雌激素抑制檸檬酸亞鐵增加小鼠腦血管內皮細胞誘導型一氧化氮上 升之分子機制探討

Molecular Mechanisms Underlying Estrogen Reduces Ferrous Citrate-Induced NOS2 Up-regulation in Mouse Cerebral Endothelial Cells

陳莉菁(Li-Ching Chen)

指導教授 ： 李文森

臺北醫學大學/醫學院/醫學科學研究所/博士(2014年)

https://doi.org/10.6831/TMU.2014.00088

摘要

出血性中風造成腦損傷處可見紅血球及其代謝產物血紅蛋白、血紅素及鐵的堆積。先前之研究指出蜘蛛膜下腔出血 (subarachnoid hemorrhage; SAH) 會造成高濃度的二價亞鐵 (ferrous) 釋出，進而引起腦血管痙攣。過去利用老鼠蜘蛛膜下腔出血動物模式的研究指出增加第二型一氧化氮合成酶 (nitric oxide synthase 2; NOS2) 在腦血管組織的表現是造成腦血管痙攣之重要因子，然而對於出血如何增加第二型一氧化氮合成酶的表現之分子機轉尚未十分明白。因此，利用檸檬酸亞鐵 (ferrous citrate; FC)來刺激小鼠腦血管內皮細胞(cerebral endothelial cells; CEC)以模擬蜘蛛膜下腔出血時高鐵濃度對血管內皮細胞的反應來探討蛛網膜下腔出血如何引發NOS2表現增加的相關議題。利用免疫細胞化學染色技術 (immunocytochemical staining technique)，我們首先證實了小鼠腦血管內皮細胞可表現第二型一氧化氮合成酶。而且投予檸檬酸亞鐵 (100 μM)可增加小鼠腦血管內皮細胞內活性氧自由基 (reactive oxygen species; ROS)的濃度、核因子κB (nuclear factor kappa-light-chain-enhancer of activated B cells; NFκB)由細胞質轉移至細胞核的量及核因子結合到第二型一氧化氮合成酶啟動子的量，進而造成第二型一氧化氮合成酶的信使核糖核酸 (mRNA) 和蛋白表現增加。然而事先投予細胞活性氧清除劑 N-乙酰半胱氨酸(N-acetyl-cysteine; NAC）則可抑制這些由檸檬酸亞鐵所引發的作用。在本研究裡，我們證實了在第二型一氧化氮合成酶啟動子上有兩個先前預測的核因子κB 結合位點分別是在-1529 bp至-1516 bp及-1224 bp至-1210 bp的範圍。有趣的是，核因子κB結合位點參與了FC-NOS2激活的轉錄活性。然而於小鼠腦血管內皮細胞細胞中，位於-1529 bp至-1516 bp處的核因子κB結合位點比位於-1224 bp至-1210 bp的結合位點發揮更大的作用。上述的發現闡述了檸檬酸亞鐵增加第二型一氧化氮合成酶在小鼠腦血管內皮細胞表現的可能分子機制。
接著，我們利用這種細胞模式來探討雌二醇（estradiol; E2）對於蜘蛛膜下腔出血引發的血管痙攣的保護作用。先前利用二次注射血液引發蜘蛛膜下腔出血的動物模式研究發現雌二醇可透過抑制第二型一氧化氮合成酶表現而顯著的減弱蛛網膜下腔出血引起的血管痙攣。本研究發現檸檬酸亞鐵會刺激第二型一氧化氮合成酶2的上升主要是因透過增加活性氧自由基的產生，而增加核因子κB由細胞質轉移至細胞核內。我們進一步探討雌二醇如何調控檸檬酸亞鐵刺激第二型一氧化氮合成酶增加的影響。研究發現，給予小鼠腦血管內皮細胞100 nM濃度的雌二醇時會減少由檸檬酸亞鐵誘導形成的活性氧自由基產生及第二型一氧化氮合成酶的信使核糖核酸和蛋白的表現量增加的現象。 此外，雌二醇也抑制了因檸檬酸亞鐵的誘導而增加小鼠腦血管內皮細胞的IκBα磷酸化、核因子κB的細胞核內遷移、核因子κB結合到第二型一氧化氮合成酶啟動子、以及第二型一氧化氮合成酶啟動子的量。然而使用RNA干擾模式 (siRNA) 阻斷雌激素受體β（ERβ），會抑制雌二醇預防檸檬酸亞鐵刺激造成的 第二型一氧化氮合成酶 信使核糖核酸和蛋白質表現增加的現象，而抑制ERα的表現則無此現象。依據現今研究的結果，我們證實了雌二醇抑制第二型一氧化氮合成酶基因表達是藉由干擾核因子κB的細胞核內遷移和核因子κB結合到第二型一氧化氮合成酶啟動子上。
本論文的研究結果或許可提供臨床上藉由調控第二型一氧化氮合成酶的表現為因蜘蛛網膜下腔出血引發血管痙攣患者提供適用的預防或治療策略。

關鍵字

雌激素、檸檬酸亞鐵、誘導型一氧化氮

並列摘要

Hemorrhagic stroke causes leakage of red blood cells, which converts to hemoglobin, heme, and iron accumulated at the bleeding brain areas. High concentration of ferrous iron from subarachnoid hemorrhage (SAH) induces cerebral vasospasm. Previous studies indicate that the nitric oxide synthase 2 (NOS2) of the brain vascular tissue in experimental SAH rats is a critical factor for inducing cerebral vasospasm. But, the underlying molecular mechanisms remain to be elucidated. Initially, ferrous citrate (FC) complexes (100 μM) were added to the primary cultured mouse cerebral endothelial cell (CEC) to mimic the SAH conditions and to address the issue how SAH-induced NOS2 up-regulation. Using immunocytochemical staining technique, this study demonstrated that NOS2 was expressed in the cultured CEC. Treatment of the CEC with FC induced increases of the intracellular level of ROS (reactive oxygen species), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) nuclear translocation as well as NFκB binding onto the NOS2 promoter, and the levels of NOS2 mRNA and protein. These effects were abolished by pre-treatment of the cells with N-acetyl-cysteine (NAC), a glutathione precursor. In the present study, two previously predicted NFκB binding sites were confirmed in the NOS2 promoter within the range from of -1529 bp to -1516 bp and from -1224 bp to -1210 bp. Interestingly, both NFκB binding sites are involved in the FC-activated NOS2 transcriptional activity; the binding site located at -1529 bp to -1516 bp played a greater role than the other binding site located at -1224 bp to -1210 bp in the mouse CEC. These findings highlight the molecular mechanism underlying FC-induced up-regulation of NOS2 in the mouse CEC.
Using the two-hemorrhage SAH model in rats, it has been demonstrated that estradiol (E2) significantly attenuates the SAH-induced vasospasm by inhibiting the NOS2 expression. To investigate the molecular mechanism underlying E2-mediated prevention in the SAH-induced vasospasm, FC complexes were added into the primary cultured mouse CEC to mimic the SAH conditions. The results showed that the NOS2 expression was up-regulated through increased nuclear translocation of NFκB induced by free radicals generation. However, treatment with E2 (100 nM) reduced the FC (100 μM)-induced increases of free radical generation and the levels of NOS2 mRNA and protein in the CEC. Moreover, E2 also prevented the FC-induced increases of IκBα phosphorylation, NFκB nuclear translocation, NFκB binding onto the NOS2 promoter, and the NOS2 promoter luciferase activity. Knock-down of the estrogen receptor β (ERβ), but not ERα, abolished the E2-mediated prevention on the FC-induced increases of NOS2 mRNA and protein. The present in vitro results suggest that E2 inhibited NOS2 gene expression by interfering with NFκB nuclear translocation and NFκB binding onto the NOS2 promoter.
Taken together the present results provide the underlying molecular basis for designing the possible preventive and/or therapeutic strategies in the regulation of NOS2 for helping SAH patients.

並列關鍵字

Estrogen、Ferrous Citrate、NOS2

參考文獻

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