| Backgrounds and ObjectivesStudies have reported that the morbidity and fatality rate of cardiovascular diseases in young women are remarkably lower than that in young men group,but in women who have menopaused,the morbidity and fatality rate of cardiovascular diseases have been increased significantly.Compared with men,the morbidity and fatality rate of cardiovascular diseases in postmenopausal women are same and the incidence of moderate and serious health problems such as hypertension,heart failure and stroke is 2-3 times higher than that in premenopausal women.Therefore,it is significant to explore the risk factors of the occurrence and development of cardiovascular events in postmenopausal women and to provide prevention and treatment strategies for women's life and health.Sympathetic hyperactivity is one of the main characteristics of postmenopausal cardiovascular disease,and the degree of sympathetic hyperactivity has a direct impact on the occurrence,development and prognosis of postmenopausal cardiovascular events.RVLM is a main center to control the level of sympathetic activity,because it has been found that the damage of RVLM nucleus will lead to the complete disappearance of sympathetic activity.The renin angiotensin system in postmenopausal RVLM is closely related to central sympathetic hyperactivity and peripheral hypertension.Ang II acts on the AT1 receptor on the presympathetic neurons in the RVLM to regulate the excitability of the presympathetic neurons.The AT1 receptor is a kind of GPCR.It has been documented that ?-arrestin is able to inhibit GPCR signal transduction,mediate the endocytosis of AT1 receptor,and reduce the sensitivity of AT1 receptor,which plays its role in cardiovascular protection.It has been reported that ?-arrestin can reduce ROS production and oxidative stress level by decreasing NADPHase in myocardial cells.In addition,it has been shown that ?-arrestin1 can also plays its cardiovascular protective role through its biased signaling pathway in preeclampsia mice model,and reduces preeclampsia hypertension.There are two main subtypes of ?-arrestin in mammals,namely ?-arrestin1 and ?-arrestin2.In addition to mediating the desensitization,endocytosis and degradation of G-protein-coupled receptor,many researchers have found that ?-arrestin can also bind with downstream signal molecules of GPCR,and they can form an unique biased signal transduction pathway.This effect is differential from the traditional signal pathway mediated by GPCR.Based on its unique biased signaling pathway,?-arrestin has become a hot topic in recent years,and has become a key molecule in the field of cardiovascular function regulation.The expression of ?-arrestin1 in the RVLM of SHR is significantly less than normatensive rats,which results in sympathetic hyperactivity and hypertension.Overexpression of ?-arrestin1 in the RVLM has the effect on reducing sympathetic hyperactivity and hypertension.We found that the expression of ?-arrestin1 in the RVLM of postmenopausal rats was significantly decreased,and it was significantly increased after estrogen supplementation.So if the decrease of estrogen level in postmenopausal rats affects cardiovascular function by affecting the expression of ?-arrestin1 in RVLM? There is no relative data to be reported.Therefore,our major objective is to determine the role of ?-arrestin1 in the RVLM in postmenopausal-associated hypertension.It has been shown that the transcription factor AP-1 can promote the transcription of ?-arrestin gene,and this transcription factor can be regulated by estrogen signaling pathway.In the classical estrogen signaling pathway,Estrogen,as a steroid hormone,can combine with its nuclear receptor through cell membrane to form complex,and then combine with specific target gene to regulate its expression.In the non-classical estrogen signaling pathway,after the transcription factor AP-1 combining to the target gene,estrogen can directly binds to AP-1,and then indirectly plays its regulatory role in the targeted gene.Therefore,our next goal is to determine whether AP-1 in the RVLM mediates the regulation of estrogen on ?-arrestin1 in postmenopausal rats.Taken together,our overall goal is to determine whether the decrease of estrogen level in postmenopausal women affects on the expression of ?-arrestin1 in the RVLM,thereby increases the levels of sympathetic hyperactivity and blood pressure level;and to further determine whether AP-1 mediates the effect of estrogen on the expression of ?-arrestin1 in the RVLM.MethodsThe rats were treated consecutively with estrogen(0.9% sodium chloride solution,30 ?g/kg/day).The duration of estrogen supplementation was 4 weeks.The blood pressure and heart rate of rats were measured by intubation of femoral artery under anesthesia.The renal sympathetic nerve was separated to be recorded.The plasma of rats was collected to detect the content of estrogen and noradrenaline by ELISA.The expression of ?-arrestin1 and AP-1 in the RVLM is detected by Western blot.The expressions of ?-arrestin1 and AP-1 were inhibited by injecting adeno-associated virus into the RVLM,and ?-arrestin1 was inhibited by injecting si RNA into the RVLM.Results1.The expression of ?-arrestin1 was decreased in the RVLM of OVX rats,and estrogen supplementation increased the expression of ?-arrestin1.The expression of ?-arrestin1 protein in the RVLM of OVX rats was decreased significantly(156.1±14.1 vs.119.9±10.3 mm Hg,n=5,P<0.05),while it was upregulated by estrogen supplemented(23.99±1.39 vs.13.17±1.57 %max,n=5,P<0.05).So,it is concluded that the expression of ?-arrestin1 protein in the RVLM decreased significantly due to the decrease of estrogen level in OVX rats,while the supplement of estrogen can promote the expression of ?-arrestin1 protein in the RVLM.2?Over expression of ?-arrestin1 in the RVLM of OVX rats reduced sympathetic hyperactivity and hypertension in postmenopausal rats.In the RVLM of OVX rats,?-arrestin1 was overexpressed by microinjection of adeno-associated virus carrying arrb1 gene into the RVLM.It was found that compared with the adeno-associated virus carrying GFP gene,the renal sympathetic nerve activity(12.82±0.87% vs.19.22±1.66%max,n=5,P<0.05)and blood pressure(98.08±3.99 vs.136.6±6.38 mm Hg,n=5,P<0.05)of OVX rats injected with arrb1 gene into the RVLM were significantly lower.It is suggested that the over expression of ?-arrestin1 protein in the RVLM of OVX rats can significantly reduces the level of sympathetic activity and blood pressure.3.The inhibition of ?-arrestin1 expression in the RVLM of OVX rats reduced the protective effect of estrogen on cardiovascular function.OVX rats injected si RNA of ?-arrestin1 in the RVLM showed the higher levels of renal sympathetic nerve activity(10.47±0.84 vs.19.68±0.97 %max,n=5,P<0.05)and blood pressure(104.8±1.83 vs.138.1±5.348 mm Hg,n=5,P<0.05).It is suggested that ?-arrestin1 inhibition in the RVLM of OVX rats prevents the protection of estrogen on cardiovascular function.4.AP-1s inhibition in the RVLM of OVX rats abolished the effect of estrogen on the expression of ?-arrestin1?The expression of ?-arrestin1 in the RVLM of OVX rats was inhibited by adeno-associated virus injection.The expression of ?-arrestin1 in the RVLM of OVX rats was not significantly changed after estrogen supplementation.It is suggested that estrogen promotes the expression of ?-arrestin1 in the RVLM through AP-1.ConclusionThe expression of ?-arrestin1 in the RVLM of postmenopausal rats was decreased significantly,which may be associated with estrogen level.This mechanism is responsible to the hyperactivity of sympathetic nerve and the increase of blood pressure in postmenopausal rats.Furthermore,the transcription factor AP-1 is suggested to mediate the regulation of estrogen on the ?-arrestin1 expression in the RVLM... |
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