Effects Of 17 Beta- Estradiol And 2-Methoxyestradiol On The State Of Oxidative Stress In Hypoxic Pulmonary Hypertensive Rats

Objective: Pulmonary arterial hypertension(PAH) is a group of clinical syndromes triggered by different etiology and pathogenesis characterized by a continual increase in pulmonary vascular resistance, which leads to pulmonary circulation higher resistance. Excessive production of reactive oxygen species(ROS) caused by hypoxia and oxidative stress caused by insufficient clearance are the basis for a variety of pathophysiological changes. ROS regulate a variety of signaling pathways through direct or indirect way, which lead to pulmonary vascular remodeling ultimately. Superoxide dismutase(SOD) is the first barrier against ROS, and manganese superoxide dismutase(Mn SOD) is the key mitochondrial antioxidant enzyme, is an important component in intracellular antioxidant system.Maintain pulmonary vascular tension depended on the balance of oxidation/antioxidant enzyme system, which has important influence in the progress of PAH. This study aims to establish a model of ovariectomized rats with long-term chronic PAH to explore the effects of 17 beta-estradiol( E2) and 2-methoxyestradiol(2ME) on the level of oxidative stress and expression of mitochondrial manganese superoxide in hypoxic pulmonary hypertensive rats.Method:1 Establishing a model: A total of 80 healthy female Sprague-Dawdley( SD) rats were divided randomly into 8 groups(n=10 each) namely, sham group, ovariectomy group(OVX), sham+hypoxia group, OVX+hypoxia group OVX+E2 group, OVX+hypoxia+E2 group, OVX+2ME group and OVX+hypoxia+2ME group. The sham group were only opened the abdominal cavity of the rats and found ovaries, which were not treated and directly returned.The OVX group were removed the bilateral ovaries.The sham+hypoxia group were placed into a low-oxygen environment(24 hour,8 weeks)after sham. The OVX+E2 group received a subcutaneous injection of E2(20 μg· kg-1·d-1).The OVX+hypoxia+E2 group had an injection of E2 and was placed into a low-oxygen environment. The OVX+2ME group received a subcutaneous injection of 2ME(240 μg· kg-1·d-1).The OVX+hypoxia+2ME group had an injection of 2ME and was placed into a low-oxygen environment.2 Changes in pulmonary artery pressure of long-term hypoxic rats: The mean pulmonary artery pressure(m PAP) was measured after bloodletting. Mean pulmonary artery pressure(m PAP) was measured with right heart catheterization, and weighing method was used to calculate right ventricular hypertrophy index(RVHI). HE staining was employed to observe hypoxic pulmonary arterial remodeling(HPSR).3 After we applied double staining with uranyl acetate and lead citrate, ultrastructural changes in various types of cells of lung tissues were observed under the transmission electron microscope.4 We used Fenton reaction and Griess color rendering principle assessment the level of ROS.5 Xanthine oxidase method was used to detect activity of SOD 、Cu/Zn SOD and Mn SOD in serum.6 Mn SOD m RNA expression in lung tissues was detected using RT-PCR method.7 Mn SOD protein expression in lung tissues was detected using Western blotting.Results:1 The general condition of rats: In hypoxia group, with prolonged time under hypoxic condition, the rats had dull coat colcr, dark iris, shortness of breath, reduced water intake and decreased activity, ultimately lying still most of the time. There was no death during 8 weeks under hypoxic condition. Compared with sham group, sham+hypoxia group and OVX+hypoxia group weight gain were obviously slow. OVX+hypoxia+E2 group and OVX+ hypoxia+2ME group weight gain were slow. There were no significant changes in weight gain of rats from ovariectomy group, OVX+E2 group, OVX+2ME group.2 Changes in pulmonary artery pressure of rats: Compared with sham group, mean pulmonary artery pressure and RVHI were significantly increased in sham+hypoxia group and OVX+hypoxia group with pulmonary arteriolar wall thickening lumen narrowing significantly. The morphological changes of OVX+hypoxia+E2 group and OVX+hypoxia+2ME group were lighter, and the RVHI had no statistic difference with controls, but their m PAP were higher than sham operation and other control groups.3 Morphological changes in pulmonary small vessels in rats: In sham group, endothelial cells in pulmonary arterioles were flat and continuous, uniformly distributed and consistent in size and thickness, without edema or necrosis. Pulmonary arterial wall structure was normal with a large lumen. In sham+hypoxia group, pulmonary arterial wall was apparently thickening; there was structural disorder, obvious thickening of red dye part, increased smooth muscle cells(SMC) in medial layer, pulmonary artery media thickness(PAMT), as well as narrowing and partially collapsed lumen; OVX+hypoxia group was more significant; wall structure of pulmonary arterioles in OVX+hypoxia+E2 group and OVX+hypoxia+2ME were better than that in OVX+hypoxia group but worse than that in sham group; wall structure of pulmonary arterioles was roughly normal in ovariectomy group, OVX+E2 group and OVX+2ME group.4 Ultrastructural changes in lung tissues of rats: In sham group, on epithelial cells of Type Ⅱalveoli of rats, there were more microvilli, but no shedding, no fracture in lamellar body, small number of mitochondria, slight swelling, and no crest break and disappearance. In sham+hypoxia group, there was cellular swelling in epithelial cells of TypeⅡalveoli, significantly reduced and less neatly arranged microvilli shedding on the surface; weight of lamellar body was reduced with visible fracture; there was mitochondrial swelling and most cristae breaks; membrane fusion disappeared and blurred, with karyopyknosis. In OVX+hypoxia group, microvilli of rats were shedding and were significantly reduced; significantly reduced lamellar bodies with visible fracture, degranulation and vacuoles could be seen; mitochondrial swelling was visible and cristae broke and membrane fusion disappeared. In OVX+hypoxia+E2 group and OVX+hypoxia+2ME, epithelial cells of TypeⅡalveoli were swelling, with moderately reduced and less neatly arranged microvilli shedding; lamellar body was moderately reduced with visible fracture; there was mitochondrial swelling and cristae breaks; membrane fusion disappeared, being myeloid. Mild ultrastructural changes could be seen in rats in ovariectomy group, OVX+E2 group and OVX+2ME group, but it was generally normal.5 SOD, Cu/Zn SOD and Mn SOD activity changes in serum: Compared with sham group, SOD activity of rats in ovariectomy group, sham+hypoxia group and OVX+hypoxia group were significantly reduced; reduction had progressive increase from these 3 groups, and OVX+hypoxia group was the most significant; In OVX+hypoxia+E2 group and OVX+hypoxia+2ME group the Mn SOD activity were reduced; in OVX+E2 group and OVX+2ME group, there were no obvious different. The Mn SOD change trend likes Mn SOD. There was no statistical difference between groups of Cu/Zn SOD active significance.6 The ROS level in serum: Compared with the sham group, the level of ROS in sham+hypoxia group, OVX+hypoxia group increase markedly; OVX+hypoxia+E2 group and OVX+hypoxia+2ME group increase. However, the OVX group, OVX+E2 group and OVX+2ME group had no obvious change.7 Mn SOD m RNA and protein expression in lung tissues: Compared with those in sham group, the expressions in ovariectomy group, sham+hypoxia group and OVX+hypoxia group were significantly reduced, and reduction had progressive increase from these 3 groups, and OVX+hypoxia group was the most significant. In OVX+hypoxia+E2 group and OVX+hypoxia+2ME group the Mn SOD activity were reduced, and OVX+hypoxia+2ME group reduced markedly; in OVX+E2 group and OVX+2ME group, there were no obvious different.Conclusisons:1 In long-term continously hypoxic environment, ROS levels in the serum of rats significantly increased and SOD、Mn SOD activity was decreased significantly, and Mn SOD protein and m RNA expression in lung tissues were obviously lowered.2 Ovariectomy group rats, Mn SOD activity of serum had no significant changes, but Mn SOD protein and m RNA expressions in lung tissues were significantly lowered.3 Compared with sham+hypoxia group and ovariectomy group rats, SOD、Mn SOD activity in hypoxic environment was significantly decreased in ovariectomized rats, and reduction in Mn SOD protein and m RNA expressions in lung tissues was the most significant, but ROS levels increased significantly.4 The E2 and 2ME can inhibit the ROS level, increase the activity of Mn SOD and expression, which is beneficial to balance the oxidation/ antioxidation, relieve oxidative stress levels, reduce pulmonary arterial hypertension. And effect of 2ME in increasing the Mn SOD is more significantly and with no difference on the level of ROS.