| ObjectiveIn 1999, China has entered an aged society. The aged population in China will increase by 3 percent per year and will reach 300 million in 2020. Structural and functional changes of blood vessel with advancing age are namely vascular aging. Clinical study indicated with advancing age, the incidence of many cardiovascular diseases such as hypertension, coronary heart disease, heart dysfunction and stroke increased rapidly. Some studies also indicated that the increased risk of cardiovascular diseases in aged population is the result of age disease interactions. The Baltimore longitudinal aging study found the age—related structural and functional changes in blood vessel were closely correlated with age—depended cardiovascular diseases. Therefore, the researches on vascular aging have been the impelling social requirement with aged society coming rapidly. The study of vascular aging, mechanism is the footstone for prevention and treatment of vascular remodeling and the related diseases. Vascular aging is the independent risk factor for cardiovascular diseases and the aging related structural and functional changes in blood vessel may be the latent target for prevention and treatment of cardiovascular diseases.The reactive oxygen species theory about aging is widely accepted. In recent years, reactive oxygen species(ROS) existing in vascular wall were discovered, related research indicated NADH/NADPH oxidase was the primary source of ROS in all kinds of vessel cells, p22phox is the essential subunit of NADH/NADPH oxidase activity, angiotensin II stimula-ted ROS production through NADH/NADPH oxidase, and possibly correlated with vascular remodification of vascular diseases, for example, it was showed NADH/NADPH oxidase contributed to the development of Ang II —dependent hypertension. As a result, ROS contributed much to the vascular aging regulation. But what is the primary mechanism for oxidation stress and chronic inflammation induced by aged organism is still unknown. To answer this question successfully will bring breakthrough for aging study and throw much light on the aging interventions. Nowadays, many studies have focused on angiotensin system, and many results showed that the local angiotensin system contribute much to the aging vascular remodeling. Unfortunately, the above theory need to be further researched.Based on above results, the following hypothesis is formed that Ang II up —regulate the expression of p22phox subunit of NADH/NADPH oxidase,so endothelial cells overproduce ROS, therefore endothelial dysfunction sets up and maintains the vascular aging process.Methods1. Animal experiment1. 1 Wistar rats were divided into three groups, the young group(3~ month old),the old control group(22~24 month old), valsartan group x treated with valsartan 30mg ? kg"1 ? d"1 for 5 months from 17 — 18 month of age).1. 2 Aorta artery structure and function were analysed.1. 3 Angiotensin H in plasma and aorta were detected.1. 4 ROS level of aortic tissues was investigated.1. 5 RT—PCR and Western blot were used to analyze the mRNA and protein expression of NADH/NADPH oxidase p22phox, angiotensin type 1 and 2 receptor (AT,R, AT2R).2. Cell experiment2.1 The cultured HUVECs in vitro were divided into 3 groups, thecontrol group, AngH group (stimulated with 10 6mol/l Angfl for 48h), Angll plus valsartan group(valsartan was added to cells lh before 10~6 mol/1 Angll ).2. 2 P—gal stain,cellcycle analysis, pi6 exprssion were used to identify cell aging status.2. 3 ROS and NO level in cells and medium were examined.2. 4 RT — PCR and Western blot were used to analysis the mRNA and protein expression of p22phox, angiotensin type 1 and 2 receptor (AT,R,AT2R).3. siRNA experiment3. 1 Design 3 kinds of siRNA and synthesis siRNA by Silencer? siR-NA Construction Kit (Ambion). Using siRNA to transfect HUVECs cultured in vitro and select the most powerful and most suitable transfec-tion concentration and time.3. 2 HUVECs were divided into 3 groups: Control group, Ang H group, siRNA group, Ang U + siRNA group.3. 3 (3—gal stain,cellcycle analysis and pi6 exprssion was used to i-dentify cell aging status.3. 4 ROS and NO level in cells and medium were examined.3. 5 RT —PCR and Western blot were used to analysis the mRNA and protein expression of p22phox, angiotensin type 1 and 2 receptor (AT,R,ATzR).Results1. Animal experiment1. 1 With advancing age, aorta artery structure and function changed, namely, aging aorta artery remodeled.1. 2 Angiontensin II and ROS level of aortic tissues increased.1. 3 mRNA and protein expression of p22phox and AT2R increased", but ATiR expression attenuated in aging aortic tissues.1.4 Valsartan treatment reduced ROS level, downregulated mRNAand protein expression of p22phox in aging aorta arteries, alleviated aorta structural and functional remodeling.2. Cell experiment2. 1 Ang II stimulation enhanced the positive cell number of |3 — gal stained HUVEC, depressed cell proliferation, and increased the protein expression of pi6, at the same time, stimulated cells producted less NO and more ROS.2. 2 The mRNA and protein expression of p22phox and AT2R in cells stimulated by AngII increased, the expression of AT]R decreased.2. 3 Valsartan treatment alleviated aging associated changes, decreased ROS production and increased NO production, and downregulated the mRNA and protein expression of p22phox.3. siRNA experiment3. 1 Among the 3 kinds of siRNA, siRNA—1 was the most powerful at silencing p22phox mRNA expression and was selected.3. 2 Silencing effect was the most powerful at 24h and 36h, and 50nM/L was the most suitable concentration.3. 3 Compared with Ang II group, the mRNA and protein expression of p22phox and AT2R in cells dereased, the expression of AT\R increased in Ang II + siRNA group.3. 4 Comparede with Ang H group, ROS level decreased and NO level increased in Ang II + siRNA group.3. 5 More cells stopped in Go —Gi stage in Ang II + siRNA group than that in Ang H group.Conclusion1. Age related vascular remodeling changes came forth in rats belonging to age group and the animal model was constructed successfully.2. AngiontensinII and ROS level of aorta increased, the mRNA and protein expression of p22phox and AT2R increased, but AT^R expression attenuated in aging aortic tissues.3. Valsartan treatment reduced ROS level, downregulated the mRNA and protein expression of p22phox in aging aorta arteries, alleviated aorta structural and functional changes.4. siRNA — 1 selected in the experiment silenced the expression of p22phox effectively, maybe siRNA construted in vitro is a readily and reliable techniques for silencing genes.5. NADH/NADPH oxidase is the primary source of ROS in endothe-lial cells, p22phox is the essential subunit of NADH/NADPH oxidase activity. After silencing the mRNA expression of p22phox by siRNA, ROS productivity decreased sharply in cells. RNAi will be the new and promising technology for prevention and treatment of cardiovascular diseases.6. AT2 R expression increased sharply in aged endothelial cells induced by Ang II and after the mRNA expression of p22phox silenced by siRNA, AT2R expression decreased markedly, which indicated the increasing expression of AT2R was an protective and regulatory mechanism for cell anti—aging process.7. Ang II inducing endothelial cells senescence is possibly important factor for vascular aging. Ang H —p22phox—ROS is the main pathway for endothelial cell senescence. Valsartan contributes some protective effects to the vascular aging by blocking ATiR.
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