氢气抗糖基化终末产物对内皮细胞凋亡的影响及机制研究
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摘要
背景与研究目的
糖尿病是一种常见的代谢性疾病，主要表现为机体胰岛素相对或者绝对不足，导致血糖水平持续性增高。目前全球2型糖尿病患者已超过1.5亿人，而且预计在未来20年内病例数还将成倍数增长态势，全球医疗负担也会因为这一数值的显著提高而明显增加[1]。不论是1型还是2型糖尿病，随着长期高血糖水平病程的逐渐进展，均可以造成不同程度的，不同靶器官的并发症，其中以心血管病变最为常见，也最具威胁性。
血管并发症是导致糖尿病患者死亡的主要原因。其中，高血压，肥胖症，动脉粥样硬化，血脂代谢异常，糖尿病肾病亦是患者预后不良的重要危险因素。在糖尿病的发生发展过程当中，血管内皮的损伤和内皮功能障碍是糖尿病重要的早期病理表现。从细胞层面来讲，内皮细胞代谢异常则是早期内皮功能受损的基本原因,伴随着内皮细胞的不断凋亡和功能受损，随之而来的则是内皮功能障碍[2.3]。
晚期糖基化终末产物(AGEs)是以体内大分子物质如脂肪，蛋白质，核酸和还原性糖(葡萄糖，果糖，戊糖)为原料，在生理环境中通过发生非酶促反应，生成的稳定共价键产物。这种非酶促反应称之为---Maillard(美拉德)。1953年Hodge等学者提出了美拉德反应，该反应主要包括如下三部分：1.初级反应阶段。还原糖的羰基与氨基之间进行加成，加成物迅速失去分子水转变为希夫碱。2.经环化形成相应的取代醛基胺，经重排后转成有反应活性的产物，其中最熟知的是糖基化血红蛋白和果糖胺。3.果糖胺等又经过一系列复杂的分解和化学重排反应最终形成了AGEs类黑精色素。AGEs一旦形成就是不可逆的，并且其具有较强的交联性和较低的溶解性。此外，AGEs还具有产生和激活ROS的能力[61]，这些特点也是其损伤血管的重要理化因素。AGEs的产生和糖尿病患者血糖水平以及高血糖暴露的时间成正相关，患者血糖水平越高，暴露高血糖的时间越久，AGEs的产生和堆积就越多。此外，局部微环境的氧化应激也是加速AGEs形成的重要因素之一[62.63]。在正常机体中，葡萄糖的糖基化反应较低。然而，在糖尿病患者体内，持续性的高血糖和局部微环境的氧化应激加速了AGEs产生和堆积。
血液循环中堆积的AGEs可进行性损害血管内皮[65]。国外研究者发现，胰岛素可通过清除AGEs来保护内皮细胞功能，胰岛素通过IRS以及PI-3K通路提高机体产生NO的能力，从而提高机体对葡萄糖的转运和利用，减少AGEs的产生和保护内皮功能。此外，胰岛素还可以提高内皮细胞清道夫受体的含量，从而加速AGEs的清除。另外，蓄积在体内的AGEs低密度脂蛋白(LDL)可以形成AGEs-LDL共价化合物，形成AGEs-LDL后，LDL在体内的循环清除受阻，LDL在循环中的比例升高，致体内脂质水平明显增加，加重内皮氧化损伤。此外，血管内皮的基质成分含有胶原蛋白，它们与循环中的还原性糖发生共价结合以后可以使胶原蛋白交联，从而使血管内皮内环境受到破坏。同时，血管内皮的AGEs还能够激活单核巨噬系统，使得它们在对AGEs发生吞噬作用的同时分泌大量的细胞因子，刺激血管内皮发生氧化，应激，变形，增生，增殖，凋亡等一系列病理反应，恶化内皮功能，最终导致动脉粥样硬化，甚至血管完全闭塞。在糖尿病患者当中，持续升高的血糖状态极易导致AGEs的过量产生[4]。我们课题组的前期研究也发现，AGEs可以加快血管内皮细胞的凋亡和内皮功能不全的病理进程；在AGEs诱导的细胞当中，其细胞间活性氧(ROS)水平较对照组是明显升高的，而且同时，这种升高的细胞间ROS水平反过来进一步刺激AGEs的不断生成，形成“氧化应激-AGEs”的恶性循环[6.7]。
因此，如何清除AGEs或减少这些AGEs的产生似乎可以成为减少糖尿病并发症的治疗策略之一。但是，AGEs一旦在血管内皮生成共价键，其就如一团黏着的基团，很难清除。那么，如果能够拮抗由AGEs介导的血管内皮功能损伤，则理论上也能预防或者延缓血管内皮损伤-动脉粥样硬化的进程[8]。所以，探寻一种拮抗AGEs介导的内皮细胞氧化应激以及凋亡的治疗策略有望成为防治糖尿病并发症的潜在切入点。
传统的抗氧化应激的制剂包括维生素C，维生素E，β胡萝卜素，超氧化物歧化酶等。由于结构和效价关系的限制，这些天然抗氧化剂的抗氧化作用较弱且作用可逆，组织穿透能力差，而且其循证学研究的结果多为阴性。另外，在已经发生氧化应激反应的细胞当中多半有代谢和功能障碍，其自身合成分泌抗氧剂的能力差。为此，寻求一种新型的，有效的，安全的，穿透力强，靶向性高的抗氧剂是拮抗机体氧化应激反应延缓动脉粥样硬化的潜在突破口之一。
氢气是一种无色，无味，无臭具有一定还原性的易燃气体。过去许多学者认为氢气是一种生理性惰性气体，不会与机体生命物质发生生理生化反应。但也有极少数的科学家认为氢气是一种具有还原性的气体。发表在1975年《Science》杂志上的一篇文章报道，在皮肤鳞状细胞癌动物模型中，连续呼吸8个大气压97.5%氢气两周，可以有效治疗鳞状细胞癌，并且研究者认为其机制可能与氢气的抗氧化作用有关[9]。之后又有实验研究发现，呼吸高压氢气能够治疗肝脏寄生虫感染引起的肝炎，并提出氢气的抗炎作用机制可能与氢气和羟自由基反应有关[10]。虽然上述研究为引起学者的广泛关注，但Ohsawa等2007年发表在《Nature》上的一篇文章提出，缺血再灌注和炎症所致的急性氧化应激可以导致严重的组织损伤。研究者通过建立大脑缺血再灌注的氧化应激大鼠模型，在非高压的条件下，应用吸入氢气作为治疗干预，结果发现氢气能够通过抗氧化作用减轻大鼠脑缺血再灌注损伤。文中还讨论到，氢气可能具有选择性的抗氧化作用，也即氢气只于与·OH和ONOO-发生反应，而并不与其它具有生理活性作用的活性氧发生生物化学反应，如O2-·、H2O2和NO·等。氢气的这种温和还原性的优点，使得它在抗氧化作用的同时对机体内部必须的生理反应过程影响极小。另外，和生理活性氧O2-·、H2O2和NO·相比，·OH和ONOO-等氧自由基的氧化性明显高于生理活性氧，而这也使得还原性比较温和的氢气只与这些强自由基的发生中和反应。除了这些特点外，氢气的高物理扩散性又是其独特的优势[11]。随后，日本科学家Hayashida等在Ohsawa等研究的基础上，在大鼠心肌梗死缺血再灌注模型中证实吸入氢气能够有助于心梗再灌注后心功能的恢复，减少心肌梗死面积，并预防心室结构重构。实验再次证实了氢气到达治疗靶点的高效性和高通透性[12]。2010年，Chen CH等学者在大鼠局灶脑缺血过程中，吸入氢气2小时能够降低基质金属蛋白-9活性并减小脑缺血面积[13]。2012年，中国学者Ge等在大鼠动物模型中发现吸入氢气可以减轻短暂脑缺血诱导的认知功能障碍，其可能的机制为氢气可以拮抗氧化应激作用从而减少神经细胞的死亡[14]。同年，Zhan Y学者等在Crit Care Med杂志发表关于吸入氢气治疗的实验研究成果。他们在130余只硬膜下出血脑损伤的大鼠模型中发现吸入氢气2小时可以明显减轻脑水肿和血脑屏障损伤[15]。同时在分子研究层面证实，氢气可以减轻大脑脂质，核酸，蛋白质的氧化损伤。正如德国学者Hardeland对该研究的评论，吸入氢气可能是将来医学领域一种颇有前景的治疗策略[16]。
不仅如此，除了吸入氢气，科学家们还利用氢气扩散特点制备成富含氢的溶液并探究其治疗作用。最开始是由Sun等学者提出假设富含氢的生理盐水是否依然具有抗氧化应激作用。他们在大鼠的心梗模型中通过注射富氢生理盐水的方法对心梗后的大鼠进行干预，结果发现富氢生理盐水仍然具有有效抗氧化应激，减少心肌死亡，改善心功能的治疗作用[17]。同期还有中国学者们发现富氢生理盐水对小肠的缺血再灌注损伤也有保护作用，并同时可以改善小肠的收缩功能[18.20]。另外Chen等在大鼠实验中发现，富氢生理盐水在急性胰腺损伤中能减轻氧化应激，减少胰腺细胞死亡，有意思的是，富氢生理盐水还能促进胰腺腺泡细胞的增生[21]。因为富氢生理盐水的制备过程并不复杂，而且有使用方便，便于运输，实用等特点，一时间将富氢生理盐水作为治疗制剂研究其治疗作用的研究迅速成为热点，相应的研究结果均提示富氢溶液作为医疗制剂的诱人前景[22.30]。
尽管如此，人们对氢的抗氧化作用的具体分子生物学机制还未能深入研究。前面我们提到，AGEs可以诱导血管内皮的氧化应激反应，从而刺激内皮细胞增生，炎症细胞侵润，凋亡，内皮功能障碍，血管粥样硬化的系列病理改变。氢能否通过抑制AGEs诱导的氧化应激，产生内皮保护作用有待实验进一步证实。基于此，我们课题组设计了“氢气抗AGEs诱导的内皮凋亡及其机制”这课题，对我们的课题假设进行初步证实。
材料和方法
SD大鼠购自于第三军医大学动物室，用于主动脉内皮细胞的原代培养。富含氢气的培养基被应用于模拟高氢气的环境。Real-time PCR和分光光度测定KIT用于抗氧化应激酶SOD和GSH-PH基因和蛋白表达量检测， DCF-DA作为荧光探针运用流式细胞仪检测细胞内的活性氧(ROS)水平；Annexin V-APC凋亡检测试剂盒和TUNEL染色法被用于内皮细胞凋亡的检测,蛋白印迹分析(WB)用于检测Bcl-2(凋亡抑制蛋白)和Bax(凋亡促进蛋白)比率分析于说明内皮细胞凋亡水平。
结果
1.AGEs可以诱导内皮细胞凋亡并且呈浓度依赖性，HRM组和正常对照组的细胞凋亡水平无显著差异。AGEs组细胞凋亡水平明显高于正常对照组和HRM组，加入HRM能够明显抑制AGEs所诱导的细胞凋亡。
2.流式细胞仪分析法检测发现，加有AGEs的内皮细胞(AGEs组)其细胞间ROS水平较正常对照组明显升高，且升高的水平成浓度依赖关系。但HRM组细胞ROS水平和正常对照组比较我显著差异。在AGEs+HRM组中发现细胞间ROS水平较AGEs组明显降低，并和正常对照组相似。
3.抗氧应激检测显示：在mRNA方面，AGEs能显著降低SOD mRNA的表达水平，而且这中降低与AGEs呈浓度相关性。而HRM干预后能够明显改善AGEs对SODmRNA表达的抑制作用。另外，在酶活性比较方面，AGEs对内皮细胞SOD和GSH-PX这两种抗氧化酶的酶活性均成抑制效应，而HRM干预则能改善AGEs的这种不良效应。
4.凋亡蛋白水平检测发现AGEs组Bcl-2表达量较正常对照组明显下降，而Bax蛋白表达量较对照组明显增加，Bcl/Bax蛋白表达量比例在改组明显下降，提示AGEs明显诱导内皮细胞凋亡。在AGEs+HRM组中，Bcl/Bax蛋白表达量比例较正常对照组无明显下降，提示HRM能够拮抗由AGEs诱导的细胞凋亡。
结论
1. AGEs可以诱导内皮细胞凋亡和氧化应激并且呈浓度依赖性.
2. HRM能够拮抗由AGEs诱导的细胞凋亡和氧化应激，起作用可能通过提高SOD和GSH-PH以及Bcl-2(凋亡抑制蛋白) Bax(凋亡促进蛋白)比率并降低ROS水平发挥抗氧化应激和抗凋亡作用.
Background and Objective
It is known that, type1and type2diabetes alike, with the gradual progression of thelong-term high blood glucose levels, can cause different degrees of complications invarious target organs, among which the cardiovascular disease is the most common andthreatening[1]. At present, the number of patients with type2diabetes has amounted to morethan150million worldwide, and is expected to grow exponentially in the next20years[2].Patients with diabetes-induced cardiovascular disease will significantly increase in number,leading to a heavier global medical burden. How to control cardiovascular complications isthe focus of current research.
In fact，cardiovascular complications in diabetes mellitus are one of the leading causesof mortality. Vascular endothelial injury and dysfunction are the important earlypathological manifestations of these complications.[31]Increasing apoptosis is an earlymanifestation of endothelial injury, which will lead to endothelial dysfunction[32].Advancedglycation end products (AGEs) are the products of non-enzymatic glycosylation of theamino groups of macromolecules, such as proteins and nucleic acids[33]. In diabetic patients,sustained high blood glucose level significantly increases the production of AGEs[33].Epidemiological studies show that the presence of AGEs is highly correlated with diabeticcardiovascular complications. Previous studies have found that AGEs increase endothelialcell apoptosis and dysfunction[34]. In addition, according to our previous study, AGEsincrease apoptosis and dysfunction of endothelial precursor cells[35].The generation ofintracellular reactive oxygen species (ROS) increases in AGEs-induced cells, and oxidativestress and ROS production, in turn, contribute to AGEs-induced apoptosis of endothelialcells and endothelial precursor cells[36].
As we know，removal of AGEs from the body is difficult. One strategy to reduce AGEs-induced endothelial injury is to antagonize AGEs-activated oxidative stress[32].Hydrogen (H2) is the smallest naturally occurring gas molecule. Studies have shown thatH2has antioxidant activities in living organisms. It can specifically neutralize the mostpotent oxidative free radicals (OH and ONOO-) and attenuate the superoxidant anion levelunder certain pathophysiological conditions[37]. Moreover, it is easy for H2to pass throughmembrane structures such as cell membranes and the mitochondrial membranes, where itcan neutralize intracellular ROS, thereby maintaining normal mitochondrial function andpreventing apoptosis[38]. Many studies have shown that H2or hydrogen-containing solutioncan alleviate ischemia-reperfusion injury[34.36.39]of the heart[40],brain[41.42], kidney, smallintestine, and liver[41]; and it can antagonize irradiation-induced cell injury[43]. Inhalation ofH2also slows down the growth of atherosclerotic plaque in apoE-/-mice[44]. Our previousstudies also found that hydrogen-rich saline prevented neointima formation after carotidballoon injury[45]. Oxidative stress plays an important role in ischemia-reperfusion injury,irradiation-induced injury, atherosclerosis and neointima formation[46]. The ability of H2toantagonize these pathological reactions is closely related to its anti-ROS effects. However,it has yet to be elucidated whether H2can ameliorate AGEs-induced ROS generation andapoptosis of endothelial cells.
The objective of the present study was to determine whether the use of hydrogen-richmedium (HRM) can protect the endothelial cells from AGEs-induced apoptosis by detectingROS production and antioxidant-related gene expression.
Materials and Methods
Two to three year old Sprague-Dawley rats purchased from the Experimental AnimalCenter of the Third Military Medical University were used in this study. The thoracic aortawas removed from these rats, and endothelial cells (ECs) were isolated and cultured. Afterculturing ECs in the presence of AGEs and/or with HRM for24h, Annexin V/7-AAD andTUNEL staining were carried out to detect apoptosis. Intracellular ROS was detected withfluorescent probes and quantified with flow cytometry. Anti-oxidative enzyme (SOD andGSH-PH) expression was determined by real-time PCR analysis and enzymatic assay. Therelative expression levels of Bcl-2and Bax were analyzed by western blotting.
Results and Discussion
1. AGEs induced ECs apoptosis in a concentration-dependent manner. After24h,evaluation of the4EC groups, including AGEs (400μg/ml) group, HRM group, HRM+AGEs group, and normal control group, revealed that the cell apoptosis rates were notsignificantly different between the HRM group and the control group when AGEs wereabsent. However, the presence of AGEs increased the rate of ECs apoptosis, and theaddition of HRM reduced the apoptosis rate of AGEs-treated ECs.
2. Treatment of ECs with AGEs significantly increased the generation of intracellularROS in a dose-dependent manner. However, HRM decreased significantly.
3. AGEs significantly reduced the level of SOD mRNA. However, after HRMintervention, the SOD expression reduction caused by AGEs was significantly attenuated.AGEs treatment significantly increased the optical density of anti-oxidative enzymes. HRMtreatment was also able to partly reduce this effect.
4. ECs co-cultured with AGEs showed a decrease in the Bcl-2/Bax ratio. ECsco-cultured with AGEs+HRM were shown to attenuate the AGEs-induced decrease in theBcl-2/Bax ratio.This indicate that endothelial cell apoptosis induced by AGEs occurs partlythrough the mitochondrial apoptotic pathway.
Conclusion
AGEs induce ECs apoptosis and oxidative stress in a concentration-dependent manner.
HRM can antagonize the apoptosis and oxidative stress induced by AGEs. It plays arole in anti-oxidative stress and apoptosis by increasing SOD, GSH-PH and Bcl-2(inhibitorof apoptosis protein)/Bax (apoptosis-promoting protein) ratio and reducing ROS levels.
糖尿病是一种常见的代谢性疾病，主要表现为机体胰岛素相对或者绝对不足，导致血糖水平持续性增高。目前全球2型糖尿病患者已超过1.5亿人，而且预计在未来20年内病例数还将成倍数增长态势，全球医疗负担也会因为这一数值的显著提高而明显增加[1]。不论是1型还是2型糖尿病，随着长期高血糖水平病程的逐渐进展，均可以造成不同程度的，不同靶器官的并发症，其中以心血管病变最为常见，也最具威胁性。
血管并发症是导致糖尿病患者死亡的主要原因。其中，高血压，肥胖症，动脉粥样硬化，血脂代谢异常，糖尿病肾病亦是患者预后不良的重要危险因素。在糖尿病的发生发展过程当中，血管内皮的损伤和内皮功能障碍是糖尿病重要的早期病理表现。从细胞层面来讲，内皮细胞代谢异常则是早期内皮功能受损的基本原因,伴随着内皮细胞的不断凋亡和功能受损，随之而来的则是内皮功能障碍[2.3]。
晚期糖基化终末产物(AGEs)是以体内大分子物质如脂肪，蛋白质，核酸和还原性糖(葡萄糖，果糖，戊糖)为原料，在生理环境中通过发生非酶促反应，生成的稳定共价键产物。这种非酶促反应称之为---Maillard(美拉德)。1953年Hodge等学者提出了美拉德反应，该反应主要包括如下三部分：1.初级反应阶段。还原糖的羰基与氨基之间进行加成，加成物迅速失去分子水转变为希夫碱。2.经环化形成相应的取代醛基胺，经重排后转成有反应活性的产物，其中最熟知的是糖基化血红蛋白和果糖胺。3.果糖胺等又经过一系列复杂的分解和化学重排反应最终形成了AGEs类黑精色素。AGEs一旦形成就是不可逆的，并且其具有较强的交联性和较低的溶解性。此外，AGEs还具有产生和激活ROS的能力[61]，这些特点也是其损伤血管的重要理化因素。AGEs的产生和糖尿病患者血糖水平以及高血糖暴露的时间成正相关，患者血糖水平越高，暴露高血糖的时间越久，AGEs的产生和堆积就越多。此外，局部微环境的氧化应激也是加速AGEs形成的重要因素之一[62.63]。在正常机体中，葡萄糖的糖基化反应较低。然而，在糖尿病患者体内，持续性的高血糖和局部微环境的氧化应激加速了AGEs产生和堆积。
血液循环中堆积的AGEs可进行性损害血管内皮[65]。国外研究者发现，胰岛素可通过清除AGEs来保护内皮细胞功能，胰岛素通过IRS以及PI-3K通路提高机体产生NO的能力，从而提高机体对葡萄糖的转运和利用，减少AGEs的产生和保护内皮功能。此外，胰岛素还可以提高内皮细胞清道夫受体的含量，从而加速AGEs的清除。另外，蓄积在体内的AGEs低密度脂蛋白(LDL)可以形成AGEs-LDL共价化合物，形成AGEs-LDL后，LDL在体内的循环清除受阻，LDL在循环中的比例升高，致体内脂质水平明显增加，加重内皮氧化损伤。此外，血管内皮的基质成分含有胶原蛋白，它们与循环中的还原性糖发生共价结合以后可以使胶原蛋白交联，从而使血管内皮内环境受到破坏。同时，血管内皮的AGEs还能够激活单核巨噬系统，使得它们在对AGEs发生吞噬作用的同时分泌大量的细胞因子，刺激血管内皮发生氧化，应激，变形，增生，增殖，凋亡等一系列病理反应，恶化内皮功能，最终导致动脉粥样硬化，甚至血管完全闭塞。在糖尿病患者当中，持续升高的血糖状态极易导致AGEs的过量产生[4]。我们课题组的前期研究也发现，AGEs可以加快血管内皮细胞的凋亡和内皮功能不全的病理进程；在AGEs诱导的细胞当中，其细胞间活性氧(ROS)水平较对照组是明显升高的，而且同时，这种升高的细胞间ROS水平反过来进一步刺激AGEs的不断生成，形成“氧化应激-AGEs”的恶性循环[6.7]。
因此，如何清除AGEs或减少这些AGEs的产生似乎可以成为减少糖尿病并发症的治疗策略之一。但是，AGEs一旦在血管内皮生成共价键，其就如一团黏着的基团，很难清除。那么，如果能够拮抗由AGEs介导的血管内皮功能损伤，则理论上也能预防或者延缓血管内皮损伤-动脉粥样硬化的进程[8]。所以，探寻一种拮抗AGEs介导的内皮细胞氧化应激以及凋亡的治疗策略有望成为防治糖尿病并发症的潜在切入点。
传统的抗氧化应激的制剂包括维生素C，维生素E，β胡萝卜素，超氧化物歧化酶等。由于结构和效价关系的限制，这些天然抗氧化剂的抗氧化作用较弱且作用可逆，组织穿透能力差，而且其循证学研究的结果多为阴性。另外，在已经发生氧化应激反应的细胞当中多半有代谢和功能障碍，其自身合成分泌抗氧剂的能力差。为此，寻求一种新型的，有效的，安全的，穿透力强，靶向性高的抗氧剂是拮抗机体氧化应激反应延缓动脉粥样硬化的潜在突破口之一。
氢气是一种无色，无味，无臭具有一定还原性的易燃气体。过去许多学者认为氢气是一种生理性惰性气体，不会与机体生命物质发生生理生化反应。但也有极少数的科学家认为氢气是一种具有还原性的气体。发表在1975年《Science》杂志上的一篇文章报道，在皮肤鳞状细胞癌动物模型中，连续呼吸8个大气压97.5%氢气两周，可以有效治疗鳞状细胞癌，并且研究者认为其机制可能与氢气的抗氧化作用有关[9]。之后又有实验研究发现，呼吸高压氢气能够治疗肝脏寄生虫感染引起的肝炎，并提出氢气的抗炎作用机制可能与氢气和羟自由基反应有关[10]。虽然上述研究为引起学者的广泛关注，但Ohsawa等2007年发表在《Nature》上的一篇文章提出，缺血再灌注和炎症所致的急性氧化应激可以导致严重的组织损伤。研究者通过建立大脑缺血再灌注的氧化应激大鼠模型，在非高压的条件下，应用吸入氢气作为治疗干预，结果发现氢气能够通过抗氧化作用减轻大鼠脑缺血再灌注损伤。文中还讨论到，氢气可能具有选择性的抗氧化作用，也即氢气只于与·OH和ONOO-发生反应，而并不与其它具有生理活性作用的活性氧发生生物化学反应，如O2-·、H2O2和NO·等。氢气的这种温和还原性的优点，使得它在抗氧化作用的同时对机体内部必须的生理反应过程影响极小。另外，和生理活性氧O2-·、H2O2和NO·相比，·OH和ONOO-等氧自由基的氧化性明显高于生理活性氧，而这也使得还原性比较温和的氢气只与这些强自由基的发生中和反应。除了这些特点外，氢气的高物理扩散性又是其独特的优势[11]。随后，日本科学家Hayashida等在Ohsawa等研究的基础上，在大鼠心肌梗死缺血再灌注模型中证实吸入氢气能够有助于心梗再灌注后心功能的恢复，减少心肌梗死面积，并预防心室结构重构。实验再次证实了氢气到达治疗靶点的高效性和高通透性[12]。2010年，Chen CH等学者在大鼠局灶脑缺血过程中，吸入氢气2小时能够降低基质金属蛋白-9活性并减小脑缺血面积[13]。2012年，中国学者Ge等在大鼠动物模型中发现吸入氢气可以减轻短暂脑缺血诱导的认知功能障碍，其可能的机制为氢气可以拮抗氧化应激作用从而减少神经细胞的死亡[14]。同年，Zhan Y学者等在Crit Care Med杂志发表关于吸入氢气治疗的实验研究成果。他们在130余只硬膜下出血脑损伤的大鼠模型中发现吸入氢气2小时可以明显减轻脑水肿和血脑屏障损伤[15]。同时在分子研究层面证实，氢气可以减轻大脑脂质，核酸，蛋白质的氧化损伤。正如德国学者Hardeland对该研究的评论，吸入氢气可能是将来医学领域一种颇有前景的治疗策略[16]。
不仅如此，除了吸入氢气，科学家们还利用氢气扩散特点制备成富含氢的溶液并探究其治疗作用。最开始是由Sun等学者提出假设富含氢的生理盐水是否依然具有抗氧化应激作用。他们在大鼠的心梗模型中通过注射富氢生理盐水的方法对心梗后的大鼠进行干预，结果发现富氢生理盐水仍然具有有效抗氧化应激，减少心肌死亡，改善心功能的治疗作用[17]。同期还有中国学者们发现富氢生理盐水对小肠的缺血再灌注损伤也有保护作用，并同时可以改善小肠的收缩功能[18.20]。另外Chen等在大鼠实验中发现，富氢生理盐水在急性胰腺损伤中能减轻氧化应激，减少胰腺细胞死亡，有意思的是，富氢生理盐水还能促进胰腺腺泡细胞的增生[21]。因为富氢生理盐水的制备过程并不复杂，而且有使用方便，便于运输，实用等特点，一时间将富氢生理盐水作为治疗制剂研究其治疗作用的研究迅速成为热点，相应的研究结果均提示富氢溶液作为医疗制剂的诱人前景[22.30]。
尽管如此，人们对氢的抗氧化作用的具体分子生物学机制还未能深入研究。前面我们提到，AGEs可以诱导血管内皮的氧化应激反应，从而刺激内皮细胞增生，炎症细胞侵润，凋亡，内皮功能障碍，血管粥样硬化的系列病理改变。氢能否通过抑制AGEs诱导的氧化应激，产生内皮保护作用有待实验进一步证实。基于此，我们课题组设计了“氢气抗AGEs诱导的内皮凋亡及其机制”这课题，对我们的课题假设进行初步证实。
材料和方法
SD大鼠购自于第三军医大学动物室，用于主动脉内皮细胞的原代培养。富含氢气的培养基被应用于模拟高氢气的环境。Real-time PCR和分光光度测定KIT用于抗氧化应激酶SOD和GSH-PH基因和蛋白表达量检测， DCF-DA作为荧光探针运用流式细胞仪检测细胞内的活性氧(ROS)水平；Annexin V-APC凋亡检测试剂盒和TUNEL染色法被用于内皮细胞凋亡的检测,蛋白印迹分析(WB)用于检测Bcl-2(凋亡抑制蛋白)和Bax(凋亡促进蛋白)比率分析于说明内皮细胞凋亡水平。
结果
1.AGEs可以诱导内皮细胞凋亡并且呈浓度依赖性，HRM组和正常对照组的细胞凋亡水平无显著差异。AGEs组细胞凋亡水平明显高于正常对照组和HRM组，加入HRM能够明显抑制AGEs所诱导的细胞凋亡。
2.流式细胞仪分析法检测发现，加有AGEs的内皮细胞(AGEs组)其细胞间ROS水平较正常对照组明显升高，且升高的水平成浓度依赖关系。但HRM组细胞ROS水平和正常对照组比较我显著差异。在AGEs+HRM组中发现细胞间ROS水平较AGEs组明显降低，并和正常对照组相似。
3.抗氧应激检测显示：在mRNA方面，AGEs能显著降低SOD mRNA的表达水平，而且这中降低与AGEs呈浓度相关性。而HRM干预后能够明显改善AGEs对SODmRNA表达的抑制作用。另外，在酶活性比较方面，AGEs对内皮细胞SOD和GSH-PX这两种抗氧化酶的酶活性均成抑制效应，而HRM干预则能改善AGEs的这种不良效应。
4.凋亡蛋白水平检测发现AGEs组Bcl-2表达量较正常对照组明显下降，而Bax蛋白表达量较对照组明显增加，Bcl/Bax蛋白表达量比例在改组明显下降，提示AGEs明显诱导内皮细胞凋亡。在AGEs+HRM组中，Bcl/Bax蛋白表达量比例较正常对照组无明显下降，提示HRM能够拮抗由AGEs诱导的细胞凋亡。
结论
1. AGEs可以诱导内皮细胞凋亡和氧化应激并且呈浓度依赖性.
2. HRM能够拮抗由AGEs诱导的细胞凋亡和氧化应激，起作用可能通过提高SOD和GSH-PH以及Bcl-2(凋亡抑制蛋白) Bax(凋亡促进蛋白)比率并降低ROS水平发挥抗氧化应激和抗凋亡作用.
Background and Objective
It is known that, type1and type2diabetes alike, with the gradual progression of thelong-term high blood glucose levels, can cause different degrees of complications invarious target organs, among which the cardiovascular disease is the most common andthreatening[1]. At present, the number of patients with type2diabetes has amounted to morethan150million worldwide, and is expected to grow exponentially in the next20years[2].Patients with diabetes-induced cardiovascular disease will significantly increase in number,leading to a heavier global medical burden. How to control cardiovascular complications isthe focus of current research.
In fact，cardiovascular complications in diabetes mellitus are one of the leading causesof mortality. Vascular endothelial injury and dysfunction are the important earlypathological manifestations of these complications.[31]Increasing apoptosis is an earlymanifestation of endothelial injury, which will lead to endothelial dysfunction[32].Advancedglycation end products (AGEs) are the products of non-enzymatic glycosylation of theamino groups of macromolecules, such as proteins and nucleic acids[33]. In diabetic patients,sustained high blood glucose level significantly increases the production of AGEs[33].Epidemiological studies show that the presence of AGEs is highly correlated with diabeticcardiovascular complications. Previous studies have found that AGEs increase endothelialcell apoptosis and dysfunction[34]. In addition, according to our previous study, AGEsincrease apoptosis and dysfunction of endothelial precursor cells[35].The generation ofintracellular reactive oxygen species (ROS) increases in AGEs-induced cells, and oxidativestress and ROS production, in turn, contribute to AGEs-induced apoptosis of endothelialcells and endothelial precursor cells[36].
As we know，removal of AGEs from the body is difficult. One strategy to reduce AGEs-induced endothelial injury is to antagonize AGEs-activated oxidative stress[32].Hydrogen (H2) is the smallest naturally occurring gas molecule. Studies have shown thatH2has antioxidant activities in living organisms. It can specifically neutralize the mostpotent oxidative free radicals (OH and ONOO-) and attenuate the superoxidant anion levelunder certain pathophysiological conditions[37]. Moreover, it is easy for H2to pass throughmembrane structures such as cell membranes and the mitochondrial membranes, where itcan neutralize intracellular ROS, thereby maintaining normal mitochondrial function andpreventing apoptosis[38]. Many studies have shown that H2or hydrogen-containing solutioncan alleviate ischemia-reperfusion injury[34.36.39]of the heart[40],brain[41.42], kidney, smallintestine, and liver[41]; and it can antagonize irradiation-induced cell injury[43]. Inhalation ofH2also slows down the growth of atherosclerotic plaque in apoE-/-mice[44]. Our previousstudies also found that hydrogen-rich saline prevented neointima formation after carotidballoon injury[45]. Oxidative stress plays an important role in ischemia-reperfusion injury,irradiation-induced injury, atherosclerosis and neointima formation[46]. The ability of H2toantagonize these pathological reactions is closely related to its anti-ROS effects. However,it has yet to be elucidated whether H2can ameliorate AGEs-induced ROS generation andapoptosis of endothelial cells.
The objective of the present study was to determine whether the use of hydrogen-richmedium (HRM) can protect the endothelial cells from AGEs-induced apoptosis by detectingROS production and antioxidant-related gene expression.
Materials and Methods
Two to three year old Sprague-Dawley rats purchased from the Experimental AnimalCenter of the Third Military Medical University were used in this study. The thoracic aortawas removed from these rats, and endothelial cells (ECs) were isolated and cultured. Afterculturing ECs in the presence of AGEs and/or with HRM for24h, Annexin V/7-AAD andTUNEL staining were carried out to detect apoptosis. Intracellular ROS was detected withfluorescent probes and quantified with flow cytometry. Anti-oxidative enzyme (SOD andGSH-PH) expression was determined by real-time PCR analysis and enzymatic assay. Therelative expression levels of Bcl-2and Bax were analyzed by western blotting.
Results and Discussion
1. AGEs induced ECs apoptosis in a concentration-dependent manner. After24h,evaluation of the4EC groups, including AGEs (400μg/ml) group, HRM group, HRM+AGEs group, and normal control group, revealed that the cell apoptosis rates were notsignificantly different between the HRM group and the control group when AGEs wereabsent. However, the presence of AGEs increased the rate of ECs apoptosis, and theaddition of HRM reduced the apoptosis rate of AGEs-treated ECs.
2. Treatment of ECs with AGEs significantly increased the generation of intracellularROS in a dose-dependent manner. However, HRM decreased significantly.
3. AGEs significantly reduced the level of SOD mRNA. However, after HRMintervention, the SOD expression reduction caused by AGEs was significantly attenuated.AGEs treatment significantly increased the optical density of anti-oxidative enzymes. HRMtreatment was also able to partly reduce this effect.
4. ECs co-cultured with AGEs showed a decrease in the Bcl-2/Bax ratio. ECsco-cultured with AGEs+HRM were shown to attenuate the AGEs-induced decrease in theBcl-2/Bax ratio.This indicate that endothelial cell apoptosis induced by AGEs occurs partlythrough the mitochondrial apoptotic pathway.
Conclusion
AGEs induce ECs apoptosis and oxidative stress in a concentration-dependent manner.
HRM can antagonize the apoptosis and oxidative stress induced by AGEs. It plays arole in anti-oxidative stress and apoptosis by increasing SOD, GSH-PH and Bcl-2(inhibitorof apoptosis protein)/Bax (apoptosis-promoting protein) ratio and reducing ROS levels.
引文
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