創傷後給予新穎Pifithrin-α 之氧類似物對於實驗性腦創傷所造成之組織傷害及行為功能缺失之改善作用

Post-trauma administration of Pifithrin-α oxygen analogue improves histological and functional outcomes after experimental traumatic brain injury

朱薏璇(Yi-Hsuan Chu)

指導教授 ： 王家儀

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

在全球，腦創傷是導致死亡和殘疾的一個重要因素。腦創傷造成許多病理上的後遺症，主要致病機轉包含生化、代謝途徑的改變、神經傳導物質大量釋放，並受到氧化壓力以及發炎反應等影響造成神經傷害。越來越多研究證實，腦創傷後細胞凋亡的情形，是造成腦部次級傷害的主因。但目前對於造成細胞凋亡的分子機制仍不清楚，因
此在臨床上，尚未有藥物能治療腦創傷的患者。
腫瘤抑制基因p53，也同時為一轉錄因子，在DNA受損時能調控細胞週期及參與細胞凋亡的過程，而抑制p53的轉錄功能，被認為是治療神經退化性疾病的有效方法。Pifithrin-α（PFT-α）可藉由抑制p53的轉錄活性、防止粒腺體損傷及活化caspase，來避免細胞走向死亡。在腦創傷的相關研究中，PFT-α 被認為具有神經保護的功能。先前研究指出，相較於PFT-α，PFT-α的環狀類似物及其前驅物在動物體內較穩定，且具有較高的藥物活性及效能。因此，在本實驗中，想知道於腦創傷後給予另一個新的PFT-α衍生物-PFT-α氧類似物 (PFT-α (O) )，是否能改善動物的行為功能缺失並減緩神經損傷的情形。
本實驗以可控制皮質撞擊 (CCI) 模型來造成大鼠腦創傷 (速度 4 m/s, 深度 2 mm) 。我們經由撕貼紙測試、擺動測試及神經學缺失分數之評估結果，發現大鼠在腦創傷後出現明顯的神經行為功能缺失。在神經行為功能測試後，大鼠經心臟灌流後斷頭犧牲，取其腦組織切片作甲酚紫染色，以檢測腦部創傷體積，發現在腦創傷後第24小時，創傷腦區相較於對侧腦區有明顯腦組織缺少的情形，因此我們在腦創傷後第1、3小時給予PFT-α (O) (2 mg/kg, i.v.) 或vehicle，並於第24小時檢測，發現其神經行為功能明顯獲得改善。腦創傷體積也於腦創傷後第24小時明顯下降，FJC染色結果也顯示，於腦創傷後第8、24小時，創傷大腦皮質區中神經退化的細胞數量明顯下降。腦創傷後第8、24小時，創傷大腦皮質區中cleaved-caspase 3 positive cells 和 53BP1 positive cells 的數量明顯增加，若於腦創傷後第3小時給予PFT-α (O)，兩者的數量皆顯著下降。即時定量聚合酶連鎖反應 (RT-qPCR) 檢測結果顯示，在創傷大腦皮質組織中，發炎性細胞激素 (IL-1β, IL-6) 與 發炎酵素 (iNOS, COX2) 的mRNA表現量，於腦創傷後第8、24小時顯著上升，但此情形在給予PFT-α (O) 後明顯下降。經由以上實驗結果，我們認為在腦創傷後給予PFT-α (O) ，能改善神經行為功能的缺失，並降低創傷腦組織發炎反應及細胞凋亡情形，而達到神經保護的效果。

關鍵字: 腦創傷 (TBI)、PFT-α 氧類似物 (PFT-α(O))、cleaved-caspase 3、p53

關鍵字

腦創傷 (TBI)；PFT-α 氧類似物 (PFT-α(O))；cleaved-caspase 3；p53

並列摘要

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Pathological consequences of TBI include alterations in biochemical and metabolic pathways, excessive glutamate release, oxidative stress and inflammation. Increasing evidence suggests apoptotic mechanisms in delayed tissue loss after TBI. Therapeutic options to stop this process are not available yet because the underlying molecular mechanisms are poorly understood.
Tumor suppressor gene, p53 can also be a transcription factor to mediate cell cycle arrest or apoptosis after DNA damage, and its inhibition could be a therapeutic approach to several neuropathologies. Pifithrin-α (PFT-α) can prevent cell death by inhibiting p53 transcriptional activity, mitochondrial damage, and caspase activation. PFT-α has been shown to exert a neuroprotective effect following TBI. Previous study indicates that PFT-α’s cyclic analogue and its open precursor are more stable and active in vivo than PFT-α. We therefore examined the effect of a new derivative analogue of PFT-α, PFT-α oxygen analogue (PFT-α (O)) on functional recovery and neuronal damage after TBI.
TBI in rats was caused by controlled cortical impact (CCI, with 4 m/s velocity, 2 mm deformation). Our results indicate that TBI caused significant functional deficits as examined by bilateral tactile adhesive removal test, elevated body swing test (EBST) and modified neurological severity score (mNSS). Animals were sacrificed after functional evaluation by transcardial perfusion and brains were removed and sectioned to measure brain contusion volume by cresyl violet staining at 24 hr. CCI injury resulted in a loss of cortical tissue in the ipsilateral (left) parietal cortex, as reflected by gross reductions in cresyl violet staining intensity. In contrast, the cytoarchitecture of the cortex remained normal in the contralateral (right) hemisphere. Rats subjected to CCI injury were injected with PFT-α (O) (2 mg/kg, i.v.) or vehicle at 1 hr and 3 hr after TBI. Functional deficits were significantly improved by PFT-α (O) administration (2 mg/kg, i.v.) at 24 hr after TBI. The contusion volume significantly decrease at 24 hr after TBI. And the number of Fluoro-Jade C (FJC) positive degenerating neurons also significantly decrease in the cortical contusion region at 8 hr and 24 hr post-injury. There was a marked rise in the number of apoptotic cells as revealed by cleaved-caspase 3 and p53 binding protein 1 (53BP1) positive cells in the cortical contusion region at 8 hr and 24 hr post-injury. PFT-α (O) administered at 3 hr post-injury rats had a significant reduction in the number of cleaved-caspase 3 and 53BP1 positive cells relative to vehicle-treated rats at 8 hr and 24 hr after TBI. Reverse transcription -quantitative polymerase chain reaction (RT-qPCR) indicated relative mRNA levels of the pro-inflammatory cytokines (IL-1β, IL-6) and inflammatory enzyme (iNOS, COX2) significantly increased at 8 hr and 24 hr after TBI. Elevated mRNA levels of pro-inflammatory cytokines (IL-1β, IL-6) and inflammatory enzyme (iNOS) mRNA at 8 hr and 24 hr were significantly reduced by PFT-α (O) administration after TBI. These data suggest that post-trauma administration of PFT-α (O) improves histological and functional outcomes after experimental TBI and reduces inflammatory responses and apoptosis.

Key words: Traumatic brain injury (TBI), PFT-α oxygen analogue (PFT-α(O)), cleaved-caspase 3, p53

並列關鍵字

Traumatic brain injury (TBI)； PFT-α oxygen analogue (PFT-α(O))； cleaved-caspase 3； p53

參考文獻

林口長庚醫院神經內科一科陳德榮編著(2002)。臨床神經學第十章。合記圖書出版。

衛生署統計室(2012)，全國事故傷害與自殺死亡概況。

U.S. Department of Health and Human Services, Centers for Disease Control and Prevention.

Becher, B., Prat, A., and Antel, J. P. (2000). Brain-immune connection: immuno-regulatory properties of CNS-resident cells. Glia 29, 293-304.

Borlongan, C. V., and Sanberg, P. R. (1995). Elevated body swing test: a new behavioral parameter for rats with 6-hydroxydopamine-induced hemiparkinsonism. J Neurosci 15, 5372-8.