自噬在雌性素對於檸檬酸亞鐵引起腦損傷的 保護作用中所扮演的角色

Role of autophagy in the neuroprotective effect of estradiol on iron-induced brain injury

陳秋瑋(Chiou-Wei Chen)

指導教授 ： 許勤

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2011年)

摘要

顱內出血後，紅血球中血紅素的降解產生過多的亞鐵離子，導致自由基及氧化壓力過多而造成腦組織受損。然而，氧化壓力同時也會引起細胞內產生自噬(autophagy)的現象，它是調控胞內蛋白質及胞器恆定的系統之ㄧ。但是在病理的情況下自噬扮演怎樣的角色目前尚未定論，因為它也可能參與細胞計畫性死亡(autophagic cell death)。我們之前的研究發現，檸檬酸亞鐵導致的腦損傷，雄鼠腦損傷的嚴重度較雌鼠為高，而且雌性素的神經保護性作用參與此性別差異性腦損傷。至於自噬是否參與雌性素的神經保護作用則仍屬未知。由於研究報告指出雌二醇的抗氧化作用可減少胞內的氧化壓力， 因此，我們假設在檸檬酸亞鐵導致腦損傷中，雌性素可透過調控細胞自噬而達到神經保護的作用。
為了探討自噬在磁性素對於檸檬酸亞鐵引起腦損傷的保護作用中所扮演的角色，我們採用雄、雌成年大白鼠(SD rat)及老鼠嗜鉻瘤細胞(PC12)進行活體內及活體外實驗。活體實驗中，我們將檸檬酸亞鐵(ferrous citrate)注入右腦基底尾狀核核區(caudate-putamen nucleus)導致神經損傷。利用西方墨點法偵測α-II Spectrin的片段量評估神經損傷程度且利用LC3-II的表現量來看自噬的表現情況。 活體外實驗，我們採用PC12細胞株，利用 siRNA方法減弱(knock down) 自噬相關基因7 (atg7)，及採用飢餓方法誘發自噬來探討自噬是否參與雌二醇的神經保護機制，更進一步的利用ERα siRNA方法減弱(knock down)ERα receptor來探討雌性素的保護作用是否透過ERα receptor。結果顯示，雌性素前處理則無論在雌鼠內或活體外皆可減低檸檬酸亞鐵引起的α-II Spectrin 裂解及LC3-II/LC3-I比例增加的現象。活體外實驗結果更顯示，抑制自噬會增加雌性素對檸檬酸亞鐵導致細胞死亡的保護作用；反之，加強了自噬作用後，會消減雌性素對檸檬酸亞鐵導致細胞死亡的保護作用。且抑制ERα receptor表現，會減少雌性素對檸檬酸亞鐵導致細胞死亡的保護作用。由這些結果可以顯示，自噬參與了雌性素對於檸檬酸亞鐵導致腦損傷的保護作用，且雌性素的保護作用透過ERα receptor。

關鍵字

顱內出血 ； 雌性素 ； 自噬 ； 腦損傷

並列摘要

Overproduction of iron, a degradation product of hemoglobin after intracerebral hemorrhage (ICH), causes free radical formation and oxidative damage in brain tissue. Meanwhile, oxidative stress also induces autophagy, which is an intracellular quality control system of proteins and organelles. The role of autophagy under pathological situation remains controversial, because it may also involves in programmed cell death (autophagic cell death), which is defined as cell death occurring with autophagic features. Our previous study showed that the severity of brain injury caused by ferrous citrate (FC) infusion in male rats was higher than that in females and estradiol (E2) plays a critical role in the sex-difference in iron-induced injury. However, whether autophagy participates in the neuroprotective effect of E2 remains unknown. Antioxidative effect of E2 has been report to decrease intracellular oxidative stress, therefore, we hypothesize that E2 protects neurons from iron-induced cell death partially via modulating autophagy. To test our hypothesis, Sprague-Dawley(SD) rats and PC12 cells were used in vivo and in vitro, respectively. Clavage of α-II Spectrin was measured to measure the severity of brain injury. Level of autophagy was quantified by western blot analysis of LC3-II, a detection marker of autophagy. Furthermore, suppression of autophagy by silencing the autophagy-related gene (Atg 7) and enhancement of autophagy by starvation were performed in PC12 neuronal cells to elucidate the role of autophagy in the neuroprotective effect of E2 on FC-induced cell death. The results show that FC-infusion in caudate nucleus significantly increased the level of 145/150 fragment cleavaged from α-II Spectrin and the ratio of LC3-II/LC3 I in vivo. Pretreatment of E2 decreases the levels of FC-induced cleavage of α-II Spectrin and the ratio of LC3-I/LC3-II both in vivo and in vitro. Furthermore, knockdown Atg7 increased the survival rate of PC12 cells incubated with FC. While, starvation decreased the survival rate of PC12 cells incubated with FC and diminished the protective effect of E2 on FC-induced cell death in vitro. These results suggest that autophagy participates in the neuroprotective effect of E2 on FC-induce brain injury.

並列關鍵字

ICH ； E2 ； Autophagy ； Brain injury

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