衰老过程中线粒体的分子病理

衰老过程中线粒体的分子病理张平线粒体几乎存在于所有组织细胞内,它是维持细胞以至整个机体生理活动的能源基础。近年来对线粒体分子生物学的深入研究,取得了重要进展,并发现其结构、功能上有许多增龄性变化（衰变）,逐步导致全身性生理功能的下降,为阐明衰老的分子...     

氧化应激损伤线粒体参与癫痫病理过程

癫痫是由脑内神经元异常放电所致,严重危害健康和影响生存质量,其致病机制主要包括:苔藓纤维芽生假说;Ca2 内流引发的细胞毒性;谷氨酸和γ-氨基丁酸及其受体结构和功能异常;氧化应激(oxidative stress,OS)损伤等.氧化应激是指活性氧(reactive oxygen species,ROS)和活性氮(reactive nitrogen species,RNS)造成的氧化损伤;即当自由基的产生过多或体内抗氧化系统出现故障,体内氧自由基代谢就会出现失衡,自由基蓄积过多,攻击机体,导致可能的损害[1].     

环孢素对癫痫大鼠海马氧化应激及线粒体能量代谢的影响

目的：探讨环孢素对癫痫大鼠海马氧化应激、线粒体膜通透性及能量代谢的影响及机制。方法：应用匹鲁卡品建立癫痫持续状态模型,检测癫痫大鼠海马在环孢素干预前后丙二醛和超氧化物歧化酶的变化;检测癫痫大鼠海马在环孢素干预前后线粒体膜通透性转换、线粒体呼吸链复合物I和III活性及ATP含量的变化。结果：环孢素抑制癫痫大鼠海马组织线粒体膜通透性转换;明显降低癫痫大鼠海马组织丙二醛的含量,提高超氧化物歧化酶的活性（P<0.05）;明显增加癫痫大鼠海马组织线粒体呼吸链复合物I活性（P<0.05）,而对粒体呼吸链复合物III活性无显著影响;明显增加癫痫大鼠海马组织ATP的含量（P<0.05）。结论：环孢素能抑制癫痫大鼠海马氧化应激反应,减轻癫痫大鼠线粒体能量代谢损伤。     

黄芪多糖对过氧化氢刺激皮肤成纤维细胞中线粒体和溶酶体的保护作用(英文)

目的：证明黄芪多糖对氧化应激皮肤细胞的保护作用。方法：原代培养皮肤成纤维细胞在过氧化氢0.5mmol/L中,孵育30min造成急性氧化损伤的模型。MTT法测黄芪多糖的剂量反应,DAPI染色法检测黄芪多糖对细胞死亡的作用;激光共聚焦法检测黄芪多糖对线粒体、线粒体膜电位以及溶酶体稳定性的保护作用。结果：经过黄芪多糖的治疗,能够明显提高细胞存活率,其作用浓度从0.5mg/L开始,到1mg/L达最大效果,呈剂量依赖性。而且,黄芪多糖能够抑制线粒体膜电位崩解保护线粒体形态,保护溶酶体膜。结论：这些结果证明了黄芪多糖在线粒体和溶酶体水平存在1个新的调节凋亡的途径。这个途径可以成为一些氧化应激以及溶酶体缺陷疾病的1个新的治疗靶点。     

衰老血管内皮细胞线粒体膜电位与活性氧的变化

背景：血管内皮细胞衰老、凋亡与再生的平衡对正常血管的功能维持具有极其重要的作用。而线粒体是机体细胞内的重要细胞器,除了合成ATP为细胞提供能量外,还控制细胞程序性死亡、以及衰老等多种病理生理的代谢过程。 目的：通过检测脐静脉内皮细胞传代过程中线粒体膜电位与活性氧的改变及其相互关系,从而探讨细胞衰老过程中所产生的功能障碍。 方法：体外培养人脐静脉内皮细胞,选取传代过程中的第2,4,6,8代细胞,采用流式细胞术检测细胞线粒体膜电位及活性氧变化。选取第2,8代细胞行透射电镜检查,观察正常及衰老细胞超微结构的改变。 结果与结论：传代衰老过程中,血管内皮细胞线粒体膜电位逐代降低,而胞内活性氧则出现由增加转而降低的过程。传代后期血管内皮细胞同早期内皮细胞相比,线粒体及内质网明显减少。说明内皮细胞在传代导致的复制性衰老过程中,线粒体膜电位降低,线粒体受损。而在早期传代过程中线粒体轻度受损,而活性氧产生增加,但在线粒体严重受损、功能严重退化过程中,活性氧产生降低。     

缺氧对大鼠心肌线粒体能量代谢和腺苷酸转位酶活性的影响

目的：探讨高原低压缺氧暴露过程中大鼠心肌能量代谢及腺苷酸转位酶活性变化特点。方法：雄性Wistar大鼠随机分为正常对照组、缺氧1d组、5d组、15d组和30d组。缺氧组于模拟海拔5000m高原低压舱内连续缺氧23h/d。提取心室肌线粒体,Clark氧电极法测定线粒体氧化呼吸活性;HPLC法测量线粒体内腺苷酸含量;［3H］-ADP掺入法测量线粒体ANT转运活性。结果：大鼠经缺氧1d、5d、15d后,ST3和RCR显著降低,缺氧30d时ST3仍显著低于对照组,ST4在缺氧1d、5d、15d时显著升高,缺氧30d时降低,RCR在30d时接近正常。缺氧1d和5d心肌线粒体ATP含量、ANT活性明显下降,缺氧15d时接近正常,缺氧30d时则再次降低。结论：缺氧对心肌线粒体氧化呼吸功能的抑制是导致线粒体内ATP含量下降的主要原因。缺氧过程中ANT活性与线粒体内ATP含量成协调变化。     

电磁脉冲辐照对大鼠脑线粒体功能的影响

为研究电磁脉冲（EMP）对大鼠脑线粒体功能的损伤，大鼠经过连续不同场强EMP辐照后测定了脑线粒体膜功能随辐照时间的变化，并分离脑线粒体，测定线粒体肿胀度、膜流动性、膜磷脂含量、呼吸功能、线粒体呼吸酶、超氧化物歧化酶（SOD）、丙二醛（MDA）、Ca^2＋等指标以显示线粒体功能、抗氧化能力的变化。结果表明，EMP辐照后大鼠脑线粒体明显肿胀，膜磷脂降解、膜流动性下降，呼吸功能衰减，呼吸酶、SOD活性降低，Ca^2＋、MDA含量升高。由此认为，EMP可造成大鼠脑线粒体功能受损，其机制可能与脑线粒体膜损伤后继发的自由基生成增加、脑线粒体能量代谢障碍有关。     

牛磺酸对大鼠草酸钙结石肾脏的保护作用

目的：观察牛磺酸在大鼠草酸钙肾结石模型中对肾脏的保护作用并探讨其作用机制。方法：采用定量灌胃2.5%乙二醇+2.5%氯化铵（4mL/d,分2次给予）并控制饮水4周诱导建立草酸钙肾结石大鼠模型。分4个组（n=8）：空白组（A组）、结石诱导组（B组）、牛磺酸干预组（C组）和牛磺酸对照组（D组）,其中B、C组定量灌胃诱石剂,A、D组灌胃相同量的饮水;A、B组喂食标准颗粒饲料;C、D组喂食含2.0%牛磺酸的颗粒饲料。每只动物饮水量均控制在20mL/d。4周后收集各组大鼠24h尿液检测草酸、肌酐和8-异前列腺素（8-IP）水平;采血测血清肌酐;取肾脏分离线粒体,比色法测定超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性,荧光比色法检测线粒体氧化损伤水平;肾组织HE和钙盐染色观察晶体沉积情况,透射电镜下观察肾小管上皮的损伤情况,免疫组化观察巨噬细胞标记物CD68在肾脏中的表达情况。结果：使用诱石剂诱导结石后大鼠尿8-IP显著增加,肌酐清除率降低,肾脏/体重比值增加,肾组织线粒体SOD、GSH-Px活性下降伴线粒体损伤;使用牛磺酸则尿8-IP生成减少,肌酐清除率增加,肾脏/体重比值减少,肾组织线粒体SOD、GSH-Px活性增加而肾小管上皮和线粒体损伤程度减轻,肾脏内晶体沉积量减少,CD68在肾脏中的表达水平也减少。结论：牛磺酸在大鼠草酸钙肾结石模型中具有明显的肾脏保护效果,其机制可能与牛磺酸的抗氧化作用有关。     

急性重复低氧对小鼠脑组织中糖酵解和线粒体氧化磷酸化以及能量负荷的影响

目的 探讨小鼠在重复急性低氧暴露后脑组织中的糖酵解、线粒体氧化磷酸化及能量负荷的变化。方法 成年Balb/c小鼠重复低氧暴露5次,测定每次低氧暴露时的平均耐受时间、体温及第0,1,3,5次低氧暴露后脑组织中的磷酸果糖激酶（PFK）、丙酮酸激酶（PK）、线粒体复合体I活性和磷酸腺苷水平。结果 重复低氧暴露使小鼠低氧耐受性增强,体温降低,脑组织中PFK和PK活性先增高后降低,复合体I活性持续降低,能量负荷保持稳定。结论 重复急性低氧使小鼠脑组织的糖酵解活性出现规律性变化,线粒体的氧化磷酸化受抑制,但能量负荷保持稳定。     

顺铂对葡萄膜黑色素瘤细胞端粒酶活性的抑制

目的 研究顺铂对人葡萄膜黑色素瘤细胞端粒酶活性的抑制作用,为临床治疗人葡萄膜黑色素瘤提供理论依据.方法 采用特定时间下不同浓度以及特定浓度下不同作用时间的端粒酶抑制剂顺铂作用于体外培养的人黑色素瘤细胞.采用多聚酶链反应--酶联免疫吸附测定(PCR-ELISA)及聚丙烯酰胺凝胶电泳法(PCR-PAGE)测定细胞中端粒酶活性的变化.结果 作用72 h,端粒酶活性在顺铂浓度达到0.10mg/L后开始下降,当浓度达到1.00mg/L后其活性下降至(0.173±0.007).当顺铂浓度固定10.00 mg/L,顺铂作用时间达到24 h后开始出现抑制作用,48 h时达到抑制高峰,端粒酶活性下降至(0.276±0.024).随着顺铂浓度的增加及作用时间的延长,端粒酶活性逐渐下降(P＜0.05).PCR-PAGE显示顺铂浓度增加及作用时间延长,端粒酶活性的显色条带越来越少.结论 顺铂可有效降低体外培养的人眼葡萄膜黑色素瘤细胞端粒酶活性,并呈浓度和时间依赖性.     

线粒体通透性转换作用Cyt C水平对慢加急性肝衰竭大鼠线粒体ATP生成的影响

目的 从线粒体损伤的角度探讨ACLF存在的能量代谢障碍.方法 雄性SD大鼠随机分三组,ACLF组、NAC组各15只,正常组10只,制作ACLF模型,NAC灌胃作抗氧化模型.留肝组织及血液标本,梯度离心分离线粒体,ELLSA法测线粒体Cyt C、荧光酶标法测MPTP RFU;酶标法测肝组织、血浆GSH、MDA、SOD.结果 ACLF组肝组织、血浆MDA明显高于正常及NAC组（P＜0.05）;NAC组MDA高于正常组（P ＜0.05）;ACLF组血浆GSH、SOD低于正常及NAC组（P＜0.05）;三组Cyt C为正常组最高、ACLF组最低（P＜0.05）;三组MPTP为ACLF组最高、正常组最低;MPTP与Cyt C、ATP呈负相关（r=-0.858,-0.799）;Cyt C与ATP呈正相关（r=0.78）.结论 ACLF大鼠处于氧化应激损伤,Cyt C外流入胞质可能是ACLF发生能量代谢障碍的原因之一;NAC可在一定程度上提高肝细胞的抗氧化能力.     

Effects of light on aging and longevity

Increasing evidence suggests an important role for light in regulation of aging and longevity. UV radiation is a mutagen that can promote aging and decrease longevity. In contrast, NIR light has shown protective effects in animal disease models. In invertebrates, visible light can shorten or extend lifespan, depending on the intensity and wavelength composition. Visible light also impacts human health, including retina function, sleep, cancer and psychiatric disorders. Possible mechanisms of visible light include: controlling circadian rhythms, inducing oxidative stress, and acting through the retina to affect neuronal circuits and systems. Changes in artificial lighting (e.g., LEDs) may have implications for human health. It will be important to further explore the mechanisms of how light affects aging and longevity, and how light affects human health.     

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

Brain energy metabolism is increased during postnatal development and diminished in neurodegenerative diseases linked to senescence. The objective of this study was to determine if these conditions could involve postnatal or senescence-related shifts in activity or expression of dihydrolipoamide dehydrogenase (DLDH), a key mitochondrial oxidoreductase. Rats ranging from 10 to 60 days of age were used in studies of postnatal development, whereas rats aged 5 or 30 months were used in the aging studies. The expression of DLDH was determined by Western blot analysis using anti-DLDH antibodies and DLDH diaphorase activity was measured by an in-gel activity staining method using nitroblue tetrazolium (NBT)/NADH. Activity of DLDH dehydrogenase was measured as NAD⁺ oxidation of dihydrolipoamide. When these measures were considered in separate groups of 10-, 20-, 30-, or 60-day-old rats, all three showed an increase between 10 and 20 days of age. However, dehydrogenase activity of DLDH showed a further, progressive increase from 20 days to adulthood, in the absence of any further change in DLDH expression or diaphorase activity. No age-related decline in DLDH activity or expression was evident over the period from 5 to 30 months of age. Moreover, aging did not render DLDH more susceptible to oxidative inactivation by mitochondria-generated reactive oxygen species (ROS). Taken together, results of the present study indicate that (1) brain DLDH expression and activity undergo independent postnatal maturational increases; (2) senescence does not confer any detectable change in the activity of DLDH or its susceptibility to inactivation by mitochondrial oxidative stress.     

Age- and tissue-specific changes in mitochondrial and nuclear DNA base excision repair activity in mice: Susceptibility of skeletal muscles to oxidative injury

In this study, we investigated age- and tissue-dependent changes in the DNA base excision repair (BER) of oxidative lesions in mitochondrial and nuclear extracts by measuring single-nucleotide (SN)- and long-patch (LP)-BER activities in five tissues isolated from 4-, 10- and 20-month-old mice. Age-dependent SN-BER and LP-BER activity was increased in the mitochondria of liver, kidney and heart, but generally decreased in skeletal muscles. In contrast, no significant changes in repair activity were observed in nuclear extracts of the same tissues, except for quadriceps, where the SN-BER activity was higher in the old animals. Moreover, the BER activities in both the nucleus and the mitochondria were significantly lower in skeletal muscles compared to liver or kidney of the same mice. The protein level of three antioxidant enzymes, Mn and Cu/Zn superoxide dismutases (SOD) and catalase, was also significantly lower in skeletal muscle compared to liver or kidney. In addition, we found higher levels of protein carbonylation in the mitochondria of skeletal muscle relative to other tissues. Thus, it appears likely that mouse skeletal muscle is highly susceptible to oxidative stress due to deficiency in both repair of oxidative DNA damage and antioxidant enzymes, contributing to age-dependent muscle loss.     

Due to reverse electron transfer, mitochondrial H2O2 release increases with age in human vastus lateralis muscle although oxidative capacity is preserved

Age-related changes in mitochondrial H2O2 release (MHR) could be responsible for an increase in oxidative stress in skeletal muscle and participate in the development of sarcopenia. We compared MHR in vastus lateralis biopsies obtained from young (23.5+/-2.0 year, n=6) and elderly (67.3+/-1.5 year, n=6) healthy sedentary men. Isolated mitochondria were incubated in the presence of glutamate/malate/succinate, with or without rotenone. Muscle fat oxidative capacity, citrate synthase, complex II, complex III, and cytochrome c oxidase activities were also measured. In parallel, we analyzed in gastrocnemius of young male Wistar rats (n=6), the impact of lidocaine (local anesthetic used in humans) on mitochondrial respiration and MHR. In humans, muscle oxidative capacity was preserved with age but muscle MHR was markedly enhanced in elderly subjects compared to young adults (+175%, P<0.05). Rotenone abolished this increase, demonstrating that it was due to a free radical release during reverse electron transfer from complex II towards complex I. Lidocaine can interfere with MHR measurements (intra-muscular injection in rats) but it can be avoided by minimizing contact with muscle (small multiple subcutaneous injections in humans). Physiologic consequences of the observed increase in muscle MHR with aging remain to be determined.     

The adversities of aging

The aging process is evolutionarily conserved and subject to quantitative modification by both genetic and environmental factors. Fundamental mechanisms of aging result in progressive deficits in the function of cells and organs, often leading to diseases that ultimately kill the organism such as cancers, cardiovascular disease and neurodegenerative disorders. Oxidative stress and damage to all of the major classes of molecules in cells are involved in aging and age-related diseases. The widely pursued approach of targeting disease-specific processes to develop therapeutic interventions has not had a major impact on healthspan. A more productive approach would be to target the fundamental mechanisms of aging throughout adult life so as to extend healthspan. Caloric restriction and regular exercise are two such approaches.     

Repair of persistent strand breaks in the mitochondrial genome

Oxidative DNA damage has been attributed to increased cancer incidence and premature aging phenotypes. Reactive oxygen species (ROS) are unavoidable byproducts of oxidative phosphorylation and are the major contributors of endogenous oxidative damage. To prevent the negative effects of ROS, cells have developed DNA repair mechanisms designed to specifically combat endogenous DNA modifications. The base excision repair (BER) pathway is primarily responsible for the repair of small non-helix distorting lesions and DNA single strand breaks. This repair pathway is found in all organisms, and in mammalian cells, consists of three related sub-pathways: short patch (SP-BER), long patch (LP-BER) and single strand break repair (SSBR). While much is known about nuclear BER, comparatively little is known about this pathway in the mitochondria, particularly the LP-BER and SSBR sub-pathways. There are a number of proteins that have recently been found to be involved in mitochondrial BER, including Cockayne syndrome proteins A and B (CSA and CSB), aprataxin (APTX), tryosyl-DNA phosphodiesterase 1 (TDP1), flap endonuclease 1 (FEN-1) and exonuclease G (EXOG). These significant advances in mitochondrial DNA repair may open new avenues in the management and treatment of a number of neurological disorders associated with mitochondrial dysfunction, and will be reviewed in further detail herein.