Effects of exercise on circadian rhythms and mobility in aging Drosophila melanogaster

Daily life functions such as sleep and feeding oscillate with circa 24 h period due to endogenous circadian rhythms generated by circadian clocks. Genetic or environmental disruption of circadian rhythms is associated with various aging-related phenotypes. Circadian rhythms decay during normal aging, and there is a need to explore strategies that could avert age-related changes in the circadian system. Exercise was reported to delay aging in mammals. Here, we investigated whether daily exercise via stimulation of upward climbing movement could improve circadian rest/activity rhythms in aging Drosophila melanogaster. We found that repeated exercise regimen did not strengthen circadian locomotor activity rhythms in aging flies and had no effect on their lifespan. We also tested the effects of exercise on mobility and determined that regular exercise lowered age-specific climbing ability in both wild type and clock mutant flies. Interestingly, the climbing ability was most significantly reduced in flies carrying a null mutation in the core clock gene period, while rescue of this gene significantly improved climbing to wild type levels. Our work highlights the importance of period in sustaining endurance in aging flies exposed to physical challenge.     

红细胞膜脂肪酸成分、膜流动性改变与糖尿病视网膜病变的关系

目的探讨红细胞膜脂肪酸成分和膜流动性与糖尿病视网膜病变（ＤＲ）之间的关系。方法采用高效液相色谱法和荧光偏振技术测定２９例正常人和６３例非胰岛素依赖型糖尿病（ＮＩＤＤＭ）患者红细胞膜脂肪酸成分和膜微粘度。结果ＮＩＤＤＭ病人红细胞膜花生四烯酸（Ｃ２０∶４ｎ－６）含量及百分组成明显低于正常对照组，而膜微粘度则明显高于对照组，且在伴有ＤＲ组其膜微粘度又明显高于不伴有ＤＲ组。患者膜微粘度与膜软脂酸（Ｃ１６∶０）和硬脂酸（Ｃ１８∶０）百分组成呈明显正相关，而与花生四烯酸和廿二碳六烯酸（Ｃ２２∶６ｎ－３）呈明显负相关。结论ＮＩＤＤＭ病人红细胞膜脂酸组成对其膜流动性降低有重要影响，而后者对ＮＩＤＤＭ微血管病变的发生和发展可能起重要作用。     

顺铂压力下HepG2细胞内活性氧及线粒体膜电位的变化

目的了解顺铂压力下人肝母细胞瘤株(HePG2)细胞内活性氧(ROS)的水平及线粒体膜电位的变化及意义。方法低剂量顺铂与HePG2细胞共孵。活性氧探针(2，7二氯荧光素)探测顺铂压力下HePG2细胞内活性氧水平的变化；用反应膜电位荧光探针罗丹明-123，在激光扫描共聚焦显微镜下，通过荧光强度反映线粒体膜电位的变化。于0、24、48、72、120、l68h动态观察HePG2细胞内活性氧和线粒体膜电位的变化。结果顺铂持续作用24h和48h时，活性氧的产生较正常明显增多，尤以24h时最明显，72h后开始下降，120h后基本接近正常；顺铂选择性压力下，24h和48h HePG2细胞内罗丹明-123的荧光强度明显减弱，以24h时最明显，72h以后逐渐升高，120h以后与未经处理的HePG2细胞无明显差异。结论HepG2细胞在低剂量顺铂的压力下，细胞内活性氧水平和线粒体膜电位的变化，提示HepG2细胞可对低剂量顺铂的持续压力发生适应性变化。     

The uptake and release of ponasterone a by the Kc cell line of Drosophila melanogaster

The association of ponasterone A (PNA) and 20-hydroxyecdysone with K_c cells is commensurate with their biological activity on this K_c cell line, the physiological activity ratio for PNA, 20-hydroxyecdysone and ecdysone is 1:50:2000, resp. Both association and release of [³H]-PNA are temperature-dependent, the activation energy was calculated as 16.7 cal (Arrhenius analysis). This association is compatible with unlabelled PNA and various ecdysteroids. The K_D for PNA (Scatchard analysis) was estimated as 3.6 × 10^?9 M, giving the number of binding sites as approx. 1800 per cell.     

Contrasted effects of suppressing live yeast from food on longevity,aging and resistance to several stresses in Drosophila melanogaster

It is often accepted that dietary restriction (DR) increases longevity in Drosophila melanogaster, but this is still a controversial issue. In the present study, adult flies were fed on a rich nutritious medium to which live yeast, a source of proteins, was added or not. Suppressing live yeast did not always increased longevity of virgin flies, but increased it in mated flies. It also decreased fecundity, delayed behavioral aging and increased resistance to heat of young females only. However, flies without live yeast suffered from a reduced resistance to cold, starvation and infection. This study thus reports some positive effects of suppressing live yeast on longevity, contrarily to previous studies of the same laboratory using other DR methods, but also shows that the absence of live yeast increases frailty. The effects of live yeast in flies are thus contrasted, and it is probable that flies without a high amount of proteins in the diet would not survive for a long time in the wild, due to their inability to resist stress and their low fecundity.     

解偶联蛋白2过表达张氏肝细胞的构建及其对线粒体膜电位和活性氧的影响

目的 构建稳定过表达人解偶联蛋白2 (UCP2)的张氏肝细胞株,观察UCP2对线粒体膜电位(MMP)和活性氧(ROS)的作用. 方法 将含有人UCP2 cDNA全长的重组质粒(pcDNA3.1-hU CP2)转染张氏肝细胞系,pcDNA3.1空载体作为对照.Zeocin筛选稳定表达UCP2的细胞株,Western blot和免疫细胞化学鉴定UCP2蛋白表达.利用不同剂量UCP2抑制剂京尼平(25、50、100μmol/L),预处理稳定表达UCP2的细胞株.荧光分光光度法检测MMP和ROS水平变化.数据分析用单因素方差分析和q检验(Newman-keuls法),P＜0.05为差异有统计学意义.结果 pcDNA3.1-hU CP2成功转染张氏肝细胞,UCP2相对表达量约为对照组的1.6倍.过表达细胞罗丹明123和2 ′,7 ′ -二氯氢化荧光素二脂荧光强度(分别为11.11±2.76和4.97±0.62)均明显低于正常对照组张氏肝细胞(分别为15.56±2.55和6.14±1.25,q值分别为4.80和3.35,P＜0.01和P＜ 0.05)和空载体对照组肝细胞(分别为16.11±2.93和6.23±1.13,q值分别为5.40和3.60,P＜0.01和P＜ 0.05);空载体组上述两指标与正常对照组相比,差异均无统计学意义.京尼平低、中、高剂量组与过表达组相比,罗丹明123的荧光强度(分别为14.89±2.89,17.89±2.93,24.00±2.55,q值分别为4.08,7.33和13.93,P值均＜0.01)和2 ′,7 ′-二氯氢化荧光素二脂的荧光强度(分别为9.16±0.78,10.84±1.09, 11.83±1.25,q值分别为12.00,16.83和19.67,P值均＜0.01)均明显升高,并呈现剂量依赖性.结论 成功构建稳定过表达人U CP2的张氏肝细胞株,UCP2表达水平及活性变化可通过MMP和ROS影响线粒体功能.     

黄绿青霉素对心肌细胞线粒体呼吸链合成相关转录调节基因mRNA表达、膜电位及活性氧的影响

目的 观察黄绿青霉素(CIT)对SD大鼠原代心肌细胞线粒体呼吸链合成相关转录调节基因mRNA表达、膜电位及活性氧的影响.方法 用不同浓度CIT[0、1、2、3、4、5、6、7、8、9、10 μmol/L]作用SD大鼠原代心肌细胞24 h,采用噻唑蓝(MTT)法测定细胞存活率,并根据检测结果,用SPSS 13.0软件计算CIT的半数抑制浓度(IC50).进一步选择心肌细胞存活率为99％、95％、90％时的CIT水平(0.715、1.234、1.650 μmol/L)作为低、中、高剂量组,同时以未加CIT作为对照组,分别作用于心肌细胞24 h,采用反转录聚合酶链反应(RT-PCR)检测心肌细胞中过氧化物酶体增殖物受体γ共激活因子1α(PGC-1α)、核呼吸因子1(Nrf1)、核呼吸因子2(Nrf2)mRNA表达;分别以阳离子荧光羰花青染色剂(JC-1)、2',7'-二氯荧光素(DCFH2-DA)作为荧光探针,用全能酶标仪检测心肌细胞线粒体膜电位(MMP)和细胞内活性氧(ROS)的变化.结果 与0μmol/L CIT组[(89.4±17.6)％]比较,2～ 10 μmol/L CIT组原代心肌细胞存活率[(80.2±20.2)％、(74.4±18.7)％、(63.2±8.9)％、(51.5±18.8)％、(39.0±15.7)％、(22.6±10.5)％、(19.9±4.9)％、(20.7±4.8)％、(18.5±3.3)％]明显下降(P均＜ 0.05).CIT作用于心肌细胞24 h的IC50为4.6 μmol/L.高、中、低剂量组PGC-1α mRNA表达(0.431±0.041、0.619±0.031、0.653±0.037)较对照组(0.776±0.081)明显降低(P均＜0.05);高剂量组Nrf1mRNA表达(0.358±0.051)明显低于对照组(0.580±0.098,P＜0.05);高、中剂量组Nrf2 mRNA表达(0.352±0.041、0.472±0.011)明显低于对照组(0.667±0.091,P均＜0.05).与对照组[(100.00±0.00)％、(100.00±0.00)％]比较,高、中、低剂量组MMP水平[(55.3±3.3)％、(69.8±4.7)％、(81.8±2.7)％]显著降低(P均＜0.05),ROS水平[(606.0±46.3)％、(275.0±53.5)％、(158.9±29.5)％]显著升高(P均＜0.05).结论 CIT可对原代心肌细胞的线粒体呼吸链生物合成产生抑制作用,并诱导细胞发生氧化应激,通过线粒体途径介导心肌细胞死亡,从而引起心肌损伤.     

Local control of mitochondrial membrane potential, permeability transition pore and reactive oxygen species by calcium and calmodulin in rat ventricular myocytes

Calmodulin (CaM) and Ca²⁺/CaM-dependent protein kinase II (CaMKII) play important roles in the development of heart failure. In this study, we evaluated the effects of CaM on mitochondrial membrane potential (ΔΨ_m), permeability transition pore (mPTP) and the production of reactive oxygen species (ROS) in permeabilized myocytes; our findings are as follows. (1) CaM depolarized ΔΨ_m dose-dependently, but this was prevented by an inhibitor of CaM (W-7) or CaMKII (autocamtide 2-related inhibitory peptide (AIP)). (2) CaM accelerated calcein leakage from mitochondria, indicating the opening of mPTP, however this was prevented by AIP. (3) Cyclosporin A (an inhibitor of the mPTP) inhibited both CaM-induced ΔΨ_m depolarization and calcein leakage. (4) CaM increased mitochondrial ROS, which was related to ΔΨ_m depolarization and the opening of mPTP. (5) Chelating of cytosolic Ca²⁺ by BAPTA, the depletion of SR Ca²⁺ by thapsigargin (an inhibitor of SERCA) and the inhibition of mitochondrial Ca²⁺ uniporter by Ru360 attenuated the effects of CaM on mitochondrial function. (6) CaM accelerated Ca²⁺ extrusion from mitochondria. We conclude that CaM/CaMKII depolarized ΔΨ_m and opened mPTP by increasing ROS production, and these effects were strictly regulated by the local increase in cytosolic Ca²⁺ concentration, initiated by Ca²⁺ releases from the SR. In addition, CaM was involved in the regulation of mitochondrial Ca²⁺ homeostasis.     

黄芩苷对体外氧化应激模型中肝型脂肪酸结合蛋白表达的影响及其意义

目的 研究黄芩苷对体外氧化应激模型中肝型脂肪酸结合蛋白(L-FABP)表达的影响及其意义. 方法 用终浓度为400 μ mol/L的过氧化氢(H2O2) 37℃避光孵育细胞20min,建立体外诱导氧化应激模型.应用甲基噻唑基四唑法(MTT)检测不同浓度黄芩苷作用细胞的存活率,确定24h、48 h黄芩苷的半数中毒浓度(TC50).流式细胞技术检测不同浓度黄芩苷(25、50、100μmol/L)作用后活性氧(ROS)的表达、细胞内超氧化物歧化酶(SOD)和谷胱甘肽(GSH)活性变化,实时PCR和Western blot检测肝细胞内L-FABP基因和蛋白表达.数据分析采用单因素方差分析.结果 根据MTT法得出25、50、100 μ mol/L的黄芩苷作用于细胞24h的存活率为83.60％±3.47％,72.36％±2.18％,70.16％±2.04％,F值为386.24,P＞0.05;作用于细胞48h的存活率为84.93％±3.11％,76.16％±2.45％,72.72％±2.31％,F值为475.92,P＞0.05.直线回归法得出黄芩苷持续作用24h和48h的TC50分别为153.2、170.6μmol/L.在此范围内,用25、50、100μmol/L浓度的黄芩苷分别作用Chang肝细胞24、48 h后,ROS含量24 h分别为37.0±3.30,22.90±3.84,29.60±2.52,F值为70.06,P＜0.05 ; 48h分别为35.77±2.35,21.80±3.10,23.87±1.98,F值为110.92,P＜0.05,而400μ mol/L H2O2组ROS含量24h和48h分别为45.50±3.47,48.80±2.70,以50μmol/L的黄芩苷作用48h效果最为显著.用50μmol/L黄芩苷处理48h后细胞内SOD活性为(51.53±1.91)μ g/mg,GSH为(49.85±1.45) U/mg;与对照组SOD为(26.36±1.23)μ g/mg,GSH为(25.11±1.74) U/mg,F值分别为93.81和92.51,P值均＜0.05).尽管50μmol/L黄芩苷处理48 h后细胞内L-FABP在mRNA水平并无明显变化,但50μmol/L黄芩苷处理48 h后细胞内L-FABP蛋白表达与对照组相比增加约80％.结论 黄芩苷能通过增强L-FABP蛋白表达,增加细胞内SOD和GSH的活性发挥抗氧化作用.     

SESN-1 is a positive regulator of lifespan in Caenorhabditis elegans

Aging is a process of gradual functional decline leading to death. Reactive oxygen species (ROS) not only contribute to oxidative stress and cell damage that lead to aging but also serve as signaling molecules. Sestrins are evolutionarily conserved in all multicellular organisms and are required for regenerating hyperoxidized forms of peroxiredoxins and ROS clearance. However, whether sestrins regulate longevity in metazoans is still unclear. Here, we demonstrated that SESN-1, the only sestrin ortholog in Caenorhabditis elegans, is a positive regulator of lifespan. sesn-1 gene mutant worms had significantly shorter lifespans compared to wild-type animals, and overexpression of sesn-1 prolonged lifespan. Moreover, sesn-1 was found to play a key role in defense against several life stressors, including heat, hydrogen peroxide and the heavy metal copper; and sesn-1 mutants expressed higher levels of ROS and showed a decline in body muscle function. Surprisingly, loss of sesn-1 did not weaken the innate immune function of the worms. Together, these results suggest that SESN-1 is required for normal lifespan and its function in muscle cells prevents muscle degeneration over a lifetime.     

不同浓度葡萄糖和游离脂肪酸对大鼠骨骼肌细胞株L6细胞胰岛素敏感性和细胞内活性氧的影响

目的研究不同浓度的葡萄糖和游离脂肪酸（FFA）对骨骼肌L6细胞胰岛素敏感性及细胞内活性氧（ROS）的影响。方法L6细胞诱导分化成熟后,分为6组,以5mmol／L葡萄糖为低糖对照组,其中加0．3或1mmol／L的混合FFA为低糖低FFA或低糖高FFA组,以25mmol／L葡萄糖为高糖对照组,其中加0．3和1mmol／L的FFA的培养基为高糖低FFA或高糖高FFA组。干预48h,^3H—D葡萄糖摄取实验（加与不加100nmol／I,胰岛素37℃30rain）验证L6细胞胰岛素敏感性,经荧光探针H2DCFDA观察细胞内ROS含量的变化。结果在5mmol／L葡萄糖组,低糖对照组基础摄糖值为22777．6±6608．7,低糖低FFA组为26027．5±4085．7,低糖高FFA组为146805．1±21099．4。胰岛素刺激后,低糖对照组为76586．5±18450．1,低糖低FFA组为43738土9203．6,低糖高FFA组为32050．6±3362．3,较低糖对照组显著降低（P〈0．01）。在25mmol／L葡萄糖组,基础葡萄糖摄取量为11793．8±551．4,高糖低FFA组11887．3±2082．3高糖高FFA组为13886．1±3872．9,差别无统计学意义（P〉0．05）,但较低糖对照组显著降低（P〈0．01）．加入胰岛素刺激后,高糖对照组为42798．8±9441．5,高糖低FFA组为23946．9±3839．6,高糖高FFA组为13886．1±3872．9,各组之间差别显著（P〈0．01）。对细胞内R0s的研究发现,低糖低FFA组为2234．2±477．2,低糖高FFA组为1969．0±480．9,高糖对照组为1969．0土229．4,高糖低FFA组为2]84．5±734．2,高糖高FFA组为2571．3±96．7,可以显著增加的L6细胞内ROS,与低糖对照组（615．3±244．3）相比差别显著（P〈0．01）。除对照组之外的各组之间差别无统计学意义（P〉0．05）。结论高糖和高FFA可以诱导L6细胞出现氧化应激,同时高糖和高FFA是导致骨骼肌细胞胰岛素抵抗的重要原因之一。     

Stable transfection of UCP1 confers resistance to hypoxia/reoxygenation in a heart-derived cell line

Mitochondrial uncoupling proteins, which secure physiological uncoupling of oxidative phosphorylation, have been proposed to serve as an oxidative-stress compensatory mechanism. Here, heart-derived H9c2 cells acquired improved resistance to injury upon transfection of the prototypic uncoupling protein UCP1. Following hypoxia/reoxygenation, stable overexpression of UCP1 provided enhanced cardioblast survival with preserved mitochondrial structure and function, while limiting reactive oxygen species formation. Thus, transfection of mitochondrial UCP1 provides a strategy for generation of a stress-resistant cardiac cell phenotype.     

Aging might augment reactive oxygen species (ROS) formation and affect reactive nitrogen species (RNS) level after myocardial ischemia/reperfusion in both humans and rats

Previous studies indicate aging results in significantly decreased cardiac function and increased myocardial apoptosis after myocardial ischemia/reperfusion (MI/R) in humans or rats. The underlying mechanisms of aging-exacerbated effects remain unknown. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to play vital roles in aging-related MI/R injury. Heretofore, the effects of aging upon ROS and RNS formation were not investigated in humans, which is the focus of the current study. Due to experimental limitations with clinical trials, an additional animal experiment was performed. All enrolled acute myocardial infarction (AMI) patients received percutaneous coronary intervention (PCI) therapy. AMI patients were assigned into two groups: adult (age <65, n = 34) and elderly (age ≥65, n = 45) AMI patients. Blood samples were obtained from all study participants at 24 h and 3 days post-PCI. Plasma/white blood cell (WBC) ROS and RNS markers (malondialdehyde (MDA), myeloperoxidase (MPO), reduced glutathione (GSH), inducible nitric oxide synthase (iNOS) activity, NOx, and nitrotyrosine) were determined. The same markers were determined in rat cardiac tissue after 24 h MI/R. Compared to the adult group, elderly patients manifested increased plasma MDA and MPO and decreased plasma GSH concentrations. No significant differences in plasma NOx or nitrotyrosine concentration existed between adult and elderly patients. Furthermore, WBC iNOS activity in elderly patients was significantly decreased compared to the adult group. The measurement of ROS markers in the rat experiments was consistent and supported human study data. Surprisingly, RNS markers (NOx and nitrotyrosine) in blood and heart tissue increased from young to middle-aged rats but decreased from middle age to old age. Aging augments ROS, which might exacerbate MI/R injury. Additionally, our data support aging-induced changes of RNS levels in humans and rats in vivo.     

Decline in oxygen consumption correlates with lifespan in long-lived and short-lived mutants of Caenorhabditis elegans

In humans, the basal energy metabolism is thought to decline linearly with age. On the other hand, in the nematode Caenorhabditis elegans, two research groups reported independently that it declined exponentially. In this study, furthermore, we used various lifespan-mutant strains to determine whether the previous conclusion is more likely to be true. We can indirectly estimate the metabolic energy by conveniently measuring the oxygen consumption rates of C. elegans using an optical apparatus. From the profile of respiratory rates as a function of age, we can quantitatively isolate the physiological decline rate, λ, that exponentially represents the decay rate of respiratory activity with age. In addition, quantitative analysis indicates that the respiratory activity of worms has a finite value in advanced age. We also show that the maximum and mean lifespans strongly correlate with the reciprocal of the λ. These findings offer crucial biochemical evidence for a molecular mechanism at work in biological aging. Consequently, we here propose a mechanism based on a chemical reaction and offer a definition of the physiological decline rate and the finiteness of respiratory activity in advanced age.     

Ganoderma lucidum (Fr.) P. Karst enhances activities of heart mitochondrial enzymes and respiratory chain complexes in the aged rat

Aging is associated with increased oxidative damage at multiple cellular levels, decline in cellular energy production and enhanced free radical status. The effect of the medicinal mushroom, Ganoderma lucidum on the activities of tricarboxylic acid (Krebs) cycle enzymes and mitochondrial complexes I–IV of the electron transport chain in aged rats were investigated. The activity of Krebs cycle enzymes, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV were determined in heart of aged male Wistar rats orally administrated with 70% ethanolic extract (50 and 250 mg/kg) of G. lucidum. DL-α-lipoic acid (100 mg/kg) was taken as the positive control. Administration of the G. lucidum, once daily for 15 days, was significantly (P < 0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complex IV activities in aged rats. The profound activity of the extract can be correlated to the significant antioxidant property of G. lucidum. The results of the study revealed that G. lucidum is effective to ameliorate the age associated decline of cellular energy status.     

Coevolution of exceptional longevity, exceptionally high metabolic rates, and mitochondrial DNA-coded proteins in mammals

Mammals' longevity is inversely related to mass-specific basal metabolic rate because the generation of reactive oxygen species constrains lifespan. Longevity increases with body mass because the latter is inversely related to mass-specific basal metabolic rates. In placental mammals the longevity residuals from the power laws that describe longevity as a function of mass-specific basal metabolic rates, or body mass, are positively correlated with the relative rates of evolution of cytochrome b, a generator of reactive oxygen species. Therefore, longevity is more accurately described as a function of both mass-specific basal metabolic rate and the relative rate of cytochrome b evolution. The longevity residuals from the power law that describe longevity as a function of body mass are positively correlated with the relative rate of evolution of most other mtDNA-coded proteins. In taxa with very high rate of cytochrome b evolution exceptional longevity is associated with an increase, rather than the predicted decrease, of basal metabolic rates. These finding are compatible with the hypothesis that, in placental mammals, the accelerated evolution of mtDNA-coded proteins, allowed the extension of lifespan by selecting mutations that reduce the generation of reactive oxygen species, mostly by increasing internal proton leak, that accelerates mitochondrial electron transport.     

糖化终末产物对MIN6细胞活力及活性氧(ROS)水平的影响

探讨糖化终末产物(AGE)对小鼠胰岛细胞株MIN6细胞活力及活性氧(ROS)水平的影响.制备BSA-AGE,用不同浓度AGE(100、200、400 mg/L)干预MIN6细胞不同时间后,MTF比色法检测细胞活力变化.以活性氧捕获剂双氢-乙酰乙酸二氯荧光黄(DCFH-DA)孵育细胞,通过流式细胞仪检测细胞内二氯荧光黄(DCF)的荧光强度而测得细胞内活性氧水平,并测定胰岛素分泌的变化.随着AGE浓度的升高和作用时间的延长,细胞活力明显下降(P＜0.05).经DCFH-DA孵育后流式细胞仪检测显示,AGE处理组细胞内DCF平均荧光强度较对照组明显升高(P＜0.05).胰岛素分泌量随着AGE浓度的增高和时间的延长,呈下降趋势(P＞0.05).提示BSA-AGE抑制MIN6活力,使细胞内活性氧生成增加,诱导MIN6细胞氧化应激.

Abstract：

To explore the effect of advanced glycation end-products(AGEs)on cell viability and level of reactive oxygen species(ROS)in MIN6 cells. After intervention of various concentrations(100,200, and 400 mg/L)of AGEs for some time, cell viability was detected by MTT assay. 2', 7'-dichlorofluorescein diacetate(DCFH-DA)was used as a reactive oxygen species capture agent. The fluorescent intensity of 2', 7'-dichlorofluorescein(DCF), which was the product of cellular oxidation of DCFH-DA, was detected by flow cytometry. The level of ROS and insulin secretion was thus measured. Viability of MIN6 cells was inhibited by AGEs in a dose and time dependent manner(P＜0.05).Intracellular fluorescent intensity of DCF was markedly elevated in the AGEs groups as compared with that in the control group(P＜0.05).Insulin secretion was decreased in the AGEs groups than that in the control group(P＞0.05). The results suggest that AGEs inhibit the viability and induce oxidative stress in MIN6 cells by overproduction of ROS.     

Effect of Ganoderma lucidum on the activities of mitochondrial dehydrogenases and complex I and II of electron transport chain in the brain of aged rats

Dysfunction of the mitochondrial respiratory chain, being direct intracellular source of reactive oxygen species (ROS), is important in the pathogenesis of number of ageing associated human disorders. Effect of ethanol extract of Ganoderma lucidum on the activities of mitochondrial dehydrogenases; complex I and II of electron transport chain have been evaluated in the aged rat brain. Aged male Wistar rats were administered with ethanol extract of G. lucidum (50 and 250 mg/kg, p.o) once daily for 15 days. Similarly dl-α-lipoic acid (100 mg/kg, p.o) administered group was kept as the reference standard. Young and aged rats administered with water were kept as young and aged control, respectively. The effect of treatment was assessed by estimating the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH), complex I and II in the mitochondria of rat brain. Results of the study demonstrated that the extract of G. lucidum (50 and 250 mg/kg) significantly (p < 0.01) enhanced the activities of PDH, α-KGDH, SDH, complex I and II when compared to that of the aged control animals. The level of the lipid peroxidation was significantly lowered (p < 0.01) in the G. lucidum treated group with respect to that of aged control. However, we could not find any statistically significant difference between the activities of enzymes in groups treated with 50 and 250 mg/kg of G. lucidum. The activity exhibited by the extract of G. lucidum in the present study can be partially correlated to its antioxidant activity. The results of the study concluded that the extract of G. lucidum may effective to improve the function of mitochondria in aged rat brain, suggest its possible therapeutic application against ageing associated neurodegenerative diseases.     

Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice

AIM： To determine the effects of allopurinol, an inhibitor of xanthine oxidase, and apocynin, an inhibitor of NADPH oxidase, on oxidant stress and liver injury caused by hepatic ischemia/reperfusion （I/R） procedure in mice.
METHODS： Mice were pretreated with a xanthine oxidase inhibitor, allopurinol, or NADPH oxidase （NOX） inhibitor, apocynin before the hepatic I/R procedure. Then treated or untreated mice underwent the hepatic I/R procedure. The effects on hepatic injury and superoxide anions were determined after starting reperfusion.
RESULTS： A standard warm hepatic I/R procedure led to a marked increase in superoxide anion production as indicated by a superoxide anion tracer, MCLA. At the same time, the procedure caused profound acute liver injury, as indicated by elevated serum alanine aminotransferase and tumor necrosis factor-α levels, reduced liver glutathione levels and elevated malondialdehyde contents, as well as a high apoptotic cell count. All these changes were reversed by the use of apocynin or allopurinol prior to the hepatic I/R procedure.
CONCLUSION： Allopurinol and apocynin exerted protective effects on hepatic ischemia/reperfusion injury. The protection is associated with blocking the generationof superoxide anions during the hepatic I/R procedure by inhibiting xanthine oxidase and NADPH oxidase activity.