Effects Of Reactive Oxygen On The Age Related Changes Of Rat's Blood Vessel And Intervention Effects Of Dehydroepiandrosterone

Aging population is a main feature in human development in the 21st century, especially in China. According to newly issued report of National Population Development Research this year, by the end of the 20th century, the ageing population more than 60 years old exceeds 10% of the total population in China. By the year 2005, the ageing population more than 60 years old approximates 0.144 billion, accounting for 11% of the total population in China. At present, the ageing population is increasing at average annual rate of 3% or so. The aging process is accelerated at the rate of net increasing 0.1 billion ageing population in every 10 years. Along with the increasing aging population, the spectrum of disease in the ageing population in China is also changing from comparatively dispersion to concentration. The former 5 diseases with higher incidence are hypertension, coronary heart disease, cerebrovascular disease, diabetes, and tumor, among which cardiovascular cerebrovascular disease, manifested as atherosclerosis (AS) has become the first killer to threaten the health of the ageing population for its higher incidence, higher fatality rate and higher disability rate. So it is urgent to develop studies on pathogenesis and prevention strategy of AS.Traditional risk factors of AS include hypertension, hyperlipidemia, diabetes, smoking, age and sex, etc. It has been previously believed that age belongs to unchangeable risk factors of AS. So the studies on the effect of age are comparatively less. Along with the developing of population ageing, more and more attention has been gradually paid on studying the role of age in the occurrence and pathogenesis of AS, which has become a new target in prevention and treatment of AS. Researches of epidemiology show that aging is an independent dangerous factor of the occurrences and development of AS. Along with the increase of age, age related changes happen in blood vessel and partial vascular cells ageing occurs, which most reflects the characteristics of the aging population, and then contribute to significant elevated occurrence of AS. Accompany with the aging of blood vessels, apparent changes take place in the structure of blood vessels, the lumen of blood vessels enlarges and its elasticity lowers, the functional disorder happens in the cells of its vessel wall, and the composition of the stroma of the vessel wall also experience great changes. These atherosclerosis-independent changes arising from the aging process transform the pathophysiological mechanisms of cardiovascular and cerebrovascular diseases, thus altering the occurrence threshold, the severity and the prognosis of vascular diseases. Why does the aging process take place in blood vessels? What are the cellular and molecular pathologic mechanisms underlying the aging of blood vessels? Are these mechanisms able to give important ideas on amelioration or delaying blood vessel aging? Because the aged population is increasing, these questions need to be answered to prevent and control cardiovascular and cerebrovascular diseases.Mechanism studies on age related changes in blood vessel first involve studies on mechanism of ageing. Now there are tens of theories associated to mechanism for ageing. Generally they can be summarized as the following two types: 1. Environment injury theory of ageing, representative by injury of reactive oxygen species (ROS), glycosylation, etc.; 2. Genetic factor programming theory of ageing, representative by telomere shortening, ageing gene, cell cycle regulatory factors, etc. These two theories clarify possible mechanisms for ageing organism, deepen study on ageing to cellular, molecular and gene level. Ageing is a complicated process, which is affected by environmental factors and genetic factors, so now studies on ageing have gradually entered the times for the cooperation of the two theories. Digging the mechanisms for ageing and efforts of delaying ageing have localized in a comparatively definite range, i.e. centralizing on range associated to injury of environmental factors induced age related changes. At present fibroblast is adopted in most ageing associated studies. But fewer studies aim at specific tissues and cells. However, advance of above associated studies provide important information for us to comprehend the mechanisms for ageing of specific blood vessel tissues and cells.We believe studies on cellular and molecular mechanism for age related changes in blood vessels should lay particular emphasis according to characteristics of blood vessels. Blood vessel is an important part of human body. Its anatomic site lies on the functional interface liable to contact with injuries. Various injuries of internal environment and external environment formed by blood circulation and blood vessel itself, especially free radicals injury mainly in ROS form, first act on vessel wall cells. ROS refers to some oxygen metabolites and their derivatives with active chemical properties (such as strong reducibility, high energy, instability) after one-electron reduction of molecular oxygen, including hydrogen dioxide, superoxide anion, hydroxyl group and lipid metabolites. Studies showed oxidative stress induced by excessive ROS play important roles in vascular lesion, especially in occurrence of AS. ROS independently or jointly participates in the formation of AS by inducing gene expression of some inflammatory factors in oxidative stress. As one grows old, continuous acumulation of injuries on blood vessel wall induced by these factors may possibly become one of the most important factors inducing the occurrence and development of blood vessel ageing, thus to make AS more liable to happen in the aged. Model of fibroblast senescence has been induced by ROS (such as H₂O₂) chronic oxidative stress in experimental studies. Then whether can chronic ROS oxidative stress induce blood vessel cell senescence? What kind of changes associated to oxidative stress in blood vessel of ageing individuals? What reaction of blood vessel cells in aged individuals to ROS produced oxidative stress and whether these cells are liable to develop to AS? A series of these questions awaits further studies. The clarification on these questions is not only helpful to understand pathological mechanism for age related changes in blood vessels, but also provide new research targets for prevention and treatment of AS in the elderly.In the elderly, besides the ageing of blood vessel itself, another risk factor with significant characteristic for the liability of AS is age related changes of sex hormone balance. In the process of aging, corresponding changes happen in endogenous sex hormone balance, among which changes in sex hormone level after climacteric period show an important role in the organism. Endogenous testosterone and bioactive testosterone level are significantly decreased in men more than 50 years. Endogenous estrogen level in women after menopause drops more rapidly. At the same time, significant changes happen in the target organs of sex hormone. Lower estrogen level in females and lower testosterone level in males have been proved to be risk factors for AS. But organisms' regulation and responsion to sex hormone level is an exquisite and complicated process with individual difference. It is not so simple as imaged by externally regulate sex hormone balance to prevent or delay the occurrence of AS. Great disputes exist in whether supplement of sex hormone, especially estrogen, can delay the occurrence and development of AS. But different from these disputes, present studies hold confirmative attitude towards a beneficial role of DHEA, the prior product of sex hormone, in the occurrence of AS and various senile diseases.DHEA, secreted from adrenal cortices, is the prior product of androgen, small amount of which converts into estrogen. DHEA is the great amount of steroid hormones contained in human bodies, and accompanied with individual growth, the amount of it keeps descending. Epidemiology studies show DHEA is closely associated to AS. Plasma DHEA level has been proved to be significantly negative correlated to the occurrence of various senile disease, such as AS, immunologic hypofunction, teoporosis, tumor, cognitive hypofunction and the lower of the quality of life, etc. Supplement of proper DHEA plays a beneficial role in postponing the occurence of these diseases. Ever since DHEA was widely applied as a food additive in foreign countries and few side effect was reported, the effects of DHEA in aging have caught more and more attention. Most present studies aim to investigate an intervention role of DHEA in a special developing process of a specific disease. It is clear that DHEA not only possesses partial sex hormone activity, but also has peculiar physiological role of its own. These actions have not been clarified. The fact that DHEA delay aging related diseases hints that DHEA probably affects the aging process of the target organ, and DHEA may delay the occurrence of senile disease through affecting the common connection of aging. Currently there are not many reports of the relations of DHEA and the aging of organs. This research further observes the impact of DHEA on the aging process of blood vessels.According to these opinions mentioned above, we suppose ROS chronic oxidative stress may induce ageing of blood vessel cells. Along with age increases, some changes might take place in antioxidase activity of blood vessel, followed by changes in ROS associated substance in blood vessel, thus to participate the occurrence of blood vessel ageing. Sensitivity of blood vessel cells to ROS enhances along with increasing age, which is more liable to develop to AS. DHEA might retard the process mentioned above. According to these presumptions, studies around the following three contents are conducted by treating with H202 as the representative for ROS.1. Tert-butyl hydroperoxide (t-BHP) induce senescence of Vascular smooth muscle cell (VSMC) and intervention effect of DHEAIn this study, the present acknowledged ageing markers, ageing associatedβ-galactosidase activity and reproductive activity of cells were adopted as main indexes. First experimental observation was conducted in the possibility of t-BHP induced VSMC senescence.β-galactosidase activity was measured with immunocytochemical method and reproductive activity of cells was measured with flowcytometry. The amount of MDA was measured with colorimetry method. Results showed after continuous treatment with 80mmol/L t-BHP for 72 hours, ageing phenotype occurred in VSMC; cell ratio of G0/G1 phase and percentage of SA-βgalactosidase staining positive cells increased; apoptosis and necrosis occurred in partial cells with significantly increased intracellular Maleic Dialdehyde (MDA), which indicated that VSMC senescence was successful induced by t-BHP. These results suggest that effect of ROS is an important mechanism in the ageing of blood vessel cells.Besides, when compared with cells treated with t-BHP alone, 100nmol/L DHEA pretreatment before t-BHP can effectively reduce VSMC ratio of ageing phenotype, reduce cell ratio in G0/G1 phase and percentage of SA-βgalactosidase staining positive cells, significantly reduce apoptotic and necrotic cells, and significantly decrease intracellular MDA content. It suggests preventive application of certain dose of DHEA can postpone ROS induced VSMC senescence, partially achieved by antioxidation, which might be a kind of common mechanisms for DHEA.2. Age related changes of ROS associated substance in rat arteries and intervention effect of DHEAIn order to further investigate the relation between ROS and age related changes of blood vessels, we observed the age related changes of ROS associated substance in rat arteries. Male Wistar rats were divided into 3 groups of 6 rats each: 12 months old roup; 18 months old rats group; DHEA interventional rats group (1mg/kg·day of DHEA was added to their fundamental fodder when they are 12 months old). The thoracic arteries extracts were subjected to following determination:western blotting analysis for determination of endothelial nitric oxide synthase (eNOS) protein level, method of Griesses for the level of nitric oxide, radioimmunoassay for the level of cGMP and method of colorimetry for the activity of SOD and level of MDA. The results showed that compared with the adult group, in the aorta of 18 months old rats group, (1) the activity of SOD was lower but the amount of MDA was higher; (2) the amounts of NO and the amount of cGMP were lower; (3) the protein expression of eNOS was lower, but after DHEA intervention, the above changes in the aged group were apparently ameliorated in the aged group. The results suggest that during the aging process in rat arteries, the activity of antioxidase in the blood vessel itself is falling and the function of oxidation resistance is weakened which reflects the descending ability to remove oxide radicals and the level of lipid over-oxidation. The results also indicate that along with aging, the protein expression of eNOS in the blood vessels has a tendency to deceased and thus directly decrease the output of NO which making the decreased ability to remove oxide radicals more apparently; due to the lack of valid activating of NO, the activity of cGMPase in vascular smooth muscle cells is falling, thus making the amount of cGMP lowered. The above changes participate the age related change of vascular function and may be one of the important mechanisms of the aging process experienced in blood vessels. DHEA is able to regulate the age related changes of ROS associated substance in rat arteries and probably an effective prescription in delaying the formation of vascular aging.3. Age related changes of sensitivity of blood vessel cells to ROS and intervention effect of DHEABased on studies of the second part, the VSMCs from the aortas of 18 months old rats and 3 months old rats were cultured using explant method to observe age related changes of sensitivity of blood vessel cells to ROS. Reproductive activity of cells was measured with MTT method. Immunocytochemical method was employed to evaluate the nuclear translocation of Nuclear factor -kappa B p65 (NF-kB p 65). The MCP-1 mRNA level was measured with Reverse transcription-polymerase chain reaction (RT-PCR) method and level of MDA was measured with method of colorimetry. The results showed that the sensitivity of VSMCs reproductive activity to ROS in 18 months old rats was more serious than that in young rats. Compared with 3 months old rats, incubation with low concentration H₂O₂, the VSMCs reproductive activity was higher in 18 months old rats while incubation with high concentration H₂O₂, the VSMCs reproductive activity was lower and the cells were more liable to occur necrosis and apotosis in 18 months old rats. Besides, the activation of NF-kB and the up-regulation of MCP-1 mRNA expression caused by 6h treatment with 50μmol/L H₂O₂ was more serious in 18 month old rats than that in 3 months old rats, which suggests that along with aging, ROS is liable to induce the expression of inflammation factors and reproductive activity in VSMC, therefore accelerate the progression of atherosclerosis. In these studies, we also observed that pretreatment with certain concentration DHEA was able to suppress the activation of NF-kB and the up-regulation of MCP-1 mRNA expression caused by 50μmol/L H₂O₂, and these results further verified that DHEA was able to ameliorate the injuries canused by ROS and then might retard the process of vascular aging.ConclusionWe may make the following conclusions from these studies: 1. Continuous treatment with 80mmol/L t-BHP for 72 hours, VSMC content of intracellular MDA, cell ratio of G0/G1 phase and percentage of SA-βgalactosidase staining positive cells increased significantly, which indicated that VSMC senescence was successful induced by t-BHP. These results suggest ROS is an important mechanism for inducing ageing of blood vessel cells.2. Along with aging, the vascular activity of SOD was lower but the amount of MDA was higher; while the protein expression of eNOS was lower and the amounts of NO and cGMP were lower; but after DHEA intervention, the above changes in the aged group were apparently ameliorated in the aged group.3. Sensitivity of VSMCs reproductive activity to ROS in 18 months old rats was more serious than that in young rats. Compared with 3 months old rats, incubation with low concentration H₂O₂, the VSMCs reproductive activity was higher in 18 months old rats while incubation with high concentration H₂O₂, the VSMCs reproductive activity was lower and the cells were more liable to occur necrosis and apotosis in 18 months old rats.4. The activation of NF-kB and the up-regulation of MCP-1 mRNA expression caused by 6h treatment with 50μmol/L H₂O₂ were more serious in 18 month old rats than that in 3 months old rats, which suggests sensitivity of blood vessel cells to ROS enhances along with increasing age.5. Certain dose of DHEA was able to delay the occurrence and progression of above changes partially achieved by antioxidation, which suggests DHEA is probably an effective prescription in delaying the formation of vascular aging.