尿苷对线粒体功能的影响

尿苷是生物体内必需的营养物之一,需将尿苷浓度维持在一定浓度水平才能维持胞内正常的生长代谢。近年来,有研究发现尿苷可通过多种机制缓解生物体炎症反应,并能参与胞内糖酵解等代谢途径,且可调节胞内糖基化、乙酰化等蛋白修饰作用。此外,其还能通过减轻胞内氧化应激、促进高能化合物合成等方式保护细胞免受缺氧性损伤。研究表明,这些保护作用与尿苷对线粒体功能的调节作用息息相关。因此,本文主要综述了尿苷及其代谢反应(物)对线粒体功能的研究进展。     

缺氧缺血性脑损伤对新生鼠脑SCN8A基因表达的影响

目的通过观察缺氧缺血致脑损伤大鼠在不同时间段SCN8A基因的表达,探讨由于缺氧缺血导致脑损伤发生的分子生物学机制。方法新生7日龄Wistar大鼠108只。随机分为对照组、假手术组、缺氧缺血组,每组分为3 h、6 h、12 h、1 d、3 d、7 d六个时点,采用Rice法制作动物模型(HIBD模型),于缺氧缺血后不同时间段处死大鼠。采用HE染色和电镜观察大鼠脑组织损伤情况;Western-blot法检测SCN8A基因表达产物Nav1.6蛋白在膜结构中含量的差异,并比较不同时点各组的差异。结果 HE染色可见缺氧缺血后大脑皮质神经元层次不清,细胞周围腔隙扩大,局部神经元坏死、崩解,形成坏死灶,周围有胶质细胞增生,毛细血管显著扩张,血管周围腔隙扩大,并有红细胞泄露到腔隙中,且损伤程度随缺氧缺血时间延长而加重。电镜观察发现缺氧缺血时间越长,脑细胞损伤愈明显,细胞膜皱褶、破碎;线粒体堆积、嵴消失、空泡化,细胞核边集化、破损。缺氧缺血组与对照组、假手术组比较,Nav1.6蛋白表达水平在缺氧缺血3 d、7 d均升高(P0.05),缺氧缺血3 h、6 h、12 h和1 d差异无统计学意义。结论缺氧缺血脑损伤后,脑组织SCN8A基因的表达提高,且在3 d、7 d时变化差异有统计学意义;缺氧缺血时Na+通道含量的改变,是造成脑细胞进一步损伤的分子生物学机制之一。     

神经节苷脂与头部贴敷式亚低温联合治疗对新生鼠缺氧缺血性脑损伤的影响

目的:研究神经节苷脂与头部贴敷式亚低温联合治疗新生鼠缺氧缺血性脑损伤时脑组织内一氧化氮(NO)和(MDA)含量的变化,为新生儿缺氧缺血性脑病的治疗提供理论依据.方法:将88只WISTAR新生鼠随机分为四组,神经节苷脂治疗组(IG)、头部贴敷式亚低温治疗组(IH)、联合治疗组(IB)以及对照组(CN).结果:CN组NO和MDA的含量明显升高,IG、IH以及IB组NO和MDA均明显下降.结论:神经节苷脂和亚低温通过降低NO和MDA的含量保护缺氧缺血神经元,二者联合效果更好.     

β－内啡肽在新生鼠缺氧缺血性脑损伤中的作用

本实验观察到新生鼠缺氧缺血后,皮层β－内啡肽（β─Ｅｐ）含量显著增多,与此同时,皮层比重显著降低,两者呈显著相关;注射适量的纳洛酮或β─Ｅｐ抗血清,可显著减轻皮层水肿;注射β─Ｅｐ,则可加重皮层水肿。提示β─Ｅｐ可能参与新生鼠缺氧缺血性脑损伤的病理过程。     

定位在线粒体的肝刺激因子对线粒体膜电势的影响

目的肝刺激因子（hepatic stimulator substance,HSS）可以保护肝细胞免受各种毒素的影响,但机制尚未清楚,研究探讨肝刺激因子保护肝细胞的可能机制。方法利用稳定转染FLAG-pcDNA3．0／hHss的肝癌细胞BEL-7402为模型,使用Alexa Flour 488、Hoechst 33342、MitoTracker 580分别将HSS、细胞核以及线粒体染色,观察HSS在细胞中的定位情况。当野生型7402细胞、转染空载体FLAG-pcDNA3．0的7402细胞以及转染FLAppcDNA3．0／hHSS的7402细胞受到线粒体膜孔道开放剂羰基氰化间氯苯腙（carbonyl cyanide m—chlorophenylhydrazone,CCCP）的损伤后,用电镜观察线粒体形态、荧光素酶检测ATP、流式细胞仪测定线粒体膜电位（mitoehondrial membrane potential,MMP）等,综合观察过表达HSS的肝细胞的抗损伤能力。结果在稳定转染hHSS基因的7402细胞中,大部分HSS与线粒体共定位;在CCCP作用下,对照组野生型7402细胞以及转染空载体的7402细胞MMP下降明显,线粒体肿胀,嵴断裂、消失,ATP下降显著;实验组稳定转染hHSS基因的7402细胞MMP下降幅度较小,线粒体肿胀与嵴形态的改变明显减轻,ATP的含量较对照组高。结论肝刺激因子HSS在细胞中主要定位于线粒体,可以稳定MMP,维持线粒体形态及细胞内ATP的水平,从而增强肝细胞抗损伤的能力。     

β—内啡肽在新生鼠缺氧缺血性脑损伤中的作用

本实验观察到新生鼠缺氧缺血后,皮层β－内啡肽（β－Ｅｐ）含量显著增多,与此同时,皮层比重显著降低,两者呈显著相关;注射适量的纳洛酮或β－Ｅｐ抗血清,可显著减轻皮层水肿;注射β－Ｅｐ,则可加重皮层水肿。提示β－Ｅｐ可能参与新生鼠缺鼠缺氧缺血性脑损伤的病理过程。     

磷酸肌酸对豚鼠心肌细胞缺氧复氧损伤膜电位的影响

本实验观察了缺氧复氧对豚鼠心肌细胞膜电位的影响及磷酸肌酸的保护作用。结果表明缺氧５－２０ｍｉｎ时ＲＰ、ＡＰＡ减小，Ｖｍａｘ减慢，ＡＰＤ１０、ＡＰＤ５０、ＡＰＤ９０缩短。复氧５－２０ｍｉｎ后，ＲＰ、ＡＰＡ和Ｖｍａｘ进一步减少，ＡＰＤ１０、ＡＰＤ５０、ＡＰＤ９０明显缩短。在灌流液中加入磷酸肌酸保持其浓度在１０－６ｍｏｌ时，ＲＰ、ＡＰＡ显著增加，Ｖｍａｘ增快，ＡＰＤ１０、ＡＰＤ５０、ＡＰＤ９０明显延长。复氧２０ｍｉｎ后膜电位各项参数趋向恢复正常。此结果提示，磷酸肌酸对缺氧复氧的心肌具有显著的保护作用。     

ATP浓度和缺氧暴露对大鼠脑线粒体RNA和蛋白质体外合成的影响

本文探讨介质中ATP浓度和急,慢性缺氧暴露对大鼠脑线粒体内RNA和蛋白质合成的影响。用差速离心法分离正常和低压舱模拟4000m高原急性连续缺氧暴露3d和慢性连续缺氧暴露40d大鼠脑线粒体,用体外无细胞（cell-free in vitro)^3H-UTP和^3H-Leucine掺入法分别测定线粒体RNA和蛋白质合成活性,结果显示,大鼠急性缺氧暴露后大脑皮质线粒体RNA体外合成活性降低40%,蛋白质合成活性降低60%;慢性缺氧暴露后线粒体RNA和蛋白质合成活性分别为对照的72%和76%;ATP对正常大鼠脑线粒体RNA以及蛋白质的体外合成活性的影响均呈双相性,大于或小于1mmol/L均可产生不同程度的抑制效应,结果提示,缺氧可在转录和翻译两个水平上影响脑线粒体mtDNA的表达,而慢性缺氧暴露时,线粒体半自主性功能的改善可能是机体对缺氧适应的细胞机制之一;ATP对脑线粒体内转录和释放活性的调节是一种经济有效的反馈调节方式。     

竹节参对力竭运动大鼠心肌线粒体ATP酶活性的影响

目的：探讨竹节参对力竭运动大鼠心肌线粒体ATP酶活性的影响。方法：建立力竭运动大鼠模型,测定心肌线粒体ATP酶的活性,研究竹节参对大强度耐力训练大鼠心肌线粒体的保护作用。结果：力竭运动引起大鼠心肌线粒体ATPase（Na＋,K＋-ATPase和Ca2＋-ATPase）活性显著下降,而运动加药组Ca2＋-ATPase有显著升高,Na＋,K＋-ATPase也有明显升高,且ATPase活性均接近于安静对照组的水平。结论：竹节参可提高力竭运动大鼠心肌线粒体内Na＋,K＋-ATP酶和Ca2＋-ATP酶的活性,提示其具有保护线粒体的作用。     

Mitochondrial Calcium Transport and Mitochondrial Dysfunction After Global Brain Ischemia in Rat Hippocampus

Here we report effect of ischemia-reperfusion on mitochondrial Ca²⁺ uptake and activity of complexes I and IV in rat hippocampus. By performing 4-vessel occlusion model of global brain ischemia, we observed that 15 min ischemia led to significant decrease of mitochondrial capacity to accumulate Ca²⁺ to 80.8% of control whereas rate of Ca²⁺ uptake was not significantly changed. Reperfusion did not significantly change mitochondrial Ca²⁺ transport. Ischemia induced progressive inhibition of complex I, affecting final electron transfer to decylubiquinone. Minimal activity of complex I was observed 24 h after ischemia (63% of control). Inhibition of complex IV activity to 80.6% of control was observed 1 h after ischemia. To explain the discrepancy between impact of ischemia on rate of Ca²⁺ uptake and activities of both complexes, we performed titration experiments to study relationship between inhibition of particular complex and generation of mitochondrial transmembrane potential (ΔΨ_m). Generation of a threshold curves showed that complex I and IV activities must be decreased by approximately 40, and 60%, respectively, before significant decline in ΔΨ_m was documented. Thus, mitochondrial Ca²⁺ uptake was not significantly affected by ischemia-reperfusion, apparently due to excess capacity of the complexes I and IV. Inhibition of complex I is favourable of reactive oxygen species (ROS) generation. Maximal oxidative modification of membrane proteins was documented 1 h after ischemia. Although enhanced formation of ROS might contribute to neuronal injury, depressed activities of complex I and IV together with unaltered rate of Ca²⁺ uptake are conditions favourable of initiation of other cell degenerative pathways like opening of mitochondrial permeability transition pore or apoptosis initiation, and might represent important mechanism of ischemic damage to neurones.     

Effects of Dichloroacetate on Brain Tissue Pyruvate Dehydrogenase

The activation of the pyruvate dehydrogenase complex (PDHC) by dichloroacetate (DCA) was studied in brain tissue. Chronic administration of DCA to rats caused no significant change of PDHC activation in brain. DCA brain concentrations were comparable to those of other tissues in which activation is known to occur. No effect of DCA on PDHC could be demonstrated from isolated brain mitochondria, whereas DCA reversed the deactivation of PDHC by ATP, alpha-ketoglutarate plus malate, and succinate in liver mitochondria. This study suggests that the regulation of PDHC activation in neural tissue differs from that in other tissues.     

Degradation of Mitochondrial Phospholipids During Experimental Cerebral Ischemia in Rats

Changes in content of brain mitochondrial phospholipids were examined in rats after 30 and 60 min of decapitation ischemia compared with controls, to explore the degradation of the mitochondrial membrane and its relation to dysfunction of mitochondria. Activities of respiratory functions and respiratory enzymes (cytochrome c oxidase; F0F1-ATPase) decreased significantly during ischemia. Considerable decreases in cardiolipin and phosphatidylinositol content were observed after 60 min, and other phospholipids showed similar but nonsignificant decreases in content. The amount of polyunsaturated fatty acids chains, such as arachidonic and docosahexaenoic acids, was reduced in each phospholipid, in some cases significantly, after 30 and 60 min of ischemia in time-dependent manners. Degradation of mitochondrial phospholipids during ischemia associated with the deterioration of mitochondrial respiratory functions suggested the significance of such changes in phospholipid content in disintegration of cellular energy metabolism during cerebral ischemia.     

Effects of Postdecapitative Ischemia on Mitochondrial Respiration in Brain Tissue Homogenates

Mitochondria isolated from ischemic brain characteristically show changes in respiratory function. As conventional procedures for mitochondrial isolation yield a subpopulation of the total population and require extensive manipulation, it is unclear to what extent these changes are representative of mitochondria in the unfractionated tissue. We previously showed that the oxygen uptake by unfractionated forebrain homogenates, measured under two different sets of incubation conditions, provided information on some aspects of the respiratory activity of both the free and synaptosomal pools of mitochondria. Forebrain homogenates from animals subjected to 30 min of postdecapitative ischemia exhibited large reductions in oxygen uptake rates measured in a high K+ (mitochondrial) buffer in the presence of either ADP (44% of control values) or an uncoupling agent (45% of control values). These reductions in respiratory activity were comparable to alterations observed under the same conditions for mitochondria isolated from the ischemic brains. Similar alterations were seen in homogenates from three subregions: neocortex, hippocampus, and striatum. In a physiological buffer, in which oxygen uptake by homogenates largely resulted from activity of mitochondria within synaptosomes, there was little or no change in basal glucose-supported rates (79-96% of control values) and small reductions in maximal rates (63-81% of control values) measured in the presence of an uncoupling agent. These results suggest that alterations of respiratory function seen in isolated free mitochondria provide appropriate estimates of the dysfunction in the total free mitochondrial pool but that synaptosomal mitochondria may be less affected. Measurements of respiratory function of isolated synaptosomes from ischemic tissue provided further support for the relative preservation of synaptosomal mitochondria during ischemic insult.     

Effect of Ischemic Preconditioning on Mitochondrial Dysfunction and Mitochondrial P53 Translocation after Transient Global Cerebral Ischemia in Rats

Transient global brain ischemia induces dysfunctions of mitochondria including disturbance in mitochondrial protein synthesis and inhibition of respiratory chain complexes. Due to capacity of mitochondria to release apoptogenic proteins, ischemia-induced mitochondrial dysfunction is considered to be a key event coupling cerebral blood flow arrest to neuronal cell death. Ischemic preconditioning (IPC) represents an important phenomenon of adaptation of central nervous system (CNS) to sub-lethal short-term ischemia, which results in increased tolerance of CNS to the lethal ischemia. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated inhibition of mitochondrial protein synthesis and activity of mitochondrial respiratory chain complexes I and IV in the hippocampus of rats. Global brain ischemia was induced by 4-vessel occlusion in duration of 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our results showed that IPC affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Repression of mitochondrial translation induced during reperfusion of the ischemic brain is significantly attenuated by IPC. Slight protective effect of IPC was documented for complex IV, but not for complex I. Despite this, protective effect of IPC on ischemia/reperfusion-associated changes in integrity of mitochondrial membrane and membrane proteins were observed. Since IPC exhibited also inhibitory effect on translocation of p53 to mitochondria, our results indicate that IPC affects downstream processes connecting mitochondrial dysfunction to neuronal cell death.     

原花青素对脑缺血再灌损伤大鼠模型的影响

目的研究原花青素对脑缺血/再灌损伤(ischemia/reperfusion,I/R)大鼠神经功能评分(neurologicaldeficit score,NDS)、脑梗死体积、脑含水量等指标的药理作用。方法采用大鼠大脑中动脉阻断(middle cerebralartery occlusion,MCAO)法复制类似人类缺血性卒中的I/R损伤模型。结果该模型各时间点内均有程度不同的神经功能缺失,原花青素给药组神经功能评分明显低于对照组(P0.05),假手术组大鼠均无神经功能缺失,脑水肿情况均较对照组明显改善(P0.05),脑梗死体积与盐水对照组相比差异有显著性(P0.05),而假手术组均未见有梗死灶。结论原花青素具有一定的保护大鼠I/R后受损脑组织的作用,可供后续研究,并可为缺血性卒中使用原花青素治疗提供确凿的理论依据。     

Regional Energy Balance in Rat Brain After Transient Forebrain Ischemia

Abstract: Phosphocreatine, ATP, and glucose were severely depleted, and the lactate levels were increased in the paramedian neocortex, dorsal-lateral striatum, and CA1 zone of hippocampus of rats exposed to 30 min of forebrain ischemia. Upon recirculation of the brain, phosphocreatine, ATP, and lactate concentrations recovered to control values in the paramedian neocortex and CA1 zone of hippocampus and to near-control values in the striatum. The phosphocreatine and ATP concentrations then fell and the lactate levels rose in the striatum after 6–24 h, and in the CA1 zone of hippocampus after 24–72 h. The initial recovery and subsequent delayed changes in the phosphocreatine, ATP, and lactate concentrations in the striatum and hippocampus coincided with the onset and progression of morphological injury in these brain regions. The results suggest that cells in these regions regain normal or near-normal mitochondrial function and are viable, in terms of energy production, for many hours before unknown mechanisms cause irreversible neuronal injury.     

Altered Mitochondrial Respiration in Selectively Vulnerable Brain Subregions Following Transient Forebrain Ischemia in the Rat

Mitochondrial respiratory function, assessed from the rate of oxygen uptake by homogenates of rat brain subregions, was examined after 30 min of forebrain ischemia and at recirculation periods of up to 48 h. Ischemia-sensitive regions which develop extensive neuronal loss during the recirculation period (dorsal-lateral striatum, CA1 hippocampus) were compared with ischemia-resistant areas (paramedian neocortex, CA3 plus CA4 hippocampus). All areas showed reductions (to 53-69% of control) during ischemia for oxygen uptake rates determined in the presence of ADP or an uncoupling agent, which then recovered within 1 h of cerebral recirculation. In the ischemia-resistant regions, oxygen uptake rates remained similar to control values for at least 48 h of recirculation. After 3 h of recirculation, a significant decrease in respiratory activity (measured in the presence of ADP or uncoupling agent) was observed in the dorsal-lateral striatum which progressed to reductions of greater than 65% of the initial activity by 24 h. In the CA1 hippocampus, oxygen uptake rates were unchanged for 24 h, but were significantly reduced (by 30% in the presence of uncoupling agent) at 48 h. These alterations parallel the development of histological evidence of ischemic cell change determined previously and apparently precede the appearance of differential changes between sensitive and resistant regions in the content of high-energy phosphate compounds. These results suggest that alterations of mitochondrial activity are a relatively early change in the development of ischemic cell death and provide a sensitive biochemical marker for this process.     

局灶性脑缺血大鼠皮层神经元超微结构及线粒体呼吸功能变化的研究

目的:研究局灶性脑缺血大鼠脑细胞超微结构及脑组织线粒体呼吸链功能的变化。方法:采用改良Zea Longa方法复制大鼠大脑中动脉缺血(MCAO)模型,透射电镜观察缺血后脑组织神经元超微结构的改变;检测呼吸链R3、R4、RCR、OPR等评价呼吸功能的指标。结果:局灶性脑缺血大鼠脑组织神经元细胞结构严重破坏;与对照组相比,脑缺血时大鼠脑线粒体ST3、RCR和OPR降低,ST4升高。结论:脑缺血急性期线粒体结构破坏,功能受损严重,随着时间延长均有所恢复;保护线粒体呼吸链可能对脑缺血损伤有保护作用。     

D-半乳糖诱导大鼠脑损伤的糖基化机制

目的D-半乳糖（D-galactose）诱导大鼠体内不同糖基化水平,研究其脑损伤发生的机理。方法采用不同剂量D-半乳糖[150、75、37．5mg／（kg·d）]分别腹腔注射（ip）处理大鼠8周,诱导糖基化状态和脑损伤。采用硫代巴比妥酸（TBA）比色法测定糖化血红蛋白,硝基四氮唑蓝（NBT）比色法测定血清果糖胺;按文献方法分别测定血红细胞醛糖还原酶活性和晚期糖基化终末产物（AGEs）含量及脑组织中AGEs含量,羟胺法和比色法分别测定SOD和GSH-Px活性,硫代巴比妥酸法测定MDA含量;以Fura-2／AM作为钙荧光指示剂,双波长荧光分光光度法检测脑海马神经细胞胞质[Ca^2＋]i的变化;透射电镜观察脑海马神经细胞线粒体的变化。结果D-半乳糖处理8周后,大鼠血红细胞醛糖还原酶活性升高,糖化产物形成增多;脑组织中AGEs及脑细胞胞质[Ca^2＋]i含量明显升高,SOD及GSH-Px活性下降,MDA含量升高（P〈0．01,P〈0．05）,海马神经细胞线粒体出现病理性改变。结论D-半乳糖通过诱导体内蛋白糖基化和脑组织AGEs大量生成,降低抗氧化能力及胞质[Ca^2＋]i超负荷等,导致脑细胞损伤。