Effects Of Atorvastatin On Age-related Endothelial Dysfunction Of Rat Aorta

Population aging is the decrease of the number of young population and the increase of the number of the elderly population in the total population, which causes a corresponding increase of proportion of the elderly population in the total population. Population aging is a worldwide problem from the second half phase of the20th century to the early phase of21st century appears. Currently the aging population of the world who aged60and above reached750million. By2050, the aging population is expected to reach2billion, and80%of old people will live in the developing countries. By2013, China’s population aged60or older had reached202million, accounting for14.8%of the total population; By2015and2020, this proportion will reach15%and18%respectively. Typically, the ratio of the population aged65or older is more than7%, it is called " aging society ", while more than14%is called " aged society." All developed countries in the world have entered the aging society, many developing countries have entered or are about to enter an aging society. In1999, China entered the " aging society ", and the physical condition of the elderly was a disturbing problem. The demand of medical treatment, health care, nursing services and living services of old people are much more than other people. Therefore, proposing healthy aging and improving the physical condition of old people is the mission of health care providers and the society. Theoretically anti-aging treatment can alter the biochemical and molecular changes of aging, rectify the physiological changes associated with aging, reduce susceptibility to aging-related diseases. American Academy of Anti-Aging Medicine Society (A4M) declared:" The age-related disability is caused by the body’s physiological dysfunction, such dysfunction can be improved by medical treatment in many cases. Human life span is expected to be extended, and the quality of life of old people is expected to be improved." Human aging can be interpreted and may be slowed down. Searching molecular targets of anti-aging and exerting corresponding intervention is the current hotspot.Because the normal operation of the whole body depends on the blood supply of circulatory system, and the normal function of blood vessels plays an indispensable role in the normal operation of the circulatory system. Therefore, the changes of vascular function in the aging process can greatly affect overall body function. With the aging of the body, the blood vessels also aged correspondingly, which is characterized by the changes of the vascular structure and function. Age-related vascular dysfunction is closely related to endothelial dysfunction, which is characterized by progressive decrease of endothelium-dependent vasodilatation capacity of the resistance and conductivity arteries. Endothelial dysfunction is a systemic dysfunction, and is a key factor of atherosclerosis, coronary heart disease and hypertension. Therefore, improving endothelial dysfunction can reduce the morbidity and mortality of cardiovascular and cerebrovascular diseases. Although in vitro experiments have demonstrated inhibition of vascular cell senescence can improve cell dysfunction, in vivo research still needs to be further explored.Statins, a3-hydroxy-3-methyl glutaric acid coenzyme A reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) inhibitors, not only have an effect of regulating lipid, but also can improve endothelial dysfunction, consequently preventing a range of vascular-related diseases, such as atherosclerosis, coronary heart disease and hypertension. However, previous studies mainly focused on the effects of in vitro or short-term statin intervention on acute pathologic vascular dysfunction models. The slow and slight pathological changes during the aging process needs a long time to accumulate to cause significant functional changes, so establishing a natural aging animal models is a better strategy to study the effect of statins on age-related vascular dysfunction.Several signaling pathways have been shown to participate in vascular aging and the inhibitory effect of statins. Nitric oxide (NO) has been recognized as a key signaling molecule in the regulation of vascular function. NO is synthesized by nitric oxide synthases (NOS), which are composed of three isoforms, namely endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). The effect of NO on vascular function depends on its environment, including the sources and amounts of NO. eNOS and eNOS-derived NO are calcium-dependent and play a useful role, but decreases with aging. iNOS and iNOS-derived NO are calcium-independent and play harmful effects, but increases with aging. Under physiological conditions, eNOS-derived NO has an effect of vasodilatation, thus maintaining normal vascular function. Under pathological conditions, iNOS is activated and produces large amounts of NO. On the one hand, excessive NO interacts with reactive oxygen species (ROS) to form peroxynitrite (ONOO-), and then induces lipid peroxidation to accelerate vascular aging, consequently results in endothelial dysfunction. On the other hand, iNOS-derived NO activates arginase I to cause eNOS substrate degradation and eNOS uncoupling which consequently results in vascular dysfunction. During the aging process, iNOS of the aorta is activated or upregulated, but the exact location and source of iNOS needs more research. The effect of Statins on NOS isoforms is also controversial, especially on iNOS. Because the natural aging animal models require long-term intervention and observation, the effect of statins on endothelial dysfunction of natural aging animal model is unclear and needs further study.SIRT1(Sirtuin-1), a nicotinamide adenine dinucleotide (NAD+)-dependent histone, can regulate endothelial cell senescence and vascular function. SIRT1can interact with eNOS, and stimulate the expression of eNOS to inhibit vascular cell senescence and improve endothelial dysfunction. Although in vitro experiments have confirmed that atorvastatin could induce SIRT1to inhibit cell senescence, but whether atorvastatin can also inhibit vascular aging by inducing SIRT1needs further study.Based on the above ideas and research status, we assume that vascular dysfunction will happen during aging, and that long-term atorvastatin intervention can improve vascular dysfunction by regulating oxidative stress injury and NOS system imbalance during aging. Meanwhile, based on the role of SIRT1in vascular function, we assume that SIRT1can interact with the NOS system changes to regulate vascular function and the effects of statins. Our previous studies have shown, after long-term feeding, blood vessels from Wistar rats of different ages developed a series of pathological changes, including oxidative stress injury and the downregulation of eNOS. These age-related changes suggested rat blood vessels developed aging, so the model can be used as a good animal model of natural aging. In this study, an improved animal model which is composed of male Wistar rats of different ages with long-term feeding was used as the natural aging model. This study focused on the following three aspects, which not only can answer the above hypothetical questions, but also can prove whether long-term atorvastatin improves age-related endothelial dysfunction by regulating classic NOS system and SIRT1.Rat age-related vascular endothelial dysfunction and the effect of atorvastatinTwo currently widely accepted indicators were used to test vascular function: The vascular endothelium-dependent function test (acetylcholine-induced vasodilatation test) and vascular endothelium-independent function test (sodium nitroprusside-induced vasodilatation test). The rats were divided into4groups: young control group (2-month-old male Wistar rats, n=8), the middle-aged control group (4-month-old male Wistar rats received a sterilized diet ad libitum until12months old, n=8); the old control group (12-month-old male Wistar rats received a sterilized diet ad libitum until20months old, n=8; At the age of19months, one rat died); the old atorvastatin group (12-month-old male Wistar rats received a sterilized diet mixed with atorvastatin at a dose of5mg/kg/day until20months old, n=8). After eight months, the rat underwent thoracotomy and the thoracic aortas were extracted, and then above indicators were measured. The results showed that: Multiple comparisons between YC, MC, and OC showed that endothelium-dependent relaxation in response to acetylcholine decreased with age (P<0.05or0.01). Multiple comparisons of endothelium-independent relaxation in response to sodium nitroprusside showed no differences between YC, MC and OC. Such results suggested that aging caused rat vascular endothelial dysfunction.In addition, the effect of long-term atorvastatin intervention on vascular function showed:Compared with OC, long-term administration of atorvastatin (OA) attenuated the reduction of endothelium-dependent relaxation (P<0.05or0.01), endothelium-independent vasodilatation did not change (P>0.05). Such results suggested that long-term atorvastatin intervention can improve age-related endothelial dysfunction.The mechanisms of age-related vascular endothelial dysfunction and atorvastatin interventionTo further investigate the mechanisms of age-related vascular endothelial dysfunction and atorvastatin intervention, we observed aortic oxidative stress and NO pathway related changes in the natural aging groups and old atorvastatin group. Oxidative stress indicators:Superoxide dismutase (SOD) activity and Malondialdehyde (MDA) content were measured by colorimetric method. NO pathway related indicators:total NO content and NOS activity of calcium-dependent and calcium-independent were measured by Griess assay. eNOS and iNOS mRNA levels were measurd by Real-time quantitative PCR assay. The expressions of eNOS and iNOS proteins were measured by Western-blotting. The localization expression of eNOS and iNOS were detected by immunohistochemistry. The results showed that:(1) Multiple comparisons between YC, MC, and OC, SOD activity decreased (P<0.05or0.01), MDA content increased (P<0.01), total NO content decreased (P<0.05or0.01), calcium-dependent NOS activity declined with increasing age (P<0.01), but calcium-independent NOS activity increased with increasing age (P<0.01). Compared with MC, the eNOS expression level in OC decreased significantly (P<.001),the iNOS expression level increased significantly (P<0.01), and the eNOS/iNOS ratio decreased significantly (P<0.01). Although the eNOS expression level in YC was not greater than that in MC, the iNOS expression level in YC was detected by neither real-time PCR nor western blotting which suggested that the eNOS/iNOS ratio declined with age. This suggests that aging leads to vascular oxidative stress and NOS system imbalance.(2) Compared with OC group, long-term atorvastatin intervention increased SOD levels (P<0.01), reduced the content of MDA (P<0.01), increased total NO levels (P<0.01), increased calcium-dependent NOS activity and reduced calcium-independent NOS activity (P<0.01). Increasing the expression of eNOS (P<0.01) and inhibition of iNOS expression (not detected) restored eNOS/iNOS ratio. This suggests that long-term atorvastatin can reduce oxidative stress and restore NOS system imbalance.(3) Immunohistochemistry showed that eNOS only expressed in aortic intima (endothelial cells), while iNOS expressed both in the intima and media (vascular smooth muscle cells), and iNOS were not detected in YC and OA groups. This suggests that iNOS is activated with increasing age, while long-term atorvastatin intervention could inhibit the expression of iNOS and restore NOS system imbalance.The effect of SIRT1on age-related vascular endothelial dysfunction and atorvastatin interventionIn order to investigate the role of SIRT1in vascular aging and the effects of atorvastatin on SIRT1, the changes of aorta SIRT1were observed. SIRT1mRNA level was measured by Real-time quantitative PCR assay. The expression of SIRT1protein was measured by Western-blotting. The localization expression of SIRT1was detected by immunohistochemistry. In order to analyze the effecs of the interaction of SIRT1and NOS system on vascular aging and the role of atorvastatin intervention, SIRT1and NOS system correlation was analyzed. The results showed that:(1) Compared with MC, the SIRT1expression level in OC decreased significantly (P<0.01). Although the SIRT1expression level in YC was not more than that in MC, it was more than that in OC (P<0.01). These results suggested that SIRT1expression gradually declined with age. Compared with all other groups, long-term administration of atorvastatin (OA) not only restored but also increased SIRT1expression (P<0.01). Immunohistochemistry showed that SIRT1expressed in both the endothelium and VSMCs.This suggested that atorvastatin was able to upregulate SIRT1expression.(2) Correlation analysis of thoracic aortic SIRT1and NOS showed that In all groups, eNOS expression was significantly positively correlated with SIRT1expression (P<0.01); the correlation coefficients reached0.956and0.523at the transcriptional and translational levels respectively. iNOS expression was significantly negatively correlated with SIRT1expression (P<0.05or0.01); the correlation coefficients reached-0.614and-0.872at the transcriptional and translational levels respectively. The eNOS/iNOS ratio was significantly positively correlated with SIRT1expression (P<0.01); the correlation coefficients reached0.987and0.905at the transcriptional and translational levels respectively. Because iNOS expression was detected only in MC and OC, correlation analysis involving iNOS expression was only conducted in these two groups. However, in view of the relatively high expression of eNOS in YC and OA, as well as no detection of iNOS in these two groups, it is reasonable to speculate that eNOS/iNOS ratio were also significantly positively correlated with SIRT1expression in these two groups.Conclusion:1During aging process, male Wistar rats developed vascular endothelial dysfunction, which suggested that aging can cause endothelial dysfunction. By increasing intracellular oxidative stress, downregulating eNOS and activating iNOS to cause NOS system imbalance, aging caused decreased NO bioavailability, consequently led to endothelial dysfunction.2Long-term atorvastatin (5mg/kg· d) intervention can improve age-related endothelial dysfunction. By reducing oxidative stress, upregulating eNOS, inhibiting iNOS to restore NOS system imbalance, atorvastatin increased NO bioavailability, consequently improved endothelial dysfunction.3During aging, vascular aging was accompanied by downregulation of SIRT1and was closely associated with NOS system imbalance. Atorvastatin maybe increase SIRT1to mantain NOS system balance and improve endothelial dysfunction.