The Effects Of Epimedium Flavonoids And Icarriin On Aging And The Mechanism Of The Interventions

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.