| [Backgroud and Objective]From 1999, China had become an aging country because the population of oldpeople increased in 3% per year and the population of the aged people will reach 300millions in 2020. With the aging population increasing steadily, the study ofaging-related diseases becomes socially popular. Some vascular ageing diseases,such as artherosclerosis, hypertension, cerebral stroke etc, have wasted a great dealof social resources because of their high incidence, high fatality rate and highdisability rate. Therefore, the research on the mechanisms of human aging and wayto intervene it turns to be markedly significant. Although there are many theoriesassociated to mechanism for ageing, we can summarize as the following two types: 1.Environmental injury theory of ageing, representative by injury of reactive oxygenspecies (ROS), glycosylation, and so on; 2. Genetic factor programming theory ofageing, representative by telomere shortening, oncogennes, cell cycle regulatoryfactors, etc. The formor is exogenous one, the latter come from the change of the body aging. Now more and more researches about aging will unify these twotheories in order to conrrespond with the real aging of body. Cutoff the pathways ofthe mechanism of aging will help us to delay the process of humanbeing aging.Among the factors contributing to human ageing, oxidative stress has beenfocusing our eyes. The ROS produced by oxidative stress not only can induce cellsenescence, but also cause a lot of cardiovascular disease. Some researchers evenconsider the long term accumulation of oxidative stress in vivo as the reasonaccounting for human aging. ROS refers to some oxygen metabolites and theirderivatives with active chemical properties (such as strong reducibility, high energy,instability) after one-electron reduction of molecular oxygen, including hydrogendioxide, superoxide anion, hydroxyl group and lipid metabolites. While oxidativestress results from an imbalance between formation and neutralization ofpro-oxidants, the free radicals such as reactive oxygen species(ROS) accumulatedduring a variety of biochemical ractions and cellular functions, the excess ROS cancause tissue patho-damage, destruction of the DNA single strand, biomembrane lipidperoxidation, denaturization of proteolytic ferment, shorten of telomere andexpression of the cell cycle regulator which can cause body aging.As discussed above, many researcher found that some kinds of physico andchemical factors such as hydrogen peroxide, tert-butylhydroperoxide(t-BHP), gammaray can induce cell senescence. These premature aging cell models were a useful wayfor aging research because aging process between premature senescence cellstimulated by ROS and the aging cell are almost the same. But the senilism cellmodel were focus on the fibroblast cells, scarcely other types. We had built thesenescence rat VSMCs whiche is called stress induced premature senescence(SIRS)model induced by tert-butylhydroperoxide (t-BHP), an inducer of hydrogenperoxide that can induce DNA single-strand breaks. This aging cell model provide us a useful way to study the vascular aging process and intervention effects of someanti-aging material. Now there is increasing evidence showing that cell senescencecontributes to aging relation diseases such as atherosclerosis(AS). So more and moreGeriatrics specialist consider that the anti-aging therapy will become an increasinglyimportant new strategy in AS treatment.The people become aging accopanied with not only the ageing of blood vessele,but also the sex hormone balance. Dehydroepiandrosterone(DHEA) is a kind ofabundant steriod that is produced by adrenal gland. The plasma concentration get tothe peak while people is 30 year old, thereafter decease steadlily after pubertythroughout life. In the epidemic researches, the negative relationship was foundbetween serum DHEA concentration and incidence of many age related diseases,including AS, immunologic hypofunction, teoporosis, tumor, cognitive hypofunctionand the lower of the quality of life, etc. Supplement of proper DHEA plays abeneficial role in postponing the occurence of these diseases. Ever since DHEA waswidely applied as a food additive in foreign countries and few side effects werereported, the effects of DHEA in aging have caught more and more attention. Wefound that DHEA has a significantly intervention effects on the nitrogen monoxidumpathway in the aging rat, which means that DHEA may play a important role on thevascular aging process in our previous study. However, there was few works on directrelation between DHEA and vascular cells senescence especially in vascular smoothmuscle cells(VSMCs). In present study, we attempted to investigate the effect ofDHEA on VSMCs senescence induced by t-BHP. Some markers of senescence weredetected such asβ-galactosidase(SAβG), telomerase and cell cycleregulators(p53,p21,p16). The conclution of this study will provide some originaltheory for vascular age related diseases.[Methods] 1,Culture rat VSMCs with blocks explanting method. VSMCs from passage 4were targeted to premature senescence by a stimulation of 80nmol/L t-BHP for72hours. Then observe the morphologic change of the premature VSMC2,VSMCs from passage 4 were divided into 4 groups: control group; t-BHP group,stimulated by 80nmol/L t-BHP for 72hours; low DHEA treated group, treatedwith 10nmol/L DHEA for 30min before a stimulation by 80nmol/L t-BHP asGroup B; high DHEA group, treated with 100nmol/L DHEA for 30min beforea stimulation by 80nmol/L t-BHP. Detecting the expression of SAβG in eachgroup through cell staining method, in which the cells were stained bypropidium iodide(50μg/ml). Then analyzing: the DNA distribution in each cellcycle with a flow cytometer.3,Group as the above mentioned. Lyse VSMCs in the cell lysis buffer. Thenmeasure the protein concentrations with a Coomassie Blue G250 stainingtechnique. Last, detect the p53,p21,p16 expression by Western blot technique.4,Group as the above mentioned, determine the activity of telomerase in eachgroup by PCR-ELISA technique.[results]:1,The VSMCs can be induced to prematureure senescence by a stimulationof t-BHP with a concentration of 80nmol/L for 72hours. The prematuresenescence cells have an appearance of enlarged, flattered, stellar shapewith increased ratio of cytoplasm/nuclear. Granules and vacuolus areseen in those cytoplasm, whereas less in the control group..2,The results of the expression of SAβG activity demonstratedasignificant difference among the four groups (P＜0.001). Respectively,(3.6±0.6)% expression in the control group, (74±1.1)% expression inthe t-BHP treated group which shows significant deviation (p＜0.001). The SaβG-positive cell is (61.8±0.8) % in the low DHEAtreated group, while in the high DHEA treated group is (46.3±0.9) %.Which shows a lower difference (p＜0.001). DHEA treatment can deceasethe expression of SA-β-gal activity, and the inhibition of 100nmol/LDHEA treatment was stronger than the 10nmol/L treatment.3,The results of the cell cycle distribution analyzed by a FACS CoulterCounter showed that most of cells in t-BHP treated group stagnated inG1/G0 phase (89.8±3.7)%, whereas in the cells treated with 10nmol/LDHEA, the G1/G0 phase decease to (82.8±3.7)% and on the 100nmol/LDHEA group was (70.7±3.5)%.4,T-BHP can induce the expression of p53, p21 and p16. And differentconcentration DHEA can make a significant different decease of the cellcycle regulation. The 100nmol/L DHEA have a much more stronginhibition than 10nmol/L.5,Detecting the telomerase activity in each group by PCR-ELISA. Theresults demonstrate a respective 10.13±0.42,9.80±0.18,9.95±0.34,10.02±0.59 in each group, there was no difference among them. t-BHPtreated group has a decreased tendency compared with Group control,but there is no significant difference. Likely the DHEA treated groupsmay have some degree of increase compared with t-BHP treated group,still no significant difference (P=0.378, P=0.226)。[Conclusion]:1,80nmol/L t-BHP can significantly induce premature senescence ofVSMCs.2,DHEA can change the expression of SA-β-gal activity and cellcycle distribution in VSMCs induced by active oxygen, in which 100nmol/L is better compared with 10nmol/L.3,DHEA can partial inhibit the expression of protein p53,p21,p16activity in VSMCs induced by active oxygen, in which 100nmol/L isbetter compared with 10nmol/L.4,There is no significant difference statistically in the expression oftelomerase in the senescence VSMCs induced by t-BHP.5,DHEA can delay the premature senescence induced by active oxygenthrought inhibiting the cell cycle effectors.
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