The Protective Effect And Preliminary Mechanism Of Estrogen On Improving Ovariectomy-induced Damages Of Vascular Endothelial Cells

Objective:1. To observe whether ovariectomy could induce the damages of aortic endothelium-dependent vasodilation and pathological changes in thoracic aorta of female Sprague-Dawley rats, and whether the corresponding vascular protective effects of estrogen therapy were related with alleviating endoplasmic reticulum stress.2. To study whether estrogen deficiency could induce oxidative stress (OS) and endoplasmic reticulum stress (ERS) in human umbilical vein endothelial cells (HUVEC), and whether the treatment effect of estrogen was related to the relief of OS and ERS.Methods:1. In vivo studies:12 weeks old of female Sprauge Dawley rats were divided into control (CON), ovariectomized (OVX) and OVX with estrogen treatment group (OVX+EST) randomly. Ovariectomized rats were used as estrogen deficiency model, and OVX+ES rats were treated with estrogen via gavage (β-estradiol 0.06mg/kg once daily), all rats were feeded for six months. Body weight was measured weekly, and the levels of serum lipid levels including TQ TC, HDL-C and LDL-C were detected at the end of experiment. HE staining was used to detect the pathological changes of thoracic aorta. Oil-red O staining was used to determine lipid deposition in thoracic aorta. Isometric tension test was used to observe the changes of vasodilation induced by cumulative concentrations of ACh.The mRNA and protein expression of GRP78 and CHOP in thoracic aorta, which were considered as ERS markers, were evaluated by RT-PCR and Western blot respectively.2. In vitro studies:Human umbilical vein endothelial cells (HUVEC) were divided into six groups, such as normal control group (CON), estrogen deficiency group (ES-D), low concentration of estrogen addition group (L-ES), medium concentration of estrogen addition group (M-ES), high concentration of estrogen addition group (H-ES) and positive control group (TM). Reacitve oxygen species were detected with flow cytometry. The mRNA and protein expression of GRP78 and CHOP were assessed by RT-PCR and Western blot respectively. Primary aortic endothelial cells of female SD rats (PRAEC) were isolated and cultured in vitro, and were also divided into six groups for CON, ES-D, L-ES, M-ES, H-ES, TM. The mRNA expression of GRP78 and CHOP were detected by RT-PCR.Results:1. In vivo studies:Compared with that in CON: ①body mass of OVX rats for six months were increased obviously, the levels of serum TC, TG and LDL-C were increased significantly, HDL-C was decreased obviously; ②HE staining showed that the thoracic aortic wall in OVX group was irregularly thickened, elastic fiber arrangement was disordered and local appeared rupture phenomenon; Oil red O staining showed a significant lipid deposition in the thoracic aortic wall of OVX group;③The aortic endothelium-dependent vasodilation of OVX group decreased significantly; ④the mRNA and protein expression of GRP78, CHOP in thoracic aorta of OVX group were significantly increased. Compared with that in OVX group, estrogen treatment could significantly decrease the body weight and invert lipid levels.There was a significant improvement of pathological changes and endothelium-dependent relaxation in the thoracic aorta of OVX+EST group. The mRNA and protein expression of GRP78 and CHOP in OVX+EST group were all reduced obviously.2. In vitro studies:Compared with that in the CON group, levels of reacitve oxygen species in ES-D group was obviously increased. The mRNA expression of GRP78 and CHOP in HUVEC, as well as the protein expression, were also significantly upregulated in ES-D group. Compared with the ES-D group, ERS was eased after incubation with different concentrations of estrogen, and the most obvious effect appeared in the H-ES group. The similar mRNA expression of GRP78 and CHOP were presented in PRAEC cells.Conclusion:1.6 months after ovariectomy successfully induced the rat model of estrogen deficiency and led to endoplasmic reticulum stress in thoracic arota.2. The mechanism of estrogen therapy on improving function of vascular endothelial cells may be related with the alleviation of oxidative stress and endoplasmic reticulum stress.