The Central Oxidative Stress Involving In Sympathetic Hyperactivity Of Ariectomized Rats

Menopause is mainly due to declining of estrogen level and the function recession of ovary. Estrogen influences the physiological function of body system as an important messenger. A lower percentage of women than men have cardiovascular disorder before menopause. The prevalence and severity of cardiovascular disorders, including hypertension and chronic heart failure, increases more markedly along with increasing age in postmenopausal women. It is indicate that estrogen can save organism from cardiovascular dysfunction. But the mechanism remains unclear. The effect of hormone replacement therapy is not satisfied.High blood pressure and increased sympathetic outflow contribute to cardiovascular diseases. It was a commonly-held belief that Rostral ventrolateral medulla (RVLM) is known as a cardiovascular center in the brainstem, where presympathetic neurons for the maintenance of sympathetic outflow and blood pressure reside. Over-excitement of the nervous in RVLM is hallmarks of sympathetic overactivity and inhibition of presympathetic neurons activity can effectively reduce sympathetic outflow and lower blood pressure.Oxidative stress results from an imbalance of generation over degradation of the reactive oxygen species (ROS), which leads to tissue damage when the organism is subjected to noxious stimuli. Oxidative stress exerts an important role in modulation the cardiovascular function both systemic circulation and in brain. Researches of peripheral cardiovascular system have documented that oxidative stress increases in postmenopausal woman and animal. Estrogen promotes a antihypertensive effect by decreasing oxidative stress activity. Other study indicate that the oxidative stress in the rostral ventrolateral medulla (RVLM), a key region for control of sympathetic outflow, has been demonstrated to be a major contributor to high blood pressure and increased sympathetic outflow in cardiovascular diseases. It is still unclear whether oxidative stress in the RVLM contributes to postmenopause -induced cardiovascular dysfunctions of OVX rates and if a loss of estrogen contributes to produce the oxidative stress in the RVLM;In animal models of hypertension and heart failure, the expression of NADPH which generate superoxide anion radical (·O2-) increases. The expression of SOD which degrade O2- decreases.·O2" can increase neuronal excitability through influencing the ion channels on cell membranes and intracellular signal transduction pathway, leading to sympathetic hyperactivity and high blood. Therefore, our concerns are whether estrogen can reduce the level of oxidative stress within the RVLM to control sympathetic outflow and blood pressure, and whether SOD or NADPH is involved in this central enhanced oxidative stress mechanism. We hope that the evidence from this work would provide a theoretical foundation for prevention and development of therapeutic strategies for post-menopause -induced hypertension.MethodsAnimal experiments use Sprague-Dawley (SD) female rats, weighting from 200 to 250g. OVX model was produced by ovariectomized as well as controls receiving sham operation. Four groups of SD rats were studied:sham+Veh,17βestradiol replaced sham (sham+E2), OVX+Veh, and 17βestradiol-replaced OVX (OVX+E2). Rats were ovariectomized at 4 weeks of age and were treated daily with 17β-estradiol (30ug/kg) (OVX+E2 group and sham+E2 group) for 4 weeks before sacrifice. The animal’s blood pressure, heart rate,24-h urinal excretion of NE, and oxidative stress within the RVLM were observed. DHE staining techniques were used to detect changes the superoxide anion content in the RVLM. Western Blot analyses were used to detect the expression of NADPH oxidase, and SOD.RESULTS1. OVX model assessmentAs expected, the relative uterine weight(0.0004±0.0001 vs 0.0018±0.0004mg/g) and plasma estrogen concentration (9.667±0.374 pmol/L versus 5.358±0.165pmol/L) of rats was significantly decreased in the OVX+Veh group as compared to the sham group. Estrogen replacement therapy increased plasma estrogen concentration and relative uterine weight.2. The cardiovascular effect of estrogen on OVX ratsAt the time of sacrifice, the mean arterial pressure, heart rate and renal sympathetic nervous activity of rats were significantly increased within 6 weeks after receiving ovariectomy procedure, the effect of which was prevented completely by administering estrogen subcutaneously for 4 weeks. The elevated level of NE in 24-h urine in OVX rats which indirectly represented the increase of sympathetic nervous activity evoked by ovariectomy, which attenuated by estrogen replacement therapy3. Detection oxidative stress in RVLMTo elucidate the effect of estrogen on ROS production in RVLM, the representative images of the RVLM with fluorescent labeling (DHE) was used to detect ROS production. The results of DHE fluorescent staining revealed that the level of ROS in the RVLM was significantly higher in OVX+Veh group than in sham+Veh group, the ROS production within RVLM of OVX rats with estrogen was reduced significantly as compared to OVX rats. The results of detecting ROS production within RVLM via Lucigenin chemiluminescence quantitative detection kit were consistent with by DHE fluorescent probe.4. E2 downregulated the protein expression of NADPH oxidase (Nox4) and upregulated SODl protein in the RVLM.To further determine the source of ROS in RVLM induced by ovariectomy and underlying mechanism of reducing ROS production by supplementing of estrogen within RVLM. Western blot analysis demonstrated that ovariectomy procedure increased Nox4 which the subunit of NADPH oxidase expression, while reducing expression of SODl in RVLM, the effects of which were abrogated or significantly attenuated by estrogen treatment.ConclusionEstrogen decreases sympathetic outflow by inhibiting oxidative stress in rostral ventrolateral medulla of ovariectomized rats.