Intervention Effects And Mechanisms Of Atorvastatin On Natural Aging Of Rat Aorta

Worldwide population aging problem specifically refers to young population reduction in the world’s total population, meanwhile the older population increase resulting in substantial increase of elderly population proportion corresponding older many social problems and contradictions, that in the 1980s the world health organization (WHO) after a survey found in the global population structure. According to the research of Europe and the United States census, worldwide population aging is a social phenomenon that never happened in the process of historical evolution. In some modern sociologists study, it is a symbol of the historical progress. Basically, the change in population structure is the social economic development, education level and medical progress brought by the achievement, and is to overcome the infectious disease, to overcome the malnutrition, to ensure the safety of drinking water and reduce maternal and infant mortality, thus is the inevitable result of the development of good health cause.Even so, according to the survey data, by 2015, over the age of 60 elderly populations in mainland China will reach 243 million, accounting for 15.2% of the total population in mainland. By 2020, this proportion will be as high as 19%. According to the National Health and Family Planning Commission (NHFPC) statistical audit practice, the population proportion of 65 years of age and older is high as more than 7% of the total population, which viewed as an aging society. Obviously, our country has entered the aging society. According to sixth national census of population, a total of 26 provinces and municipalities directly under the central government in Chinese mainland have stepped into an aging society stage. According to experts’speculation, by the end of the 12th five-year, the growth of those provinces aging population will also be at a speed of more than 3%. This means that the aging process in our country is still in further, and followed many such as elderly health care, rehabilitation, nursing and public health service problems will become increasingly outstanding and serious. At present, the whole society is advocating to promote healthy aging, promote the elderly health and improve the quality of life of older population, which are the shared mission of the broad masses of medical workers and the whole society of industry participants. American Geriatrics Society (AGS) in the 2013 science convention clearly pointed out:"the body’s aging caused by disorder of relevant function is usually ascribed to the body itself physiological dysfunction, can largely be treated to improve by internal corresponding treatment medicine". In theory, when changing the senescence caused by molecular biological and biochemical changes, changing age-related physiological situation, improving the tolerance of age-related disorder situation, the elderly people’s quality of life will improve, human life can be extended. Exploring the body’s aging related molecular targets and effectively intervention has become the current research hot spot and focus.With the growth of the global aging population, the development trend of the senile correlation diseases, such as stroke, coronary heart disease, high blood pressure, diabetes in our country and developed countries is also the centralized dynamic evolution. For Atherosclerosis (AS), the main pathophysiological characteristics of cardio-cerebrovascular disease, its morbidity, mortality and morbidity is gradually rising. One of the important reasons is the social phenomenon of an aging population growing. As the growth of the age, morbidity rates of heart and cerebral vascular diseases continue to rise, thus the research of AS pathogenesis and prevention and control of strategy is especially important.There are some main risk factors caused AS, including obesity, high blood pressure, high cholesterol, diabetes, smoking, age, etc. Because of the increasingly prominent social aging problem, ages viewed as the key role in the AS pathogenesis of research has begun to widely attention, and gradually become the new targets for intervention and treatment of AS. Epidemiological studies have found that with the growth of the age, the incidence of the AS will be sharply increased. The main reason for this is that blood vessels with the whole body is accordingly in aging, mainly characterized by vascular structure and function of vascular cells recession, gradually losing its functions of blood vessels, leading to the decrease of the arterial compliance and lumen expand and elastic recovery ability drop, changing matrix composition in the blood vessel walls. Therefore single from the atherosclerosis, the pathological changes of vascular aging changes further to promote the cardiovascular pathological physiology evolution, thus to some extent, has increased the risk of cardiovascular disease, for what the elderly also increase the illness severity, reduce the prognosis of patients with recovery. In current society, under the background of excessive aging population structure, prevention and treatment of the AS is even more critical. How to clarify that the age growth will bring blood vessels in a wide range of such change, and why should there be aging cells of the blood vessels, what is the mechanism hidden behind the vascular aging cell and molecular biology? All these need further research to answer them one by one.Atorvastatin, viewed as 3-hydroxy-3-methyl glutaric acid single acyl-coenzyme A reductase (HMG CoA reductase) selective inhibitor, could reduce the levels of plasma cholesterol and lipoprotein through by inhibiting the HMG CoA reductase in the liver and cholesterol biosynthesis, thus could help body reach the role of regulating blood lipid. In addition, with the patients suffered from diabetes and asymptomatic atherosclerosis diseases such as coronary heart disease risk factors combined with abnormal blood lipid level treated with atorvastatin, it will reduce the risk of nonfatal myocardial infarction, reduce the risk of stroke and vascular reconstructive approach, so as to achieve the prevention and control of related disease of heart and cerebral vessels. In the study of vivo or vitro, vascular dysfunction intervention mode, as the main research model, is mainly to impose intervention in acute vascular dysfunction. This research model is obviously not well simulated biological organism aging, a slow and gradual process of development, thus establishing the natural aging in vitro and in vivo model is a key problem we need to solve.The current study found that besides of lowering blood lipids, atorvastatin also have other functions of cardiovascular protection. The diversity of atorvastatin plays its role in the protection function of vascular wall structure, mainly due to anti-oxidation, anti-inflammatory, and by promoting the biological activity of Nitric oxide (NO) to improve endothelial function, and so on. In the myocardial infarction model, atorvastatin can promote the formation of new blood vessels in myocardium of mice. In cellular and molecular levels, atorvastatin could activate endothelial nitric oxide synthase (eNOS) and increase the production of the NO. Whereas in the past finding of acute vascular damage model, another kind of isomorphic isomers of Nitric oxide synthase (NOS), induced Nitric oxide synthase (iNOS), could increase its expression level, and decrease the production of NO that regulate diastolic blood vessel. Thus we speculate that eNOS/iNOS-NO normally plays a very important role in maintaining vascular function. For slow and gradual vascular aging model, what a kind of situation need to further study.According to the reports, there is a lot of the signal pathways involved in the process of aging in the information control. A lot of individual components through genetic control of these pathways are confirmed and regarded as new targets for drug development. Silent Information Regulator 1 (SIRT1) is one of the targets. Based on the past studies, the location of SIRT1 is at the intersection of signaling pathways constituted with induction and regulation of stress tolerance, and its coding gene is known as the "longevity genes". During the restrictions on energy (Calorie restriction, CR), it would be better to regulate life, thus some scholars adopt several methods to stimulate the expression of the activity of SIRT1, including hunger and high fat diet, nutrition stress and other ways. So in the case of natural aging, it is not in a position to exactly know what kind of effect on the aging of the blood vessels and cellular senescence SIRT1 has. What is kind of the relations between SIRT1 and NOS-NO system? Based on the research background mentioned above, we conceive of the natural aging process that could cause vascular tissue and blood vessel cells aging, is accompanied by oxidative stress and NOS of the system disorders with aging, therefore, easily develop to the AS stage. Atorvastatin may delay the progress of aging, and SIRT1 in the process can be activated to interact with NOS-NO system to play the essential role in the protection of blood vessels and vascular cells. This study used natural aging model implement the following three steps.一、Natural doubling induced the aging of Vascular smooth muscle cells (VSMC) and the intervention effect of atorvastatinStudy first of all, according to previous research of cultivation experiment in senile rat vascular smooth muscle cell, we could obtain in vitro cell culture aging models based on the improved in vitro to extend aging. Experiments were divided into four groups:young group (6PDL), middle-aged group (14PDL), senile group (24PDL), senile drug intervention group (starting to add atorvastatin quantitative dissolved drug in the cell cultures of the middle-aged generation, dose of 10μmol/L). According to the drug and each group intervention time, the activity of age-related beta galactose glucoside enzyme was observed by immunohistochemical staining, the cell proliferation ability were reflected by CCK-8 method and flow cytometry detecting the cell cycle. The cell apoptosis was detected by JC-1 and TUNEL method. Then the cell oxidative stress damage indicators:the content of Malondialdehyde (MDA), the activity of Superoxide dismutase (SOD) and NOS system related indexes (the total content of NO and the activity of NOS) were measured by colorimetric assay and Griess method, respectively. The content of NOS system (eNOS and iNOS) mRNA was determined by real-time quantitative PCR assay. The protein expression of eNOS and iNOS was measured by western blotting. The cellular localization and expression of eNOS and iNOS were observed by immunofluorescence stain. The results showed that:(1) with the cells sub-cultured, VSMC were gradually appeared aging phenotype(F= 156.751, P<0.001). Compared with young rat group and middle-aged group, SA-beta galactose glucoside enzyme staining positive percentage of cells and cell cycle detection G0/G1 phase ratio increased significantly in senile group (P<0.05). (2) the intracellular MDA content increased (F=38.640, P<0.001), SOD activity decreased (F=191.888, P<0.001), suggesting that the in vitro culture passaged model successfully induced VSMC aging caused by oxidative stress, which is one of the important mechanisms of the vascular smooth muscle cell senescence. NO were decreased (F=28.736, P<0.001), the activity of calcium dependency NOS declined with the increase of cell number of batches (F=67.070, P<0.001), the calcium dependency NOS activity increased (F=67.070, P<0.001). Along with the gradually increase the number of batches, the expression of eNOS decreased the ratio of eNOS/iNOS gradually declined, indicated that NOS system disorder is one of the important mechanisms of blood vessel cells aging. (3) compared with senile group, the activity of SOD increased through a quite long period of atorvastatin intervention in senile rat drug intervention group (P<0.05), MDA content decreased (P<0.05), the content of NO and calcium dependent NOS activity increased (P<0.05), the calcium dependency NOS activity decreased (P<0.05). Immunofluorescence found that NOS expression mainly located in the cytoplasm. Along with the increasing the number of batches, the intensity of fluorescence of eNOS decreased, iNOS fluorescence intensity increased, senile drug intervention group can maintain the balance of NOS system by inhibit the expression of iNOS and increasing the expression of eNOS.二、Natural age increase induced rat aortic vascular aging and the intervention effect of atorvastatinIn order to further investigate oxidative stress and NOS system imbalance in the role of vascular aging, we observed the process of the increase of age changes of the aortic blood vessels and the corresponding effects on atorvastatin intervention from the perspective of natural aging animal models. Experiment is divided into four groups:young age rat group (2 months of age, n=8), middle age rat group (12 months of age, n=8), the elderly rat group (20 months of age, n=8), senile rat drug intervention group (starting to add compound feed contained atorvastatin from 12 months of age to 20 months, n=8, dose of 5mg/kg·d). According to the end of intervention time of each group, all rats were killed following the certain weeks of feeding, the thoracic aorta were harvested by using thoracotomy, detecting vascular ring tension changes, adopting immunohistochemical staining methods to observe the activity of age-related beta-galactose glucoside enzyme, measuring the cell oxidative stress indicators (the content of MDA and the activity of SOD), and detecting the NOS system related indexes(the content of total NO and the activity of "NOS). The content of eNOS and iNOS mRNA was measured by real-time PCR assay, the protein expression of eNOS and iNOS was determinated by western blotting method, and the location and expression of eNOS and iNOS were observed by immune histochemical staining method. Results showed that:(1) Along with the increase of age change, endothelium dependent vasodilatation function decreased significantly (F=70.346, P<0.001), the endothelial dependent diastolic function also has a downward trend. The intervention of Atorvastatin can improve vasodilatation function (P<0.05). (2) along with the age growth, compared with young rat group and middle-aged rat group, the IOD of positive cells using SA-beta-galactose glucoside enzyme dyeing increased in senile rat group (F=79.486, P<0.001). (3) In the specimens of aorta, MDA content of senile rat group increased (F=132.420,P<0.001), SOD activity decreased (F=241.340, P<0.001), indicated that the change of living tissue age successfully induced aortic vascular aging caused by oxidative stress, which was one of the important mechanisms of the vascular aging. The content of total NO decreased (F=27.208, P<0.001), the activity of calcium dependency NOS decreased (F=86.555, P<0.001) and the activity of calcium dependency NOS increased with aging (F=86.555, P<0.001). Along with the age growing, the expression of eNOS decreased (F=141.618, P<0.001), while the expression of iNOS increased (t=-10.903, P<0.001), the ratio of eNOS/iNOS gradually declined, indicated that prompting NOS system disorder is one of the important mechanisms of vascular aging. (4) Compared with senile rat group, the activity of SOD increased in senile rat drug intervention group followed with atorvastatin long-term intervention (P<0.05), the content of MDA decreased (P<0.05), the content of NO and calcium dependent NOS activity increased (P<0.05), and the calcium dependency NOS activity decreased (P<0.05). Immunohistochemical found that along with the increasing of the age, the expression of eNOS gradually fell, and the expression of iNOS was the highest in the elderly group, long-term intervention of atorvastatin can maintain the balance of NOS system by restraining the microscopic expression of iNOS and increasing the expression of eNOS in senile rat drug intervention group.三、The effect of SIRTl on the natural aging induced blood vessels of rats and the aorta smooth muscle cells and the intervention mechanism of atorvastatinIn order to further explore the role of SIRT1 and the effect of atorvastatin in the natural aging model, the content of SIRT1 expression was detected for each group of aorta and vascular smooth muscle cells.1、In vascular smooth muscle cells, the content of SIRT1 mRNA was determined by the use of real-time quantitative PCR method, the protein expression of SIRT1 was detected by western blotting method, the cell area and situation of SIRT1 expression were located using immunofluorescence. Results indicated:(1) the SIRT1 expression was mainly in the nucleus. With extend aging of cells(F=505.650,P<0.001), the expression of SIRT1 in the elderly rat group was significantly lower than the middle-aged rat group (P<0.05) and young rat group (P<0.05). And through the intervention of atorvastatin, compared with senile rat group, the SIRT1 expression of senile rat drug intervention group increased (P<0.05). (2) In the vascular smooth muscle cells, correlation analysis between SIRT1 and NOS system showed that the expression of SIRT1 was positively correlated with the expression of eNOS (r=0.933, .P<0.001), and negatively correlated with iNOS relationship (r=-0.776,P<0.001). And SIRT1 and the ratio of NOS system was positively correlated relationship (r=0.840, P<0.001).2、In the aortic blood vessels, the content of SIRT1 mRNA was determined by using real-time quantitative PCR method, the expression of SIRT protein was detected by western blotting method, and the aortic area and situation of SIRT1 expression were located by immunohistochemical staining method. Results indicated: (1) As the aorta age gradually aging (F=168.199, P<0.001), the expression of SIRT1 in the elderly rat group was significantly lower than both the middle-aged rat group (P<0.05) and young rat group (P<0.05). And through the intervention of atorvastatin, compared with senile rat group, the expression of SIRT1 increased in senile rat drug intervention group (P<0.05). (2) In the rat thoracic aorta, correlation analysis between SIRT1 and NOS system showed that the expression of SIRT1 were positively correlated with the expression of eNOS (r=0.975, P<0.001), and negatively correlated with iNOS relationship (r=-0.972, P<0.001). The SIRT1 expression and the ratio of NOS system was positively correlated relationship (r=0.989, P<0.001).Through the tests mentioned above for aortic vascular tissue in vivo and cell model of aging in vitro, it could be rational to conclude that there was positive correlation between the ratio of eNOS/iNOS and SIRT1.Conclusion:Through this research, we can draw the following conclusion:1. By in vitro to extend simulation cell natural aging, there will be the aggravation of oxidative stress degree and the disorder of NOS system. These results indicated that cell aging can increase the damage of the intracellular oxidative stress, while decreasing the expression of eNOS and increasing the expression of iNOS, which in turn cause the disorders of NOS ratio balance.2. Through natural age simulating aortic aging in the body, it could cause the obstacle of vasodilation function. These results indicated that aging can aggravate the degree of oxidative stress in vascular, increase the disorder of NOS system, reduce the NO bioavai lability, thus cause the change of vasodilatation function in the aging rat aorta.3. Both in vivo and in vitro experiments with long-term atorvastatin intervention, it not only could reduce the damage of oxidative stress, restore the balance of NOS in the cells, and also could improve the bioavailability of NO, improve the diastolic function of blood vessels in the aorta vascular.4. Blood vessels in the process of aging and senescence, the gradual decline of SIRT1 expression is related to the disorder of NOS system balance. Atorvastatin can increase the expression of SIRT1, and maintain the balance of the eNOS/iNOS, so as to achieve the purpose of protecting and delaying vascular senescence.