快速老化模型小鼠组织脑抗氧化酶活性及其基因表达的增龄变化

目的　探讨增龄对快速老化模型小鼠 (SAM P/ 10 )脑组织抗氧化酶活性及基因表达的影响。　方法　采用生化及分子生物学技术 (NorthernBlot方法 ) ,观察了不同月龄SAM P/ 10小鼠脑组织丙二醛含量 (MDA)、超氧化物岐化酶 (SOD)活性及其基因表达水平的改变。　结果　模型动物自由基代谢产物MDA含量从 3月龄的 1 74± 0 37nmol/mg增至 9月龄的 4 2 7± 1 5 9nmol/mg·蛋白抗氧化酶活性从 3个月的 4 5 2 4± 3 2 5nU/mg ,蛋白降至 39 4 5± 3 12nU/mg·蛋白 ,并对SODmRNA表达有下调作用。　结论　提示增龄可在基因调控水平影响抗氧化酶活性 ,增加自由基代谢产物 ,从而导致脑老化的发生。     

远志总苷对快速老化模型（SAM—P／8）小鼠学习记忆能力的作用及其机理研究

目的探讨远志总苷对快速老化模型小鼠学习记忆能力的作用及其机制。方法以SAM—P／8小鼠为对象，给予远志总苷，连续灌胃30d，采用水迷宫方法逐日测定小鼠的学习记忆能力，用化学比色法测定脑组织总蛋白含量、总抗氧化能力和乙酰胆碱酯酶活性，用放免法测定血清中IL-2含量。结果远志总苷能明显提高SAM—P／8小鼠的学习记忆能力，缩短到达目的地的时间；显著提高脑组织总蛋白含量、总抗氧化能力、降低乙酰胆碱酯酶活性及血清中IL-2含量。结论远志总苷可提高SAM—P／8小鼠的学习记忆能力，其作用机理可能与降低脑组织氧化损伤有关。     

四逆汤含药血清对早老性痴呆小鼠神经生长因子的影响及其作用机制

目的 探讨四逆汤含药血清治疗早老性痴呆的作用机制.方法 60只SAM-P/8小鼠随机分为实验组、模型组和治疗组;20只健康昆明种小鼠为空白对照组.实验组给予四逆汤含药血清混浊液2 ml/d灌胃;治疗组给予盐酸多奈哌齐混悬液400 mg·kg-1·d-1,空白对照组和模型对照组用等量生理盐水.21 d后,各组小鼠进行行为学测试,应用免疫组化法检测脑组织神经生长因子(NGF)改变及电镜观察形态学改变.结果 四逆汤含药血清能够改善SAM-P/8小鼠的学习和记忆能力,减少早老性痴呆模型小鼠的包涵体数量,增加大脑皮层及海马区NGF的含量,对SAM-P/8小鼠具有较好防治作用.结论 四逆汤含药血清能够明显改善早老性痴呆小鼠的学习能力和相关蛋白的表达.     

长期接触低剂量氯化甲基汞对老化模型小鼠脑蛋白激酶C活性的影响

目的 探讨氯化甲基汞(Methylmercury chloride,MMC)对12月龄快速老化模型小鼠(Senescence-accelerated mouse,SAM)的快速老化亚系SAMP-prone/8(SAMP8)及抗快速老化亚系SAM-resistance/1(SAMR1)小脑组织蛋白激酶C(Protein kinase C,PKC)活性的影响.方法 实验小鼠随机分为3组MMC各剂量组小鼠6月龄开始连续喂饲含有不同剂量(1.00、2.00和3.00mg/kg)MMC的普通饲料至12月龄建立SAM快速老化亚系SAMP8和抗快速老化亚系SAMR1汞中毒模型,对照组给予普通饲料,提取鼠小脑组织胞浆和胞膜PKC.采用改良Takai法观察MMC对鼠小脑PKC活性的影响.结果 MMC各剂量组鼠小脑组织胞浆和胞膜PKC活性明显高于对照组(P＜0.05,P＜0.01),与对照组比较其脑汞含量随接触剂量和时间的延长不同程度地明显高于对照组(P＜0.01,P＜0.001).结论 MMC可能通过影响小鼠小脑PKC活性介导其神经毒性作用,在老化进展中具有重要作用.     

快速老化鼠和自然衰老鼠中超氧化物歧化酶的比较研究

目的通过比较快速老化鼠（SAM—P／8）和昆明种自然衰老鼠中超氧化物歧化酶（SOD）表达情况，探讨两种衰老鼠之间异同及SOD在衰老过程中所起的作用。方法选用快速老化鼠（SAM—P／8）3月龄和6月龄各10只，昆明种小鼠3月龄和15月龄各10只，采用生物化学方法检测SOD蛋白活性和MDA含量；免疫组化对SOD蛋白的表达定位；蛋白印迹法检测SOD蛋白的含量变化；RT—PCR方法检测SOD基因的表达变化。结果两种衰老小鼠肝脏中SOD基因表达，SOD蛋白表达和活性均呈增龄性降低，MDA含量均呈现增龄性增加。结论快速衰老鼠和自然衰老鼠的SOD变化趋势相同，SOD在衰老过程中起了重要作用。     

山楂对D-半乳糖致衰小鼠抗氧化系统及钙稳态影响的实验研究

目的 观察衰老与细胞内Ca^2 含量的关系和山楂对D-半乳糖衰老小鼠抗氧化作用。方法 本实验以D-半乳糖衰老小鼠为研究对象，分别以大、中、小剂量的山楂水煎剂灌胃45d，测定了青年、老年小鼠血清总抗氧化能力(TAA)，红细胞内超氧化物歧化酶(SOD)活性、脑组织内丙二醛(MDA)含量，红细胞膜Na^ -K^ ATPase，脑组织Ca^2 含量变化，同时，观察不同剂量山楂对老年小鼠上述指标的影响。结果小鼠血清TAA，红细胞内SOD活性，红细胞膜Na^ -K^ -ATPase活性随增龄而下降；脑组织MDA含量，脑组织Ca^2 含量随增龄而上升。山楂可提高小鼠血清TAA，红细胞内SOD活性，红细胞膜Na^ -K^ -ATPase的活性，并能降低脑组织Ca^2 含量，脑组织MDA含量。在各给药组中以山楂中剂量组的抗氧化作用最强。结论 山楂能增强机体抗氧化能力，具有一定的抗衰老作用，同时，脑组织Ca^2 含量可作为衰老的一个指标。     

当归红芪超滤物对衰老大鼠脑组织氧化损伤及线粒体DNA缺失的影响

目的探讨当归红芪超滤物延缓老龄大鼠脑衰老的作用及其机制。方法选用3月龄大鼠12只作为青年组,选用24月龄大鼠36只随机分为老年组、当归红芪超滤物组、维生素E组。给予相应处理后检测各组学习记忆功能、脑指数测定,组织形态学观察,脑组织中超氧化物歧化酶(SOD)活力,丙二醛(MDA)含量测定及脑组织线粒体DNA(mt-DNA)缺失的表达。结果与青年组大鼠相比:老年组大鼠学习能力下降,脑组织形态学存在差异,脑指数存在差异(P<0.05),脑组织SOD活力明显降低,MDA含量明显升高,脑缺失mt-DNA/内参mt-DNA比例明显增高(P<0.05);与老年组相比:用药组大鼠学习能力明显增加,脑组织形态学存在差异,脑指数无明显差异,脑组织中SOD活力显著增高,脑组织中MDA含量显著降低,脑缺失mt-DNA/内参mt-DNA比例降低(P<0.05)。与维生素E组相比,用药组大鼠学习能力、脑指数、脑组织SOD活力、MDA含量、脑缺失mt-DNA/内参mt-DNA比例均无显著差异(P>0.05)。结论当归红芪超滤物可能通过清除自由基,防止mt-DNA片段丢失发挥延缓老龄大鼠脑组织衰老的作用,其功效与维生素E相当。     

加味温胆汤对日本快速老化模型鼠肝细胞核活性基因的影响

目的：探讨加味温胆汤对日本快速老化模型鼠（SAM-P/10)肝细胞核活性基因的影响。方法：采用DAN酶I活性基因探针,酶解DNA。结果：经治疗后各组肝细胞核基因组经DAN酶消化的肝细胞核酶解片段缩小,接近R／1系水平。结论：加味温胆汤具有提高SAM－P／10鼠肝细胞核活性基因水平,并增加转录活性的作用。推测其机制与其改善了SAM－P／10 鼠肝细胞染色质构象,从而有利于DNA酶的消化作用有关。     

增龄对小鼠心肌氧自由基浓度、超氧化物歧化酶活性及丙二醛含量的影响

以心肌氧自由基(OFR)浓度、超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量为指标,观察了增龄对心肌自由基反应的影响。结果表明:(1)心肌OFR浓度随增龄上升,至13、17月龄时达到显著水平(P<0.05):(2)心肌SOD活性随增龄下降,至13、17月龄时达到显著水平(P<0.05);(3)心肌MDA含量随增龄而增加,至13、17月龄时增加显著(P<0.05,P<0.01);(4)老年小鼠(17月龄)心肌SOD活性与OFR浓度呈高度显著的负相关关系(P<0.01),而MDA含量与OFR浓度呈显著的正相关关系(P<0.05)。上述结果表明,随增龄心肌自由基损伤性因素(OFR—MDA)增加,而保护性因素(SOD)减弱,这可能是老化心脏在结构、功能、代谢方面发生衰退的原因之一。     

增龄对大鼠心肌线粒体DNA氧化损伤的影响

目的　探讨增龄对大鼠心肌线粒体 DNA(mt DNA)缺失、线粒体呼吸链酶复合体及 ATP合成的影响。方法　 Wistar雄性大鼠分为 3组 :幼年组 (1月龄 )、青年组 (6月龄 )各 1 2只和老年组 (2 4月龄 ) 1 0只。心肌线粒体 DNA缺失、线粒体呼吸链酶复合体及腺苷三磷酸 (ATP)合成分别用聚合酶链反应 (PCR)、酶动力学和生物发光技术进行测定。结果　 3组大鼠均有不同程度 mt DNA缺失。老年组 mt DNA缺失 (2 .0 9± 1 .62 )较幼年组 mt DNA缺失 (0 .77± 1 .1 6)明显增高 (P<0 .0 5) ;较青年组 mt DNA缺失 (1 .54± 1 .1 7)也有升高的趋势 (P>0 .0 5)。老年组心线粒体呼吸链复合酶 活力 (2 347.2 1± 62 3.33)均较青年组 (3 859.1 2± 70 3.53)、幼年组 (4776.90± 548.63)明显降低 (P<0 .0 1 )。老年组心线粒体 ATP合成量 (1 96.95± 33.2 6)较青年组 (337.53± 62 .1 8)明显降低 (P<0 .0 1 )。心线粒体呼吸链复合酶 活力 3组间无明显性差异 (P>0 .0 5)。结论　大鼠线粒体 DNA氧化损伤与衰老有一定的相关性。     

Age-related changes in barrier function in mouse brain I. Accelerated age-related increase of brain transfer of serum albumin in accelerated senescence prone SAM-P/8 mice with deficits in learning and memory

The time course of brain accumulation of radiolabelled human serum albumin ((125)I-HSA) injected intravenously and the transfer of (125)I-HSA from blood to brain were evaluated in DDD mice using a double isotope technique. The brain accumulation of (125)I-HSA at 3 and 9 h but not at 24 h postinjection and the brain transfer rates were significantly higher in 22-month-old DDD mice than in 4-month-old ones. The brain transfer rates of (125)I-HSA were measured also in senescence accelerated prone mice (SAM-P/8) with age-related deficits in learning and memory, and in senescence accelerated resistant mice (SAM-R/I) without these deficits. The brain transfer rates were significantly higher in 13-month-old SAM-P/8 and 22-month-old SAM-R/1 than in 3-month-old mice of the same strains, respectively. The mean brain transfer rates in five regions observed in 22-month-old DDD mice, 22-month-old SAM-R/1 and 13-month-old SAM-P/8 increased by 31%, 41% and 51% compared with corresponding values in 3- or 4-month-old mice of the same strains. DDD mice and SAM-R/1 mice with normal characteristics of aging showed similar age-related significant changes in brain transfer rates. Age-related increase in the brain transfer rate was manifested at the youngest age in SAM-P/8 among the three strains examined. These findings show that the transfer of human serum albumin into the mouse brain increases with aging and suggest that the barrier function in the mouse brain against macromolecules changes with aging.     

The senescence-accelerated mouse (SAM-P8) as a model for the study of vascular functional alterations during aging

We studied vascular function in quiescent aortas from senescence-accelerated resistant (SAM-R1) and prone (SAM-P8) mice. Myographical studies of thoracic aorta segments from 6–7 month-old mice showed that the contractility of SAM-P8 aortas was markedly higher than that of SAM-R1 after KCl depolarization or phenylephrine addition. Acetylcholine dose-response relaxation curves revealed that SAM-R1 vessels were slightly more sensitive than those of SAM-P8. In the presence of the NO synthase inhibitor, L-NAME, all vessels displayed contractions to acetylcholine, but these were more distinct in the SAM-R1. Phenylephrine plus L-NAME displayed stronger contractions in both animal strains, but were markedly more pronounced in SAM-R1. The cyclooxygenase inhibitor, indomethacin did not change the vessel responses to acetylcholine or phenylephrine. These data indicate that NO synthase, not cyclooxygenase, was responsible for the differences in contractility. Standard histology and immunohistochemistry of endothelial NO synthase revealed no differences in the expression of this protein. In contrast, increased levels of malondialdehyde were found in SAM-P8 vessels. We conclude that SAM-P8 vessels exhibit higher contractility than those of SAM-R1. Furthermore, our results suggest that the endothelium of SAM-P8 vessels is dysfunctional and lacks normal capability to counteract smooth muscle contraction. Therefore, our findings support SAM-P8 as a suitable model for the study of vascular physiological changes during aging.     

康欣口服液对老年小鼠肾线粒体DNA缺失突变的影响

目的 研究康欣口服液对老年Balb/c小鼠肾线粒体DNA（mtDNA）缺失突变的影响.方法 老年Balb/c小鼠随机分为空白组和康欣口服液组,分别给予生理盐水和康欣口服液4个月.采用聚合酶链反应（PCR）技术和光密度扫描检测两组mtDNA的缺失突变情况.结果 与空白对照组比较,康欣口服液能显著减少老年Balb/c小鼠肾mtDNA的缺失（P＜0.001）.结论 康欣口服液可以抑制老年小鼠mtDNA的缺失突变,对mtDNA有保护作用.     

Hypertriglyceridemia and hepatic steatosis in senescence-accelerated mouse associate to changes in lipid-related gene expression

Aged rodents show increasing plasma and tissue triglycerides, and reductions in liver peroxisome proliferator-activated receptor alpha (PPARalpha) and its target genes. We determined whether a similar situation is present in a model of accelerated aging, the senescence-accelerated prone (SAM-P8) mouse. Five-month-old SAM-P8 mice were hypertriglyceridemic, and exhibited hepatic steatosis and reduced fatty acid oxidation versus control 5-month-old senescence-accelerated resistant (SAM-R1) mice, with no differences in PPARalpha expression and binding activity; in fact, fenofibrate administration to SAM-P8 mice induced a clear PPARalpha-driven response. Complementary DNA (cDNA) microarray analysis (Affymetrix Mouse Genome 430A 2.0 GeneChip array), Western blot, and electrophoretic mobility shift assay (EMSA) experiments indicated, among other changes, a deficit in farnesoid X receptor (FXR) expression and binding activity in the livers of SAM-P8 mice with respect to SAM-R1 controls. Triglyceride accretion and a deficit in hepatic fatty acid oxidation, features of the aging process in mammals, associate to a deficit in hepatic FXR activity in the SAM-P8 mice.     

Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice

PurposeTo investigate whether mitochondrial DNA (mtDNA) damage, specifically deletion, contributes to the development of atherosclerosis or is simply a secondary effect of the primary factors causing atherosclerosis.Materials and methodsmtDNA deletion was detected by PCR in the aortic root of atherosclerosis-prone C57BL/6J apolipoprotein (Apo) E gene deficient (−/−) mice and control C57BL/6J mice at different ages. Atherosclerotic plaques in the Apo E−/− mice were assessed using frozen sections of the aortic root. The protein levels of COX III and 8-oxoguanine glycosylase (OGG1) were determined.Resultswhile mtDNA deletions accumulated significantly in mice as young as 2- month-old, atherosclerotic plaques were not detected until mice were 6 months old or older, suggesting that mtDNA deletion occurs prior to the formation of atherosclerotic plaques in the aortic root of these mice. Moreover, the expression levels of mtDNA-encoded COX III protein in both 2-month-old and 16-month-old C57BL/6J ApoE−/− mice were significantly lower than those in C57BL/6J mice (p < 0.05). Additionally, the protein level of 8-oxoguanine glycosylase (OGG1), a mitochondrial enzyme that functions in DNA excision repair, decreased with age in these mice, indicating that age-related down-regulation of mtDNA excision repair also contributes to atherosclerosis in C57BL/6J ApoE−/− mice.ConclusionThese results reveal that mtDNA deletions occur during the early “initiation” stage of atherosclerosis in C57BL/6J ApoE−/− mice and have the potential to promote atherosclerosis.     

Characteristics of cardiac aging in C57BL/6 mice

The specific processes that cause aging of the cardiac tissue remain elusive. C57BL/6 (B6) mice are commonly used for investigating age-related diseases in mammals. We thus sought to evaluate the cardiac aging process in B6 mice. Cardiac tissues from the newborn (B6 NB), 2 month-old (B6 2M) and 21–27 month-old B6 mice (B6 aged) were used for the investigation. Several age-related cellular processes were evaluated, including telomere shortening, changes in p53 and p16 expression, changes in mitochondria DNA expression and DNA deletion, and alteration of mitochondria. We found that the aging of the B6 mice cardiac tissue is associated with the maintenance of telomere length, increased expression of p53 and p16, mild changes in mitochondrial DNA expression but widespread DNA deletion, and significant alterations of the mitochondrial ultrastructure within the cardiac tissue. The results of our studies suggest that mitochondrial DNA deletions, which affect the mitochondrial ultrastructure, cytochrome C oxidase activity, and p53 expression, are significantly associated with cardiac aging and may be a source of age-related heart failure.     

Tissue-specific deletion patterns of the mitochondrial genome with advancing age

Aging is a multifactorial process and a lot of theories have been put forward to explain the deterioration of organ function with advancing age. The free radical hypothesis developed by Harman is amongst the most prominent today and has been focused on mitochondrial aging in the last decades. Applying a long PCR approach we screened human skeletal muscle, heart, caudate nucleus and cerebellum of 50 individuals for large-scale deletions of mitochondrial DNA (mtDNA). The most important observation of our study was the detection of age dependent tissue specific deletion patterns of mtDNA. The pattern of the same tissue of different individuals was more similar than the pattern of different tissues of the same individuals. Whereas deletions were barely detectable in cerebellar tissue, in caudate nucleus a specific banding pattern with deletions of 4-8 kb was already observed around the age of thirty. However, the increase of these large-scale deletions in number and variety over lifetime was more pronounced in skeletal muscle or heart. Our data support the notion that different tissues accumulate mtDNA damage in a specific manner. Although functional consequences of mitochondrial deletions are clearly supported by experimental data on the single-cell level in model organisms and mammals, their role regarding impaired function of organs with advancing age in humans remains unresolved.