【作者】 吴铁军；

【导师】 刘维永； 金振晓；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【副题名】附乌拉坦对离体大鼠心脏保存作用的探讨

【摘要】 O-连接N-乙酰氨基葡萄糖基化(O-linked N-acetylglucosamine, O-GlcNAc)是发生在细胞浆与细胞核内的、仅单个N-乙酰氨基葡萄糖通过O-糖苷键连接到丝氨酸或苏氨酸(Ser/Thr)羟基上、一种广泛动态的蛋白质翻译后修饰现象,蛋白质的O-GlcNAc可以敏锐地感受环境变化,调节蛋白质间的相互作用,影响细胞的信号级联通路。在多种形式的应激状态下,细胞活化己糖胺生物合成途径(HBP),增加蛋白质的O-GlcNAc修饰水平,从而提高细胞存活,减轻细胞损伤。红景天苷是从藏药红景天分离出来的一种苯乙醇类化合物,具有抗缺氧、抗肿瘤、抗衰老、抗肝纤维化等作用。在体动物实验及临床资料证实,红景天苷具有心肌保护作用,但对红景天苷减轻心肌缺血/再灌注损伤机制的研究尚不多。本实验通过建立大鼠心肌细胞培养缺血/再灌注损伤模型和离体心脏灌注缺血/再灌注损伤模型,观察红景天苷对心肌缺血/再灌注损伤的保护作用,并通过与四氧嘧啶(O-连接N-乙酰氨基葡萄糖转移酶抑制剂,能够抑制O-GlcNAc的形成)作用比较,探讨O-GlcNAc在这种保护机制中的作用。实验一红景天苷对培养大鼠心肌细胞缺血/再灌注损伤的的保护作用方法:取1～2 d龄SD乳鼠,分离、培养原代乳鼠心肌细胞,细胞培养3 d后建立缺血/再灌注损伤模型:用缺氧液置换正常培养液,置入三气培养箱(95% N2 + 5% CO2)缺氧培养4 h,然后细胞再置入37℃、21% O2和5% CO2培养箱中继续培养16 h。培养的心肌细胞分为5组(n=6):①正常对照组:用正常培养液培养细胞;②正常培养+红景天苷预处理:红景天苷80 mol/L预处理细胞24 h,然后用正常培养液培养细胞20 h;③缺血/再灌注:细胞正常培养3 d后按照缺血/再灌注模型培养;④缺血/再灌注+红景天苷+四氧嘧啶预处理:红景天苷80 mol/L加四氧嘧啶2.5 mmol/L预处理细胞24 h,然后按照缺血/再灌注组培养;⑤缺血/再灌注+红景天苷预处理:红景天苷80 mol/L预处理细胞24 h,然后按照缺血/再灌注组培养。处理完毕,检测各组细胞存活率、乳酸脱氢酶(LDH)释放量、细胞凋亡、细胞ATP含量、细胞内游离Ca2+水平、葡萄糖摄取率及细胞O-GlcNAc水平。结果:与对照组比较,单纯缺血/再灌注损伤导致心肌细胞存活率显著降低为64.7±4.5%,红景天苷预处理组心肌细胞缺血/再灌注后存活率增加至85.8±3.1%,与单纯缺血/再灌注组比较差异显著;加入四氧嘧啶后心肌细胞存活率减少为66.5±2.9%,与红景天苷预处理组比较差异显著。缺血/再灌注损伤导致心肌细胞LDH释放量从9.3±1.5%增加至38.5±2.1%,红景天苷预处理显著降低心肌细胞LDH释放量至21.2±1.7%,与单纯缺血/再灌注组比较差异显著;加入四氧嘧啶后心肌细胞LDH释放量增加至39.3±1.6%,与红景天苷预处理组比较差异显著。缺血/再灌注后心肌细胞凋亡率从6.5±1.2%显著增加至27.2±3.2%;红景天苷预处理组心肌细胞凋亡率为12.4±1.9%,与单纯缺血/再灌注组比较差异显著;加入四氧嘧啶后心肌细胞凋亡率为26.2±2.4%,与红景天苷预处理组比较差异显著。单纯缺血/再灌注损伤导致心肌细胞ATP含量从24.2±2.0 mol/g proteion显著下降至17.9±2.8 mol/g protein,红景天预处理组心肌细胞ATP含量为21.3±3.5 mol/g protein,虽然高于单纯缺血/再灌注组,但是两组比较无显著性差异;加入四氧嘧啶后心肌细胞ATP含量为16.7±1.6 mol/g protein,与红景天苷预处理组比较无显著性差异。与对照组比较,缺血/再灌注损伤导致心肌细胞内Ca2+水平增加到147.2±10.5%,红景天苷预处理后心肌细胞内Ca2+水平从147.2±10.5%显著降低至121.9±9.9%,与单纯缺血/再灌注组比较差异显著;加入四氧嘧啶后心肌细胞内Ca2+水平增加至138.5±9.2%,与红景天苷预处理组比较差异显著。在正常氧和缺血/再灌注培养条件下,红景天苷预处理均可显著增加心肌细胞对葡萄糖的摄取率,分别为对照组的1.8倍和2.5倍,与对照组比较差异显著;缺血/再灌注损伤亦可增加心肌细胞葡萄糖摄取率,为对照组的1.5倍,但与红景天苷预处理组比较差异显著,四氧嘧啶组心肌细胞葡萄糖摄取率为对照组的2.4倍,表明四氧嘧啶不抑制红景天苷预处理对心肌细胞葡萄糖摄取率的增加。缺血/再灌注损伤增加心肌细胞O-GlcNAc水平,为对照组的1.4倍,红景天苷预处理可显著增加心肌细胞中O-GlcNAc水平,为对照组的2.2倍,与单纯缺血/再灌注组比较差异显著;加入四氧嘧啶后心肌细胞O-GlcNAc水平显著下降,为对照组的0.98倍,与红景天苷预处理组比较差异显著。结结论:红景天苷能够显著地减轻心肌细胞的缺血/再灌注损伤,红景天苷预处理能够提高细胞存活率、降低LDH漏出、抑制细胞凋亡、减轻细胞内Ca2+超载、抑制ATP的消耗,促进细胞对葡萄糖的摄取和提高细胞O-GlcNAc水平。OGT抑制剂四氧嘧啶抑制了红景天苷对O-GlcNAc水平的提高,同时消除了红景天苷对心肌细胞的保护作用,表明红景天苷对心肌细胞缺血/再灌注损伤的保护作用主要是通过促进细胞对葡萄糖的摄取和提高细胞O-GlcNAc水平介导,提高细胞ATP含量亦可能是其作用机制之一。实验二红景天苷对离体灌注心脏缺血/再灌注损伤的保护作用方法:采用Langendorff非循环灌注,灌注液为37℃、95% O2及5% CO2混合气体饱和的KH液。建立缺血/再灌注损伤模型:平衡灌注30 min,然后停止灌注30 min,再复灌注30 min。18只SD大鼠随机分为三组(n=6):①单纯缺血/再灌注组:停止灌注30 min,复灌注30 min;②缺血/再灌注+红景天苷+四氧嘧啶预处理:红景天苷80 mol/L及四氧嘧啶2.5 mmol/L在停灌注前20 min加入灌注液,然后按照缺血/再灌注组灌注;③缺血/再灌注+红景天苷预处理:红景天苷80 mol/L在停灌注前20 min加入灌注液,然后按照缺血/再灌注组灌注。记录左心室内压最大上升和下降速率(±dP/dt)、左室发展压(LVDP)及冠状动脉流量(CF),收集再灌注后冠状动脉流出液检测LDH释放量,检测心肌ATP含量、心肌细胞凋亡、O-GlcNAc水平及心肌超微结构变化。结果:各组心脏停灌前LVDP、+dP/dt、-dP/dt和CF无显著差异,再灌注30 min后单纯缺血/再灌注组、四氧嘧啶组和红景天苷预处理组LVDP的恢复百分率分别为76.4±5.2%、73.8±5.2%和85.9±4.7%,+dP/dt的恢复百分率分别为75.2±6.3%、72.9±5.8%和88.2±6.1%,-dP/dt的恢复百分率分别为79.1±4.4%、74.4±7.2%和87.4±6.8%,红景天苷预处理组与单纯缺血/再灌注组和四氧嘧啶组差异显著。再灌注30 min后单纯缺血/再灌注组、红景天苷预处理组和四氧嘧啶组CF的恢复百分率分别为76.2±5.1%、78.1±5.4%和79.6±4.9%,三组比较无显著差异。与单纯缺血/再灌注组比较,红景天苷预处理显著降低LDH释放量,两组比较差异显著。加入四氧嘧啶后LDH释放量增加,与红景天苷预处理组比较差异显著。TUNEL染色显示单纯缺血/再灌注组21.3±3.5%的细胞发生凋亡,而红景天苷预处理组9.5±2.6%的细胞发生凋亡,两组比较差异显著;加入四氧嘧啶后细胞凋亡增加到19.8±3.1 %,与红景天苷预处理组比较差异显著。再灌注30 min后三组心肌ATP含量分别为1.48±0.18 mol/g tissue、1.42±0.13 mol/g tissue和1.51±0.17 mol/g tissue,三组比较无显著差异。红景天苷预处理可显著增加心肌中O-GlcNAc水平,为单纯缺血/再灌注对照组的1.9倍,加入四氧嘧啶后抑制了红景天苷对细胞O-GlcNAc水平的增加,O-GlcNAc水平为单纯缺血/再灌注对照组的1.2倍,红景天苷预处理组与单纯缺血/再灌注组和四氧嘧啶组差异显著。电镜观察显示红景天苷预处理组心肌结构清晰,肌节结构清楚,细胞无水肿,线粒体形态、大小正常;单纯缺血/再灌注组心肌超微结构大致正常,部分细胞肌纤维挛缩,线粒体略肿胀;四氧嘧啶组肌丝收缩较明显,部分线粒体肿胀明显,细胞内致密颗粒增多,提示可能存在钙超载。结论:用离体心脏灌注模型可排除麻醉药物、神经、激素等影响心肌机械功能因素的干扰,本实验进一步观察了红景天苷对离体灌注心脏缺血/再灌注损伤的保护作用,红景天苷能够显著地提高左心功能恢复率、降低LDH释放量、抑制细胞凋亡和更好的保存心肌组织的超微结构,而且红景天苷亦能显著提高离体灌注心脏的O-GlcNAc水平。OGT抑制剂四氧嘧啶抑制了红景天苷对O-GlcNAc水平的提高,消除了红景天苷的心肌保护作用,进一步表明红景天苷对心肌细胞缺血/再灌注损伤的保护作用是通过提高细胞O-GlcNAc水平介导。更多还原

【Abstract】 Objective: The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is increasingly recognized as an important posttranslational modification that modulates cellular function. Recent studies suggested that augmentation of O-GlcNAc levels increase cell survival following stress. Salidroside, one of the active components of Rhodiola rosea, shows potent anti-hypoxia property and protective effects on myocardial ischemia/reperfusion injury in isolated rat heart. However, the mechanisms have yet to be elucidated. In the present study, we reported the cardioprotection of salidroside from ischemia and reperfusion and discussed the relationship between protective effect of salidroside and O-GlcNAc levels.PartⅠ:Protective effects of salidrosides via increased protein O-GlcNAc on ischemia and reperfusion injury in cardiomyocytesMethods: Cardiomyocytes were isolated from 1-to 2-day-old Sprague- Dawley rats. Within 2 days of isolation, cardiomyocytes were exposed to 4 h of ischemia and 16 h of reperfusion, and then cell viability, apoptosis, glucose uptake, ATP levels and cytosolic Ca2+ concentration were determined, and O-GlcNAc levels were assessed by Western blotting. Salidroside (80 M), salidroside (80 M) plus alloxan (2.5 mM) were added 24 h before ischemia/reperfusion were induced.Results: Compared to untreated cells following ischemia/reperfusion, treatment with salidroside markedly improved cell viability from 64.7±4.5% to 85.8±3.1%, decreased LDH release from 38.5±2.1% to 21.2±1.7%, reduced cell apoptosis from 27.2±3.2% to 12.4±1.9%, reduced cytosolic Ca2+ concentration from 147.2±10.5% to 121.9±9.9%, increased ATP content from 17.9±2.8 mol/g protein to21.3±3.5 mol/g protein, as well as significantly improving cardiomyocytes glucose uptake by 1.7-fold and increasing O-GlcNAc levels by 1.6-fold. However, alloxan, an inhibitor of O-GlcNAc transferase that should block the formation of O-GlcNAc, markedly decreased cell viability to 66.5±2.9%, increased LDH release to 39.3±1.6%, increased cell apoptosis to 26.2±2.4%, elevated cytosolic Ca2+ concentration to 138.5±9.2%, and reversed the increase in O-GlcNAc levels seen with salidroside to 98.8±9.8%. Conclusions: Salidroside significantly improved cell viability, decreased LDH release, attenuated cells apoptosis, as well as attenuating cytosolic Ca2+ elevation, stimulating glucose uptake and increasing O-GlcNAc levels compared to untreated cells following ischemia/reperfusion. Alloxan prevented salidroside-induced increase in O-GlcNAc and also blocked the protective effects of salidroside. These findings suggested that cardioprotection of salidroside was associated with enhanced glucose uptake and increased protein O-GlcNAc levels. PartⅡ:Cardioprotective effects of salidroside with increased O-GlcNAc on ischemia and reperfusion injury rat hearts in vitroMethods: Rat hearts were excised and perfused at a constant pressure of 75 mm Hg with KHB equilibrated with 95% O2 and 5% CO2, and then were subjected to 30 min of global, no-flow ischemia followed by 30 min of reperfusion. There were three experimental groups (n=6):①I/R groups: ischemia and reperfusion;②Salidroside groups: salidroside (80 M) added 20 min before the start of ischemia;③Alloxan groups: salidroside (80 M) plus alloxan (2.5mM), an inhibitor of OGT, added 20 min before the start of ischemia. Cardiac function, ATP content, LDH release, cardiomyocyte apoptosis and ultrastructural alterations were examined.Results: There was no significant difference in contractile function among three groups before ischemia. After 30min of ischemia and 30min of reperfusion, salidroside significantly increased cardioal functional recovery compared with the I/R group. The recovery rates of LVDP in I/R groups, salidroside groups and alloxan groups were 76.4±5.2%, 73.8±5.2% and 85.9±4.7%, respectively; The recovery rates of +dP/dt were 75.2±6.3%, 72.9±5.8% and 88.2±6.1%, respectively; The recovery rates of -dP/dt were 79.1±4.4%, 74.4±7.2% and 87.4±6.8%, respectively. Treatment with salidroside markedly reduced cell apoptosis from 21.3±3.5% to 9.5±2.6%, significantly increased O-GlcNAc levels by 1.9-fold compared to untreated hearts following ischemia/reperfusion. However, alloxan, an inhibitor of O-GlcNAc transferase, blocked the increase of O-GlcNAc induced by salidroside, markedly increased cell apoptosis to 19.8±3.1%. Electron microscopy studies demonstrated better cardiomyocyte structure and mitochondrial integrity in salidroside group than those in I/R group and alloxan group.Conclusions: Salidroside showed protective effects from ischemia and reperfusion injury in perfused rat isolated hearts. It increased hemodynamic recovery rate, attenuated cardiomyocytes apoptosis, and increased O-GlcNAc levels. Alloxan prevented the salidroside-induced increase in O-GlcNAc and also blocked the protective effects of salidroside. Salidroside cardioprotection from ischemia and reperfusion injury in perfused rat hearts was associated with increased O-GlcNAc.更多还原