磺胺胡蘿蔔素對於鐵誘導小鼠紋狀體損傷的神經保護機制

The Mechanism Underlying the Neuroprotective Effect of Sulforaphane Against Iron-induced Striatal Injury in Mice

王證瑋(Jheng-Wei Wang)

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(2014年)

https://doi.org/10.6832/KMU.2014.00020

摘要

研究背景：
　　患者發生顱內出血(intracerebral hemorrhage; ICH)之後，有近半數倖存的患者常得承受嚴重的殘障，但目前仍未見有效的治療策略被研發出來。ICH之後因血色素溶解導致鐵的累積，會持續生成氫氧自由基(hydroxyl radical; •OH)，而氫氧自由基造成的氧化壓力使得腦組織受到損傷。再者，接續的脂質過氧化反應(lipid peroxidation)會摧毀掉細胞膜的完整性。此外氧化壓力下heme oxygenase 1 (HO-1)會被誘導表現，但HO-1在鐵引起的腦損傷中扮演的角色仍屬未知。既然磺胺胡蘿蔔素(sulforaphane; SF)是青花菜中主要的抗氧化成分，因此本研究擬驗證SF的抗氧化性質是否可藉著抑制鐵引起的HO-1過度表現，而保護紋狀體(striatum)免於鐵的毒性。

實驗設計與方法：
　　為了模擬ICH過後鐵引起的氧化傷害，我們採用微量注射器(micro-infusion pump) 將3μl的1mM檸檬酸亞鐵 (ferrous citrate; FC)注射到雄性小鼠的右側紋狀體。再以溶於玉米油的SF注射到腹腔三次(每次劑量為5mg/kg體重)，時間依序為注射FC的24小時前、15分鐘後和24小時後。注射FC過後的第二天，進行前肢不對稱測試(forelimb use asymmetry test)評估各別的肢體障礙程度，接著犧牲小鼠，取下含紋狀體的腦組織樣本，再以西方墨點法(Western blot)分析並量化分子量145kDa與150kDa的αⅡ-spectrin breakdown product (SBDP 145/150) (腦損傷指標)、4-hydroxynonenal (4-HNE，脂質過氧化反應的最終產物)以及HO-1的蛋白質含量。

實驗結果：
　　給予SF能夠減少FC引起的行為障礙程度以及FC引起的αⅡ-spectrin裂解成SBDP 145/150。另外，給予SF能夠降低FC引起的脂質過氧化程度與FC誘導的HO-1過度表現。

結論：
　　SF可以保護紋狀體免於鐵累積引起的損傷。而減低FC引起的脂質過氧化反應和HO-1的誘導程度，可能參與SF的神經保護作用。

關鍵字

磺胺胡蘿蔔素 ； 亞鐵 ； 顱內出血 ； 紋狀體 ； 脂質過氧化作用 ； 第一型血基質氧化酶

並列摘要

Background:
　　After intracerebral hemorrhage (ICH), half of the survived patients were suffered from significant disability. However, no effective therapeutic strategy was developed until now. The brain is damaged by oxidative stress caused by hydroxyl radicals (•OH) that are generated continuously by iron overload due to the lysis of hemoglobin after ICH. Moreover, the integrity of cell membrane is destroyed by consequent lipid peroxidation. Heme oxygenase 1 (HO-1) is induced under oxidative stress, but the role of HO-1 in iron-induced brain injury remains unknown. Since sulforaphane (SF) is a major antioxidant component of broccoli, this study was designed to examine whether the antioxidative property of SF protects striatum from iron toxicity through inhibiting the over-induction of HO-1.

Materials & Methods:
　　For simulating oxidative damage caused by iron overload after ICH, 3μl of 1mM ferrous citrate (FC) was infused into the right striatum of the male mouse with micro-infusion pump. SF dissolved in corn oil was injected intraperitoneally at a dosage of 5mg/kg B.W. for three times at 24 hours before, 15 minutes and 24 hours after FC-infusion. Two days after FC-infusion, the behavioral deficit was evaluated by forelimb use asymmetry test. Then, the brain tissue containing striatum was sampled after sacrificed. The protein levels of αII-spectrin breakdown product with molecular weights of 145 or 150 kDa (SBDP 145/150), a marker for brain injury, 4-hydroxynonenal (4-HNE, a final product of lipid peroxidation), and HO-1 were quantified by Western blot analysis.

Results:
　　SF treatment decreases the FC-induced behavioral deficit, and the level of FC-induced cleavage of αII-spectrin into SBDP 145/150. In addition, SF treatment reduces the levels of lipid peroxidation and HO-1 induction by FC-infusion.

Conclusion:
　　SF treatment protects the striatum from brain injury caused by iron accumulation. The decreases of lipid peroxidation and HO-1 induction may mediate the protective effect of SF on FC-induced striatal injury.

並列關鍵字

Sulforaphane ； Ferrous ； Intracerebral hemorrhage ； Striatum ； Lipid peroxidation ； Heme oxygenase-1

參考文獻

1. Ferro, J.M., Update on intracerebral haemorrhage. J Neurol, 2006. 253(8): 985-99.

2. Brott, T., J. Broderick, R. Kothari, W. Barsan, T. Tomsick, L. Sauerbeck, J. Spilker, J. Duldner, and J. Khoury, Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke, 1997. 28(1): 1-5.

3. Balami, J.S. and A.M. Buchan, Complications of intracerebral haemorrhage. Lancet Neurol, 2012. 11(1): 101-18.

4. Inagawa, T., Diurnal and seasonal variations in the onset of primary intracerebral hemorrhage in individuals living in Izumo City, Japan. J Neurosurg, 2003. 98(2): 326-36.

5. Inagawa, T., N. Ohbayashi, A. Takechi, M. Shibukawa, and K. Yahara, Primary intracerebral hemorrhage in Izumo City, Japan: incidence rates and outcome in relation to the site of hemorrhage. Neurosurgery, 2003. 53(6): 1283-97; discussion 1297-8.