Effects Of Reactive Oxygen On The Senescence Of Human Umbilic Vein Endothelial Cells And Intervention Effects Of Testosterone

[Objective]The structure and fuction of vessel wall change gravely with advancing age, which includes luminal enlargement, initimal and medial thickening, increased vascular stiffness, partial vascular cells aging, and the endothelium-dependent vasodilator respones which are estimated of endothelial function declines. Age-associated remodeling of the vascular wall changes the pathophysiological of cardiovascular and Cerebrovascular diseases, thus alters the occurrence threshold, the severity and the prognosis of vascular diseases, it may contribute to many age-related diseases, including atherosclerosis (AS ).The vascular endothelium is situated at the interface between the blood and the vascular wall/tissue and is more than a protective barrier since it possesses anticoagulatory properties and generates a number of autocoids that regulate vascular tone and homeostasis. There is a intimate relationship between endothelium dysfunction and the beginning and progress of many diseases, and endothelium dysfunction exists in the early stage of many diseases. Recently, some research indicated the senescence vascular endothelial cells cultured in vitro exhibit a flattened and enlarged morphology, secrete less endothelial nitrogen monoxidum, and present a phenotype of pro-imflammation and pro-throm, it may contribute to the age-related ahterothrombotic diseases.Oxidative stress is one of the most classic mechanism of aging. Some evidences supported that reactive oxygen species ( ROS ) induce the DNA of cells injured by oxidative stress, when the DNA repair decompensation happen, the express of cell genes change, then cells enter an irreversible growth arrest and being senescence. In the research of the pathogenesis of AS, scientists discovered ROS induce the expression of some inflammation factors which take part in the process of AS independently or jointly. With advancing age, the damage induced by those factors accumulates, it may be one of the most important factors that induces the occurrence and development of vascular aging, thus to make AS more liable to happen in the aged.In the elderly, besides the age-related remodeling of vessel, another risk factor why the AS liable to happen in the aged is the age-related change of sex hormone balance. In the progress of aging, corresponding change happen in endogenous sex hormone balance. Research showed endogenous testosterone level are significantly decreased in men more than 50 years. Is it any relation between the age-related change of endogenous testosterone level and the age-related vascular remodeling in men? At present, there is few research about it, and it is lack that the research about the effects of testosterone on the senescence of vascular endothelial cells and its mechanism of action.So we induced human umbilical vein endothelial cells ( HUVECs ) senescence by hydrogen peroxide ( H₂O₂ ) at the low concentration, then detected a few senescence biomakers of HUVECs, such as the proliferation of cells and senescence associatedβgalactoside ( SA-β-gal ) activity, and the intracellular oxidative stress status and the expression of hypophosphorylated Rb protein ( a cell cycle regulating factor), and approached the aging mechanism of HUVECs, then intervened with testosterone and sex receptor antagonist, and detected the effects of testosterone on the senescence of HUVECs induced by ROS and approached its possible mechanism of action. So as to identify the effect of testosterone on the pathogeny of vascular aging, and provide new theory and experiment evidence to its mechanism of action, it may provide new target for the prevention and cure of age-related vascular diseases.[Methods]1. Culture HUVECs, the cells of passages of 4-6 were used in the experiments. Inducing HUVECs senescence by 60μmol/L H₂O₂ for 72h.2. Groups: 1) nomal control group: cultured with low serum ( 2% ) medium. 2) H₂O₂ control group: cultured with low serum medium and H₂O₂. 3) testosterone intervention group: cultured with testosterone at different concentrations ( 3nmol/L, 30nmol/L, 300nmol/L, 3μmol/L ) for 30min respectively, then added H₂O₂. 4) mechanism research groups: cultrued with estrogen receptor antagonist ICI182, 780 or androgen receptor antagonist flutamide ( 1μmol/L) for 30min respectively, and added testosterone ( 30nmol/L ) for 30min, then added H₂O₂. Or cultured with antioxygen N-acetyl-L-cysteine ( 1μmol/L) for 30min, then added H₂O₂.3. Detecting the proliferation of cells by MTT method. Detecting the SA-μ-gal activity by cells staining method. And detecting intracellular ROS level by DCF fluorescence coloration. Detecting the hypophosphorylated Rb protein expression by Western blot method[results]1. Effects of testosterone with or without sex receptors antagonists on the proliferation of HUVECs stimulated by H₂O₂.The proliferation of HUVECs in H₂O₂ was lower ( P<0.001 versus nomal control group ). And the proliferation of HUVEC in the testosterone at low concentration ( 3nmol/L, 30nmol/L and 300nmol/L ) intervention groups was higher, most significantly at the concentration of 30nmol/L ( P=0.335, P=0.058, P=0.127 versus H₂O₂ control group, respectively ), whereas it was opposite in the testosterone at the higher concentration ( 3μmol/L ) intervention group ( P<0.001 versus H₂O₂ control group ). The estrogen receptors antagonist ICI182, 170 ( P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ) rather than the androgen antagonist flutamide ( P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ) at the concentration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L.2. Effects of testosterone with or without sex receptors antagonists on the percentage of SA-β-gal staining positive of HUVECs stimulated by H₂O₂.The percentage of SA-β-gal positive cells was higher in H₂O₂ control group ( P<0.001 versus normal control group ). And the percentage of SA-β-gal positive cells was lower in testosterone at the low concentrations ( 3nmol/L, 30nmol/L and 300nmol/L) intervention groups, most significantly at the concentration of 30nmol/L ( P=0.464, P=0.094, P=0.115 versus H₂O₂ control group, respectively ), whereas it was opposite in the testosterone at the higher concentration ( 3μmol/L) intervention group ( P<0.001 versus H₂O₂ control group ). The estrogen receptors antagonist ICI182,170 ( P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ) rather than androgen antagonist flutamide ( P=0.038 versus testosterone at the concentration of 30nmol/L intervention group) at concentration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L. 3. Effects of testosterone at the physiologic concentration with or without sex receptor antagonist on the intracellular oxidant status of HUVECs stimulated by H₂O₂.The relative DCF fluorescence intensity of H₂O₂ control group was higher (P<0.001 versus nomal control group ). And the relative DCF fluorescence intensity in the NAC group was lower ( P<0.001 versus H₂O₂ control group ). And it was lower in testosterone at the concentration of 30nmol/L (physiologic concentration ) intervention group ( P=0.362 versus H₂O₂ control group ). The estrogen receptor antagonist ICI182,170 ( P = 0.002 versus testosterone at the concentration of 30nmol/L intervention group) rather than androgen antagonist flutamide ( P=0.004 versus testosterone at the concentration of 30nmol/L intervention group ) at the concerntration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L.4. Effects of testosterone at the physiologic concentration with or without sex receptor antagonist on the hypophosphorylated Rb protein expression in HUVECs stimulated by H₂O₂.The hypophosphorylated Rb protein expression was more in HUVECs of H₂O₂ control group ( P<0.001 versus nomal control group ). And it was less in the cells of NAC intervention group ( P<0.001 versus H₂O₂ control group ). And it was less in the cells of testosterone at the concentration of 30nmol/L ( physiologic concentration ) intervention group ( P=0.132 versus H₂O₂ control group ). The estrogen receptors antagonist ICI182,780 ( P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ) rather than androgen antagonist flutamide ( P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ) at the concentration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L.[Conclusion]1. Cultured the cells in low concentration of H₂O₂ ( 60μmol/L ) for 72h, increased the ROS level and the expression of hypophosphorylated Rb protein in HUVECs, its proliferation was depressed and the percentage of SA-β-gal staining positive cells was increased significantly vs the nomal control group, cells became senescence. These demonstrated that ROS plays an important role in the process of HUVECs senescence.2. Testosterone at low concentrations ( 3nmol/L, 30nmol/L and 300nmol/L) had the trend of delaying the depression of proliferation and the increase of SA-6-gal staining positive cells percentage of HUVECs stimulated by H₂O₂, whereas higher concentration (3μmol/L) had opposite effect. These demonstrated that testosterone modulate the senescence of HUVECs induced by H₂O₂ in a dose-related manner.3. Estrogen receptor antagonist ICI182,170 rather than androgen antagonist flutamide inhibited the delaying trend induced by testosterone at the concentration of 30nmol/L, such as increased the intracells ROS level and hypophosphorylated Rb expression in HUVECs, and depressed the proliferation and increased the positive cells percentage of SA-6-gal staining of HUVECs stimulated by H₂O₂. We deduced that testosterone at the physiologic concentration may transform to estrogen which acts on estrogen receptor, resulting in decreasing the intracellular oxidative stress and hypophosphorylated Rb protein expression, then finally interfering in the cell cycle and cell senescence.