雌二醇減緩大鼠實驗性蜘蛛膜下腔出血引發腦血管痙攣的角色

The role of 17beta-estradiol in attenuating experimental subarachnoid hemorrhage-induced cerebral vasospasm in rats

施慧娟(Huei-Chuan Shih)

指導教授 ： 洪純隆

高雄醫學大學/醫學院/醫學研究所/博士(2008年)

摘要

蜘蛛膜下腔出血(subarachnoid hemorrhage, SAH)後的腦血管痙攣是自發性顱內動脈瘤破裂的病人特有的後遺症之一。其高的死亡率與神經性的損傷，被深信與SAH後的腦血管痙攣有密切相關。在臨床上血管擴張劑被廣泛使用在緩解痙攣性的腦血管；然而這些藥物的全身性血管擴張作用可能進而使腦血流量減少。在停經前的婦女，女性賀爾蒙雌二醇(17??-estradiol,E2)，是透過活化內皮素一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)，來達到血管保護的作用，但E2並不會影響其心臟血管正常的功能，E2可能是一種治療SAH後的腦血管痙攣的理想藥物。我們將一條充滿E2的矽膠管包埋於公鼠皮下，使其血清中E2濃度能維持在生理範圍內達七天。實驗性SAH引發的腦血管痙攣，且會抑制基底動脈eNOS的表現及引起誘導型一氧化氮合成酶(inducible nitric oxide synthase, iNOS)的表現。外源性的E2能緩解公鼠SAH後的腦血管痙攣經由恢復基底動脈eNOS的表現及抑制iNOS的表現。研究顯示iNOS扮演很重要的角色在SAH後的腦血管痙攣， 利用Aminoguanidine可阻斷iNOS的表現，且緩解SAH後的腦血管痙攣。所以E2抑制SAH後基底動脈iNOS表現的角色研究，成為我們實驗主要的議題。利用電泳遷移率分析(Electrophoresis mobility shift assay, EMSA)研究顯示，在SAH組iNOS特異性NF?羠核內DNA結合活性顯著增強，但SAH加上E2處理組其結合活性則降低。我們實驗顯示：E2處理組沒有影響細胞質NF?羠的核轉位。利用雙重免疫沉澱(Immounoprecipitation, IP)研究顯示，SAH加上E2處理組在核內會增加雌激素接受器(estrogen receptor,ER)/NF?羠免疫複合體的量。我們相信E2抑制SAH引起iNOS基因表現是經由干擾NF?羠的轉錄活性。
E2的效果可緩解SAH後的腦血管痙攣，提昇我們探索SAH的作用在腦血管ER的表現。我們顯示在正常狀態下公鼠的基底動脈有兩種ER亞型(ER?悀咶R??)皆會表現，且SAH後的基底動脈ER?悛漯穛{顯著上升，但ER?猁漯穛{沒有改變。我們進一步顯示E2緩解SAH後的腦血管痙攣，經由恢復eNOS的表現及抑制iNOS的表現，並透過ER?悃抰鄔妐纁|。
SAH引發腦血管痙攣及同時誘發某些反應為了對抗致命性的作用，在SAH後的基底動脈的ER?悛穛{上升是SAH所引起的反應之一，如此可能解釋外源性E2的效果緩解SAH腦血管後的痙攣。ER?悛穛{的增加可能確保ER足量的供給，能同時使eNOS恢復是透過活化的ER?悝@用於eNOS基因的啟動子上；而抑制iNOS則是經由干擾NF?羠的轉錄活性，也是透過活化的ER?恁C我們的結論是：SAH後ER?悛漯穛{上升，是一種保命的過程，且確保外源性E2能完全發揮其保護血管的作用；ER?悝@用劑可替代E2，是一種治療SAH後的腦血管痙攣具有療效的候選藥物。

關鍵字

蜘蛛膜下腔出血 ； 雌二醇 ； 誘導型一氧化氮合成酶內皮素一氧化氮合成酶雌激素接受器

並列摘要

Post subarachnoid hemorrhage (SAH) cerebral vasospasm is one of the characteristic consequences in patients who suffer from spontaneous rupture of intracranial aneurysm. The high mortality and neurological morbidity are believed to be closely related to the post SAH cerebral vasospasm. Vasodilators are currently used to relieve spastic cerebral blood vessels in clinical practice; however systemic vasodilatory effects of these agents may further compromise the cerebral blood flow. 17??-estradiol (E2) is a female sex hormone that exerts its vasoprotective effects through activation of endothelial nitric oxide synthase (eNOS) in pre-menopausal women without compromising their cardiovascular functions. E2 becomes a potential candidate for the treatment of post SAH cerebral vasospasm. We implanted a Silastic tube filled with E2 in male rats and their serum levels of E2 could be maintained within the physiological range for at least 7 days after implantation. Our previous report demonstrated that experimental SAH induced cerebral vasospasm, suppressed eNOS expression and induced inducible nitric oxide synthase (iNOS) expression in the basilar artery. Exogenous E2 could relieve post SAH cerebral vasospasm in male rats, restore eNOS expression and suppress iNOS expression in the basilar artery. We also found that iNOS played an important role in triggering post SAH cerebral vasospasm and blockade of iNOS expression by aminoguanidine relieved post SAH cerebral vasospasm. So the study of the role of E2 in suppressing iNOS expression in the basilar artery after SAH became the main theme of our experiment. The electrophoretic mobility shift assay (EMSA) study demonstrated that the nuclear DNA binding activity of iNOS specific NF?羠 in SAH group was significantly enhanced and the binding activity was reduced in the E2 treated group after SAH. We showed that nuclear translocation of cytosolic NF?羠 was not affected by E2 treatment. The double immunoprecipitation study demonstrated the increased amount of estrogen receptor/NF?羠 immunocomplex in the nuclear fraction of the E2 treated group after SAH. We believe that E2 inhibits SAH-induced iNOS gene expression by interfering with the NF?羠 transactivation.
The effectiveness of E2 in relieving the post SAH cerebral vasospasm raised our interest in exploring the effect of SAH in the expression of estrogen receptors in cerebral blood vessels. We demonstrated both estrogen receptor subtypes (ER?? and ER??) are expressed in the basilar artery under normal condition in male rats and ER?? expression is significantly upregulated after SAH in the basilar artery, but not ER?? expression. We further showed that E2 relieves post SAH cerebral vasospasm, restores eNOS expression and suppresses iNOS expression through an ER?? dependent pathway.
SAH induces cerebral vasospasm and at the mean time it triggers certain reactions in order to counteract its detrimental effects. Upregulation of ER???nexpression in the basilar artery after SAH is one of the responses to SAH. This may explain the effectiveness of exogenous E2 in relieving post SAH cerebral vasospasm. The increased ER?? expression may guarantee the adequate supply of estrogen receptors for both eNOS restoration through activated ER?? acting on eNOS gene promotor and iNOS suppression through interfering NF?羠 transactivation by activated ER??. In conclusion, upregulation of ER?? after SAH is a kind of life saving process and it ensures exogenous E2 can fully exert its vasoprotective effects. ER?? agonist instead of E2 is a promising candidate for the treatment of post SAH cerebral vasospasm.

並列關鍵字

subarachnoid hemorrhage ； 17beta-estradiol ； inducible nitric oxide synthase ； endothelial nitric oxide synthase ； estrogen receptor

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