节点文献
淫羊藿总黄酮和淫羊藿苷对衰老的干预及机制研究
The Effects of Epimedium Flavonoids and Icarriin on Aging and the Mechanism of the Interventions
【作者】 张素琴;
【导师】 沈自尹;
【作者基本信息】 复旦大学 , 中西医结合临床, 2010, 博士
【摘要】 背景与目的:衰老是机体成熟后必然会发生的退行性改变,属生理性改变。但是衰老与老年病的病理生理改变没有本质的区别,如不进行干预则必然发展为老年病的病理生理改变,导致老年病的发生。在全球范围内,人口老龄化成为了本世纪最为突出的问题之一,给全球各国社会和经济长远发展带来严峻挑战。因此抗衰老研究成为当今生物医学研究领域的热点,也是衰老研究领域的焦点。目前被证实能够延长低等动物到哺乳动物等模式生物平均寿命和最大寿命的最有效干预手段是限制饮食,但是限制饮食要在临床中实施还存在许多困难,并且可能引起多种健康问题。所以寿命虽然是衡量干预措施是否能够延缓衰老的重要指标,但是衰老领域的研究目标并非只是简单的延长寿命,而是同时改善生存质量,达到延长健康寿命。中医药在抗衰老研究中具有悠久的历史和明显的优势,发现许多中药具有延缓衰老的作用,但是多数尚缺乏确实有效的实验证据。本课题组在前期的研究中发现中药淫羊藿提取物淫羊藿总黄酮(EF)能够延长低等动物果蝇和线虫的寿命,体外实验也证实EF能够延长二倍体细胞的传代次数。在此实验基础上,本研究进一步在哺乳动物小鼠上评价淫羊藿总黄酮(ED及其单体成分淫羊藿苷(ICA)对寿命和生存质量的干预作用。目前研究认为DNA损伤引起的基因组不稳定性在衰老发生过程中起到重要作用。DNA分子中遗传信息的完全正确,对机体生命的维持十分重要,体内外环境多种有害因素均可造成DNA损伤,这种损伤的累积,造成的基因组不稳定是导致衰老的核心环节。γ-H2AX是迄今所研究的最重要的DNA损伤感应分子,被认为是检测DNA双链断裂的特异性指标。本研究进一步从基因组稳定性探讨EF及ICA干预衰老的作用机制。材料与方法:动物:8~9周龄雄性C57BL/6小鼠正常喂养至12月龄时进行随机分组药物:淫羊藿总黄酮(EF):含20%淫羊藿苷,用于制备含0.06%EF的小鼠饲料。淫羊藿昔(ICA):纯度98.5%,用于制备含0.02%ICA的小鼠饲料。分组分组一:观察EF、ICA对小鼠寿命的影响。197只12月龄雄性C57BL/6小鼠随机分为四组:正常对照组(Control,52只)、限食对照组(CR,47只)、淫羊藿总黄酮组(EF,49只)、淫羊藿苷组(ICA,49只)。小鼠从12月龄开始药物及限食干预。其中正常对照组小鼠给予正常饲料喂养,限食组小鼠给予60%正常饲料量喂养,淫羊藿总黄酮组小鼠给予含0.06%淫羊藿总黄酮的饲料喂养,淫羊藿苷组小鼠给予含0.02%淫羊藿苷的饲料喂养。详细记录每个小鼠的自然死亡日期,观察各组小鼠的平均寿命、最大寿命。分组二:评价EF、ICA对小鼠老年时期生存质量的影响,并进一步探讨EF、ICA干预衰老的作用机制。49只12月龄雄性C57BL/6小鼠随机分为四组,分组及药物、限食干预同上,即正常对照组(Control。11只)、限食对照组(CR,13只)、淫羊藿总黄酮组(EF,13只)、淫羊藿苷组(ICA,12只)。该组小鼠喂养至24月龄时进行如下指标的观察,同时取3月龄小鼠作为青年对照。1)转棒实验:观察各组小鼠从转动棒上掉下的时间(转棒潜伏期时间),比较各组小鼠神经肌肉协调能力,该实验是评价小鼠生存质量的重要指标。2)Morris水迷宫实验:进行定位航行实验及空间探索实验,比较各组小鼠学习记忆能力。小鼠完成上述实验后处死,取标本进行如下实验:3)股骨骨密度测定:采用双能X线骨密度仪测定各组小鼠股骨骨密度。4)主要组织病理观察:取心、肝、脾、肺、肾、脑等组织固定包埋后,制成蜡块,利用利用组织阵列仪按照阵列设计将上述各组织制作在同一张组织芯片上,进行H&E染色,观察各组小鼠主要组织的病理改变情况。5)组织中DNA损伤标志物γ-H2AX表达情况检测:取各组小鼠心、肝、脾、肺、肾、脑等组织固定包埋后,制成蜡块,利用组织阵列仪按照阵列设计将上述各组织制作在同一张组织芯片上,采用免疫组织化学方法检测各组织中γ-H2AX的表达。6)基因芯片检测:利用基因组稳定及DNA损伤反应功能分类芯片检测与基因组稳定性相关的基因表达差异。7)肝组织匀浆上清超氧化物歧化酶(SOD)、丙二醛(MDA)含量测定。结果与讨论1) EF、ICA对小鼠平均寿命和最大寿命的影响。正常组小鼠的平均寿命为705天,EF组和ICA组小鼠的平均寿命分别为745天、754天,EF、ICA使小鼠的平均寿命分别延长5.7%、7.0%;采用国际上常用方法计算小鼠最大寿命(即90百分位上的小鼠寿命),正常对照组小鼠的最大寿命为890天,EF组小鼠的最大寿命为924天,ICA组小鼠的最大寿命为949天,EF、ICA使小鼠最大寿命分别延长3.8%、6.6%。上述的结果提示:EF、ICA能够延长小鼠的平均寿命和最大寿命,且ICA的作用略优于EF的作用。2) EF、ICA对小鼠老年时期生存质量的影响:a、转棒实验结果显示:24月龄小鼠的转棒潜伏期时间明显低于3月龄小鼠(P<0.05),提示老年小鼠神经肌肉协调能力下降。而EF组、ICA组和CR组小鼠的转棒潜伏期时间明显高于老年对照组小鼠(P<0.05),提示EF、ICA及限制饮食都能够提高老年小鼠的神经肌肉协调能力。b、Morris水迷宫实验结果显示:与青年组小鼠比较,老年小鼠水迷宫逃避潜伏期明显延长,空间探索实验穿越平台次数明显减少,说明老年小鼠的学习记忆能力下降;与老年对照组比较,EF组、ICA组和限制饮食组小鼠的穿越平台的平均次数增加,但逃避潜伏期无明显缩短,这可能与该次实验的实验强度和时间不够有关。c、小鼠股骨骨密度测定结果显示:24月龄小鼠的股骨骨密度较3月龄小鼠明显降低,而ICA能明显提高老年小鼠股骨骨密度(P<0.05),其作用优于EF组和限食组。上述结果提示:EF、ICA能够减轻老年相关性改变,提高老年时期的生存质量,且ICA的作用能够代替EF的作用,甚至在提高骨密度方面其作用优于EF和限食的作用。3)组织芯片H&E染色的病理观察结果显示:老年对照组小鼠肝组织出现水样变性,柯氏细胞增生,部分肝细胞出现坏死,并有少量炎症细胞呈小灶性浸润等病理改变,而EF、ICA和限食能够减轻肝组织中的上述病理改变。在心、肾、脾、肺、脑等组织病理变化中,EF组、ICA组、限食组与老年对照组之间无明显差异。上述结果提示:长期服用EF、ICA不仅对机体不会产生毒副作用,而且能够减轻肝组织随增龄而发生的病理损害。4)心、肝、脾、肺、肾、脑等组织中DNA损伤标志物γ-H2AX表达情况:与青年组比较,老年对照组小鼠心、肝、脾、肺、肾、脑等组织中γ-H2AX的表达都明显增加(p<0.05),而EF、ICA和限食都能够不同程度的降低心、肝、脾、肺、肾、脑等组织中的γ-H2AX的表达。如肝、肾组织中EF、ICA和限食都能够显著降低γ-H2AX的表达(p<0.05);在肺组织中EF、ICA能够显著降低γ-H2AX的表达(p<0.05);而在心脏组织中γ-H2AX的表达,则是以ICA的降低效果最为明显(p<0.05);脾脏组织中γ-H2AX的表达是以限食的降低效果最为明显(p<0.05);但是γ-H2AX在脑组织中的表达,EF组、ICA组、限食组与老年对照组无显著性降低(p>0.05)。上述结果提示:老年小鼠体内DNA损伤增加,而EF、ICA能够减少机体DNA损伤的发生,从而提高基因稳定性,这可能是其能够延长小鼠寿命的重要原因。5)基因组稳定性与DNA损伤反应功能分类基因芯片结果显示:与3月龄青年组小鼠比较,老年对照组小鼠的DNA损伤反应相关基因明显上调,在检测的与DNA损伤反应相关的128个基因中有40个基因发生上调。而与老年对照组比较,EF组、ICA组和限食组小鼠DNA损伤反应相关基因则明显下调,其中EF下调21个基因,ICA下调14个基因,限食下调10个基因。这一结果与组织中γ-H2AX表达情况的检测结果相一致,提示老年小鼠体内DNA损伤增加,从而激活了DNA损伤反应相关基因表达增加,而EF组、ICA组和限食组小鼠体内DNA损伤减少,则DNA损伤反应相关基因的表达也较老年对照组明显下调。同时结果还显示:DNA损伤检验点上的关键基因ATM、Brcal基因,老年对照组与青年组比较明显降低(p<0.05),而EF、ICA及限制饮食则能明显提高老年小鼠ATM、Brcal基因的表达(p<0.05)。提示EF、ICA和限食能够提高DNA损伤检验点上关键基因的表达,从而增强DNA损伤检验点功能,这可能有利于增强DNA损伤修复,减少DNA损伤发生,从而提高基因组稳定性。6)肝组织匀浆上清中的SOD活力及MDA含量测定结果显示:老年对照组的SOD活力明显低于青年对照组(p<0.05),而药物干预组和限食组的SOD活力老年对照组升高,其中以ICA组尤为明显(p<0.05);老年对照组小鼠肝组织匀浆上清中MDA含量与青年对照组比较明显升高(p<0.05),而与老年对照组比较,EF组、ICA组和限食组的MDA含量则明显降低(p<0.05),其中ICA的作用与EF的作用相当。上述实验结果提示老年小鼠体内清除氧自由基的能力降低,从而使氧化损伤的产生增加,而EF、ICA和限制饮食能够提高小鼠体内的抗氧化能力,减轻机体细胞受到氧化损伤的程度,在某种程度上也减轻氧化应激对DNA造成的损伤。结论1)EF及ICA能够延长小鼠平均寿命和最大寿命,这为EF及ICA能够延缓衰老提供了重要证据。EF、ICA使小鼠的平均寿命分别延长5.7%、7.0%; EF、ICA使小鼠最大寿命分别延长3.8%、6.6%。2) EF、ICA能够减轻衰老相关性改变,提高老年时期的生存质量,延长健康寿命。衰老领域的研究目标并非只是简单的延长寿命,而是延长健康寿命。本研究实验结果显示EF及ICA能够提高老年小鼠神经肌肉协调能力及股骨骨密度,减轻老年小鼠肝组织的病理损害,提示EF、ICA能够减轻小鼠随增龄而发生的机体功能减退,提高老年时期的生存质量。3) EF、ICA能够减少衰老过程中DNA损伤发生,提高基因稳定性。DNA损伤引起的基因组不稳定性在衰老过程中起到核心作用,本研究发现EF、ICA能够减轻体内DNA损伤的发生,提高基因组稳定性。这一作用可能与EF、ICA能够提高机体抗氧化能力,减少氧化损伤和提高DNA损伤检验点功能,增强损伤DNA修复这两个方面的作用密切相关。4)ICA是EF的主要有效药理成分。本研究发现ICA延缓衰老作用完全可以代替EF作用,为ICA是EF的主要有效药理成分提供了重要证据。同时ICA是一单体成分,具有“结构明确,质量可控,疗效确切,减少服用剂量”等优点,有利于从作用深处探寻其作用靶点,同时也有利于在临床上的开发运用。
【Abstract】 Background and objective:Aging is the degenerative change of mature body, which is a physiological change. However, there is no essential difference between the pathophysiology of aging and age-related diseases. If not to intervene, the aging would inevitably develop into the pathophysiological changes of age-related diseases. Population aging is one of the most prominent problems in this century around the world, and it challenges the long-term social and economic development of all countries. Anti-aging research becomes a hot area of biomedical research, but also the focus of aging research. So far, restrict diet has been the most effective means to extend the average life expectancy and maximum life span of the lower animals to mammals model organisms. But there are still many difficulties in the clinical practice of restricted diet which may cause many health problems. Therefore, although life expectancy is an important indicator of whether the anti-aging intervention is effective, but the field of anti-aging research is not just a simple extension of life span, but also to improve the quality of life, to extend the healthy life. Chinese medicine in anti-aging research has s long history and a clear advantage, and it has been found that many Chinese herbals have a role of anti-aging, but most still lack credible empirical evidence. In our previous studies, Epimedium flavavone (EF) which is of Chinese herbal Epimedium has been shown to extend the life span of lower animals such as fruit and nematodes; and it also has been found that EF could extend the passages of the diploid cells in vitro. On the basis of the ability of EF to extend lifespan in simple organisms, we further evaluated the effects of EF and icariin (ICA) which is monomer composition of EF, on the lifespan and quality of life in mice.Current studies suggest that genomic instability induced by DNA damage plays an important role in the aging process. DNA molecules which contain the genetic information play an important role in the maintaining life. The vitro and vivo factors can cause a variety of harmful DNA damage, and this DNA damage accumulation will result in genomic instability which inevitably leads to aging. So far,γ-H2AX has been studied to be the most important DNA damage sensor molecule, and the presence ofγ-H2AX foci in cells has been thought to reflect sites of DNA double-strand breaks. So this study further explored the possible anti-aging mechanisms of EF and ICA on the basis of genomic instability hypothesis.Material and Methods:Animal:eight to nine-week-old male C57BL/6 mice.Drugs:Epimedium flavavone (EF), Icariin (ICA)Part 1:To observe the effect of EF and ICA on lifespanOne hundred and ninty seven 12-month-old male C57BL/6 mice were randomly divided into four groups:Control group (n=52), CR group (n=47), EF group (n=49), ICA group (n=49). The mice were treated with drugs and diet restriction from 12 months. The mice in control group were given normal diet; the mice in CR group were given 60% normal diet; the mice in EF group were given the diet containing 0.06% EF; and the mice in ICA group were given the diet containing 0.02% ICA. The death data of each mouse were recorded to observe the mean lifespan and maximum lifespan of each group.Part2:To observe the effects of EF and ICA on the lifespan and life quality in mice and further explore its treatment mechanisms. Forty nine 12-month-old male C57BL/6 mice were randomly divided into four groups: Control group (n=11), CR group (n=11), EF group (n=13), ICA group (n=12). The drug and diet restriction intervention were the same as the above. The mice were fed to 24 months old when the following experiments were done, and the 3-month-old mice were used as young control.1) Rotarod experiment:To observe the latency of mice to fall from the rotarod and evaluate the ability of neuromuscular coordination in mice.2) Morris water maze experiment:The place navigation and space exploration experiments were done and to evaluate the learning and memory ability in mice.After the above experiments, the mice were sacrificed and the samples were collected for the following tests.3) Measurement of femur bone mineral density.4) Histophathological observation:Heart, liver, spleen, lung, kidney, and brain from each group mice were fixed and embedded. And tissue array was made accordance to the experimental design. Then the tissue array was stained with haematoxylin and eosin (H&E) for histological examination.5) Detection ofγ-H2AX expression in tissues. The tissue array containing the tissues such as heart, liver, spleen, lung, kidney and brain was stained by immunohistochemistry for the detection of theγ-H2AX expression in tissues.6) Microarray test:The genes expression related to the genetic stability were detected by such functional microarray as genomic stability and DNA damage response microarray.7) Superoxide dismutase (SOD), malondialdehyde (MDA) levels determination in the supernatant of live tissue.Results and discussion:1) The effect of EF and ICA on the mean lifespan and maximum lifespan:The mean lifespan of mice in control group was 750 days, the mean lifespan of mice in EF group and ICA group was 745 days and 754 days respectively. So mean lifespan increase for EF group was 5.7% and the mean lifespan increase for ICA group mice was 7.0%. Maximum lifespan (the ages at the 90th percentile) of control group mice was 890 days, however the maximum lifespan in the EF group and ICA group was 924 days and 949 days respectively. So EF increased the maximum lifespan by 3.8% and ICA increased the maximum lifespan by 6.6%. The above data showed that both EF and ICA could increase the mean lifespan and maximum lifespan in mice.2) The effects of EF and ICA on the life quality of mice. a) The results of the rotarod experiment:The latency to fall from the rotarod in the 24-month-old control mice was significantly less than 3-month-old mice (p<0.05), suggesting that the neuromuscular coordination declined with aging. This decline was significantly attenuated in EF group、ICA group and CR group mice which presented much longer latency time to fall from rotarod compared with the old control group mice. b) Morris water maze experiments:the escape latency in old control group mice was significantly longer than the young control mice, and the number of across the platform deceased, indicating that learning and memory in aged mice decreased. The average number of cross platform in EF group、ICA group and CR group increased, but not yet reached statistical significance. This may be related the fact that the intensity and duration of experiments were not relevant. c):Measurement of femur bone mineral density: The femur bone mineral density of 24-month-old mice was significantly lower than that of 3-month-old mice, however the ICA could increase significantly the femur bone mineral density in the aged mice(p<0.05). The above data indicated that EF and ICA could reduce the age-related changes to improve the quality of life in old age. 3) Histophathological observation:The histopathological changes in the liver of old control group mice showed that water-like degeneration, coriolis cell proliferation, part of the liver cell necrosis, small focal inflammatory cells infiltration and so on, and EF、ICA and CR could reduced such pathological changes. There was no significant difference of the histophathological changes in the heart, kidney, spleen, lung and brain among the observed groups.4) Detection ofγ-H2AX expression in tissues:γ-H2AX expressions in the heart, liver, spleen, lung, kidney, and brain increased significantly in elderly control group, compared with the young control group. However, EF、ICA and CR could decrease theγ-H2AX expressions in the above tissues. For example, EF ICA and CR could significantly reduced theγ-H2AX expression in the liver and kidney tissue(p<0.05); and EF、ICA could reduceγ-H2AX expression in the lung tissue(p<0.05); in the spleen, theγ-H2AX expression in CR group was much lower than old control group (p<0.05), however, EF、ICA and CR could not reduced theγ-H2AX expression in brain. These data suggested that DNA damage increased in old mice, while the EF, ICA could reduced DNA damage and enhanced the genome stability, which might be important reason why they could prolong the lifespan.5) Microarray test:The genes related to the DNA damage response were up-regulated in the 24-month-old mice, compared with 3-month-old mice, with 40 genes of 128 genes were up-regulated. Compared with the old control group, the genes related to DNA damage response were down-regulated in the EF group, ICA group and the CR group. The results of gene expression were consistent with the results ofγ-H2AX expression in the tissues, indicating that the DNA damage increased in the aged mice, which activated the DNA damage response-related gene expression. While EF, ICA and CR could decrease the DNA damage, then the DNA damage response-related genes were down-regulated. The results of microarray also showed that the ATM and Brcal which were the key genes of the DNA damage checkpoint decreased significantly in the aged control group mice compared with the young control group. But EF, ICA and CR up-regulated the ATM, Brcal expression in the aged mice. So it indicated that EF, ICA, and CR could attenuate the down-regulation of key genes expression in the aged mice, thereby enhancing the DNA damage checkpoint function, which might enhance the DNA damage repair to reduce the DNA damage in the old organisms. 6) Superoxide dismutase (SOD), malondialdehyde (MDA) levels determination in the supernatant of live tissue. The SOD activity in the old control group was significantly lower than the young control group (p<0.05), while ICA could increased the SOD activity of old mice (p<0.05). The MDA level in the supernatant of live tissue increased significantly in the old control group, compared with the young control group(p<0.05). And EF, ICA and CR could decrease the MDA levels (p<0.05). These results suggested that the capacity of scavenging oxygen free radicals decreased with age, allowing producing more oxidative damage. And EF, ICA and CR could increase the antioxidant capacity, reduce the oxidative damage, and to some extent reduce the oxidation stress on DNA.Conclusion:1) EF and ICA could extend the mean lifespan and maximum lifespan in mice, which provided important evidence for the anti-aging effect of EF and ICA. EF and ICA extended mean lifespan of mice by 5.7%,7.0% respectively; and increased the maximum lifespan of mice by 3.8%,6.6% respectively.2) EF, ICA could reduce the age-related changes and improve the quality of life to extend the healthy life. The field of anti-aging research is not just to extend lifespan, but to extend the healthy life. The results of this study showed that EF, ICA could improve the neuromuscular coordination in aged mice, increased the bone mineral density and reducing the pathological changes of aged mice liver, suggesting that EF, ICA could improve the quality of life in old age.3) EF, ICA could reduce the DNA damage occurring with age, improve the genomic stability. Genomic instability induced by DNA damage plays a central role in the aging process. In this study, it was found that EF, ICA could reduce the occurrence of DNA damage with age, and enhance the genome stability. This effect might be related to the following two aspects:1) EF, ICA could improve antioxidant capacity, reduce the oxidative stress on DNA;2) EF, ICA could improve the function of DNA damage checkpoint and enhance the DNA damage repair.4) ICA was the main effective pharmacological composition of EF. The anti-aging effects of ICA could replace the anti-aging effects of EF. ICA is a monomer component which has such advantage as "clear structure, quality control, effective, and reduced dose". So it is beneficial to explore its treatment targets, and more conducive to the development of clinical application.
【Key words】 Aging; Epimedium flavonoids; Icarriin; lifespan; genome instability;