Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/99396
標題: HMGB1在腦缺血中的病理作用
The pathological effects of HMGB1 in cerebral ischemia
作者: 董佳雯
Chia-Wen Tung
關鍵字: HMGB1;缺血性腦中風;發炎;血腦屏障;微膠細胞;星芒狀細胞;石蓮花水萃物;HMGB1;Cerebral ischemia;inflammation;BBB;Microglia;Astrocyte;WGP
摘要: 
缺血性腦中風為一種神經退化性疾病,除腦細胞明顯受損外,亦會造成血腦障壁(Blood-brain barrier/ BBB)的破壞及嚴重的腦組織發炎現象。導致上述病理變化的分子及作用機轉極為多元,包括HMGB1。HMGB1近年來被認為可由受損的腦細胞釋出,造成發炎反應,因此HMGB1本身既是一可以調控基因表現或DNA複製的核蛋白,亦是一個促發炎的分泌性蛋白。雖然已知HMGB1可促進缺血腦組織的發炎反應,但詳細的作用機轉及可能的防禦策略仍有待釐清。因此,本論文的研究目標主要在探討HMGB1在腦缺血過程中對發炎反應及BBB通透性的調控機轉,及其作為腦缺血治療標的蛋白的可能性。實驗中主要利用體外缺血(缺糖-缺氧-缺血清=GOSD)及體內缺血(動物腦血管結紮)兩種模式,伴隨不同抑制劑的處理,作為主要研究平台。使用的分析技術包括,Western blotting、ELISA、EMSA、體外BBB通透性測試、膠原蛋白降解分析、TTC染色及IHC染色等。研究結果顯示GOSD(2h)可透過抑制PPARγ/p-Akt/HO-1來活化NF-κB或提升NOX2來抑制HDAC4/HDAC5的兩種訊號路徑,提升微膠細胞中HMGB1的生成及釋出,進而促進微膠細胞中發炎因子(TNF-α及IL-1β)的生成及釋出。GOSD(24h)亦可提升星芒狀細胞中HMGB1的生成及釋出,進而提升MMP-9的生成及釋出,但抑制β-catenin的蛋白表現,而MMP-9的提升最終可促進Collagen IV(BBB基底膜蛋白)的降解及BBB通透性的提高。甘草酸(Glycyrrhizic acid/Gly)明顯可抑制GOSD提升的發炎反應及BBB通透性。除了GLY,Rosiglitazone (ROSI;PPARγ促進劑)、Apocynin (Apo;NOX2抑制劑)及WGP均被證明可有效抑制HMGB1的生成;WGP對GOSD微膠細胞中HMGB1的抑制是透過抑制NF-κB及NOX2,但提升p-Akt、HDAC4及HDAC5的蛋白表現所致。在缺血/再灌流之動物模式中,GLY的腦部注射可有效抑制缺血腦組織的腦水腫程度,而長期餵食石蓮花水萃物(Water-extracted graptopetalum paraguayense /WGP)亦可抑制缺血/再灌流(I/R)大鼠腦組織中微膠細胞HMGB1、TNF-α及IL-1β及星芒狀細胞中HMGB1及MMP-9,但提升β-catenin的蛋白表現。本論文主要貢獻在於進一步釐清HMGB1對缺血腦組織的傷害機轉,並藉此找出更多具抗中風潛力的HMGB1抑制劑(ROSI、Apocynin、WGP),有助於抗中風藥物的開發。

Cerebral ischemia is a neurodegenerative disease, which can injure brain cells, blood-brain barrier (BBB) and lead to brain infarction (inflammation). Many molecules and mechanisms contribute to ischemia-induced brain injury, including high mobility group box 1 (HMGB1). HMGB1 is a nuclear protein also a pro-inflammatory secretory protein due to the fact it can be released from injured brain cells to promote inflammation. Although HMGB1 has been indicated in ischemia-caused brain inflammation or even BBB injury, the underlying mechanisms and strategies to control of HMGB-mediated pathological effects still require further investigation. The primary goal of this study was therefore aimed to investigate how HMGB1 protein regulates the brain inflammation and blood-brain barrier (BBB) permeability in cerebral ischemia and to know if HMGB1 can serve as a therapeutic target in this disease. Briefly, both the in vitro ischemia (GOSD: glucose-oxygen-serum deprivation) and in vivo focal cerebral ischemia animal model were used in the study, with or without specific chemical blockers as indicated. Techniques applied include, the Western blot analysis, ELISA, EMSA, transwell assay for BBB permeability, collagen degradation assay, TTC stain and IHC staining. The results showed that GOSD was likely to inhibit the signaling pathway PPARγ/p-Akt/HO-1 to activate NF-κb or stimulate NOX2 to inhibit HDAC4/HDAC5 expression,and than inducing the expression and release of HMGB1 from microglia that further contributed to the increased expression and release of TNF and IL-1β. GOSD also increased the expression and release of HMGB1 from astrocytes to inhibit β-catenin but stimulate the expression or release of MMP-9, the latter further degraded collagen IV (basement membrane protein in BBB) and increased the BBB permeability. GLY was able to block the GOSD-induced inflammatory response and BBB permeability. Besides GLY, Rosiglitazone (PPARγ agonist) and Apocynin (NOX2 inhibitor) and WGP, were all newly proven as HMGB1 inhibitors as well. It appeared that WGP could inhibit NF-κb and NOX-2 but stimulate HDAC4/HDAC5 expression to down regulate the HMGB1 expression of GOSD microglia. In ischemic animal model, intracisternal injection with GLY could inhibit ischemia/reperfusion (I/R)-induced brain infarction; and long-term feeding with WGP could inhibit microglial HMGB1, TNFα and IL-1β and astrocytic HMGB1 and MMP-9 expression but increase β-catenin expression in ischemic brain tissues. In overall, the major contributions of this study was to further delineate the pathological roles of HMGB1 in cerebral ischemia; and through the mechanistic study to find new HMGB1 inhibitors (rosiglitazone, apocynin, WGP) with therapeutic potential in the control of ischemic stroke.
URI: http://hdl.handle.net/11455/99396
Rights: 同意授權瀏覽/列印電子全文服務,2022-02-12起公開。
Appears in Collections:生命科學系所

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