zeng XH, et al. Protective effect of vitamin C on rat heart mitochondria injury induced by oxygen radicals [J].HarbinYike Daxue Xuebao(J Harbin Med Univ),2001,35(2):82-84.
[39]Benard G, Bellance N, James D, et al.Mitochondrial bioenergetics and structural network organization [J]. J Cell Sci,2007,120(5):838-848.
[40]Chen ZH, Saito Y, Yoshida Y, et al. Effect of oxygen concentration on free radical-induced cytotoxicity[J]. Biosci Biotechnol Biochem.2008,72(6): 1491-1497.
[41]Sodha NR, Clements RT, Feng J, et al.The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia-reperfusion injury[J].Eur J Cardiothorac Surg,2008,33(5):906-913.
[42]Billman GE.The calcium channel antagonist, flunarizine, protects against ventricular fibrillation [J].Eur J Pharmacol,1992,212(2-3):231-5.
[43]Bognar Z, Kalai T, Palfi A, et al. A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death [J].Free Radic Biol Med,2006,41(5):835-848.
[44]Singal PK, Khaper N, Palace V, et al. The role of oxidative stress in the genesis of heart disease [J]. Cardiovasc Res,1998,40(3):426-432.
[45]Sehirli O, Tozan A, Omurtag GZ, et al.Protective effect of resveratrol against naphthalene-induced oxidative stress in mice [J]. Ecotoxicol Environ Saf,2008,71(1):301-308.
[46].Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].Br J Cancer,1972;26(4):239-257.
[47].JAmes TN. Normal and abnormal consequences of apoptosis in the human heart.From postnatal morphogenesis to paroxymal arrhythmias [J]. Circul-ation, 1994,90(1):556-573.
[48].吴青,陶宏凯,陶大昌,等.灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究[J].中国心血管病研究杂志,2004,2(11)：905.
[49].宋治远,李永华,姚青,等.缺血再灌注对在体兔窦房结细胞凋亡及凋亡相关基因表达的影响[J].中国病理生理杂志,2003,19(12)：1606-1609.
[50].Xie Z.Koyama T, Suzuki J, et al.Coronary reperfusion following is hemia: different expression of Bcl-2 and Bax proteins,and cardiomyocyte apoptosis[J].Jpn heart J,2001,42(6):759-770.
[51].McClintock DS,Santore MT,Lee VY, et al.Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death [J].Mol Cell Biol,2002;22(1):94-104.
[52].Antonsson B, Conti F, Ciavatta A, et al. Inhibition of bax channel forming activity by bcl-2 [J]. Science,1997,277 (5324):370.
[53].Naumovski L, Cleary ML. The p53-binding 53 BP2 also inferacts with bcl-2 and impedes cell cycle progression at G2/M [J].Mol Cell Biol,1996,16:3884.
[54].Saleh A, Srinivasula SM, Balkir L,et al. Negative regulation of the Apaf-lapoptosome by Hsp70[J].Nat Cell Biol,2000,2(8):476.
[55].Skulachev VP.Cytochrome C in the apoptotic and antioxidant cascades [J]. FEBS Lett,1998,423(3):275.
[56].Misao J. Hayakawa Y,Ohno M, et al. Expression of bcl-2 protein, an inhibitor of apoptosis, and Bax, an accelerator of apoptosis, in ventricular myocytes of human hearts with myocardial infarction[J]. Circulation,1996,94(7):1506-1512.
[57]Gonzalez-Gonzalez E, Lopez-Casas PP, Del Mazo J. Gene silencing by RNAi in mouse Sertoli cells[J].Reprod Biol Endocrinol.2008,6(1):29.
[58]Takei Y, Nemoto T, Mu P, et al. In vivo silencing of a molecular target by short interfering RNA electroporation:tumor vascularization correlates to delivery efficiency[J].Mol Cancer Ther.2008,7(1):211-221.
[59]Elbashir SM, Lendeckel W, Tusch T. RNA interference is mediated by 21-and 22-nucleotide RNAs[J]. Gene,2001,15:188-200
[60]McManus MT, Petersen CP, Haines BB, et al. Gene silencing using micro-RNA designed hairpins[J].RNA,2002,8:842-850.
[61]Brummelkamp T R,Bernards R,Agami R.A system for stable expression of short interfering RNAs in mammalian cells[J].Science,2002,296:550-553
[62]Sui G C,Soohoo C,Affar EIB.A DNA vector-based RNAitechnology to suppress gene expression in mammalian cells[J]. Proc Natl Acad Sci USA,2002, 99:5515-5520
[63]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.[J]. Nature.1998, 391(6669):806-811.
[64]Hammond S M, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. [J]. Nature. 2000,404(6775):293-296.
[65]Sakoda T, Kasahara N, Kedes L, et al. Calcium phosphate coprecipitation greatly enhances transduction of cardiac myocytes and vascular smooth muscle cells by lentivirus vectors.[J]. Exp Clin Cardiol.2007,12(3):133-138.
[66]Rinne A, Littwitz C, Bender K, et al. Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.[M].2009:107-123.
[67]Movassagh M, Philpott A. Cardiac differentiation in Xenopus requires the cyclin-dependent kinase inhibitor, p27Xic1[J], Cardiovasc Res.2008,79(3): 436-447.
[68]Frias M A, Rebsamen M C, Gerber-Wicht C, et al. Prostaglandin E2 activates Stat3 in neonatal rat ventricular cardiomyocytes:A role in cardiac hypertrophy.[J]. Cardiovasc Res.2007,73(1):57-65.
[69]Fluiter K, Mook O R, Baas F. The therapeutic potential of LNA-modified siRNAs:reduction of off-target effects by chemical modification of the siRNA sequence.[J]. Methods Mol Biol.2009,487:189-203.
[70]Feigner P L, Gadek T R, Holm M, et al. Lipofection:a highly efficient, lipid-mediated DNA-transfection procedure. [J]. Proc Natl Acad Sci U S A. 1987,84(21):7413-7417.
[71]Almofti M R, Harashima H, Shinohara Y, et al. Cationic liposome-mediated gene delivery:biophysical study and mechanism of internalization.[J]. Arch Biochem Biophys.2003,410(2):246-253.
[72]Nakanishi M, Noguchi A. Confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cationic cholesterol derivative.[J]. Adv Drug Deliv Rev.2001,52(3):197-207.
[73]Wrobel I, Collins D. Fusion of cationic liposomes with mammalian cells occurs after endocytosis.[J]. Biochim Biophys Acta.1995,1235(2):296-304.
[74]van der Woude I, Visser H W, ter Beest M B, et al. Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system.[J]. Biochim Biophys Acta.1995,1240(1):34-40.
[75]Gopalakrishnan B; Wolff J. siRNA and DNA transfer to cultured cells.[J]. Methods Mol Biol.2009,480:31-52.
[76]Liu D, Ren T, Gao X. Cationic transfection lipids.[J]. Curr Med Chem.2003, 10(14):1307-1315.
[77]Tseng Y C, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA.[J]. Adv Drug Deliv Rev.2009,61(9):721-731.
[78]De Rosa G, La Rotonda M I. Nano and microtechnologies for the delivery of oligonucleotides with gene silencing properties.[J]. Molecules.2009,14(8): 2801-2823.
[79]Zhang S, Zhao B, Jiang H, et al. Cationic lipids and polymers mediated vectors for delivery of siRNA.[J]. J Control Release.2007,123(1):1-10.
[80]Peng C, Knebel A,Morrice N A,et al. Pirn Kinase Substrate Identification and Specificity [J]. J Biochem (Tokyo),2007,141 (3):353-362.
[81]Dai H,Li R,Wheeler T,et al. Pim-2 Up regulation:Biological Implications Associated with Disease Progression and Perinueral Invasion in Prostate Cancer [J]. Prostate,2005,65 (3):276-286.
[82]Muraski JR., Rota M, Misao Y, et al. Pim-1 regulates cardiomyocyte survival downstream of Akt[J]. Nat Med,2007,13(12):1467-1475.
[83]Jung J I, Lim SS, Choi HJ, et al. Isoliquiritigenin induces apoptosis by Depolarizing mitochondrial membranes in prostate cancer cells [J]. J Nutr Biochem,2006,17 (10):689-696.
[84]Kim BC, Mamura M, Choi KS, et al. Transforming growth factor betal induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells [J]. Mol Cell Biol,2002,22 (5):1369-1378.
[85]Grethe S, Coltella N, Di Renzo MF, et al. p38 MAPK downregulates phosphorrylation of Bad in doxorubicin-induced endothelial apoptosis[J]. Biochem Biophys Res Commun,2006,347 (3):781-790.
[86]Korsmeyer SJ, Wei MC, Saito M, et al. Pro2apop totic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c [J]. Cell Death Differ,2000,7 (12):1166-1173.
[87]Bin Y, Marina Z, Sheldon H, et al. The Pim-2 Kinase Phpsphorylates BAD on Serinel 12 and Reverses BAD-induced Cell Deat h[J]. J Biol Chem,2003,278 (46):45358-45367.
[88]White E. The Pims and Outs of Survival Signaling:Role for the Pim-2 Protein Kinase in the Suppression of Apoptosis by Cytokines[J]. Genes Dev,2003,17: 1813-1816.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |