- 學位論文
Pifithrin-μ對於實驗性腦創傷的神經保護效果:細胞與動物實驗
Neuroprotective effect of pifithrin-μ against traumatic brain injury : Cellular and animal studies
摘要
腦創傷是世界上造成死亡與神經功能缺失的主要原因，因此治療及預防腦創傷是醫學研究上的重要議題。沒有特定的藥物治療是目前可用於防止繼發性腦損傷的發展，創傷後的短時間內，興奮性胺基酸神經傳導物質-麩胺酸 (glutamate)過度釋放，導致麩胺酸受體過度活化，進而細胞的鈣離子失衡造成細胞的興奮性毒性，進而使得神經細胞死亡。 腫瘤抑制因子p53被認為是神經細胞凋亡的重要調控因子之一。Pifithrin-α (PFT-α) 可以藉由抑制p53的轉錄活性、粒線體受傷害、凋亡蛋白酶(caspase)活化來避免細胞死亡。有研究指出p53抑制劑pifithrin-μ (PFT-μ)和PFT-α不同的是PFT-μ可以抑制和p53有關的細胞凋亡(apoptosis)，而不影響p53的轉錄活性。且PFT-μ在缺氧缺血性腦傷的神經保護效果比PFT-α更好。我們的研究想比較抑制p53的藥物PFT-μ 相較於PFT-α對於TBI後神經保護的效果。 首先我們檢視這些藥物在體外對於細胞毒性的保護，我們利用大鼠大腦皮質神經/膠質混和細胞原代培養之細胞給予glutamate (100 mM)及不同濃度的PFT-μ。細胞死亡率以LDH assays測量，免疫細胞染色觀察神經元的表現。結果發現PFT-μ在大鼠腦皮質神經膠質細胞細胞可保護神經免於麩胺酸造成的神經細胞死亡。為了近一步比較PFT-α和PFT-μ神經細胞在活體研究中的影響，成熟大鼠被施予可控制的大腦皮質撞擊。受傷後5小時或7小時給予大鼠靜脈注射PFT-α (2 mg/kg) 或PFT-μ (2 mg/kg)。受傷後的功能以撕貼紙測試、擺動測試及神經學缺失分數、走平衡木測試評估。神經行為功能測試後於TBI後24小時大鼠斷頭犧牲，取腦組織切片做甲酚染色，檢測腦部創傷體積。結果發現受傷後5小時給予PFT-α或PFT-μ，可以減少腦部創傷體積、Fluoro-Jade C (FJC)染色陽性的退化神經元，及Annexin V、p53抗體染色陽性之神經元，在受傷後24小時，但受傷後5小時給予PFT-α或PFT-μ則沒有以上效果。 經由以上實驗結果，我們認為在腦創傷後給予PFT-μ，能改善神經行為功能的缺失、藉由抑制p53減緩神經細胞凋亡，而達到神經保護的效果，且效果比PFT-α要顯著。
並列摘要
Traumatic brain injury (TBI) is a major cause of death and neurological disorders worldwide. Therefore, the prevention and treatment of TBI is an important issue of medical research. No specific pharmacological therapy is currently available that prevents the development of secondary brain injuries. Following TBI, extracellular concentration of glutamate, the principal excitatory neurotransmitter, markedly increases, leading to glutamate-receptor overstimulation and unbalanced calcium ions to cause excitotoxicity resulting in cell death. The tumor suppressor p53 is a crucial regulator of apoptotic neuronal death. Pifithrin-α (PFT-α) can prevent cell death by inhibiting p53transcriptional activity, mitochondrial damage, and caspase activation. Comparing to PFT-α, Pifithrin-μ (PFT-μ) is a compound that inhibits p53-dependent apoptosis without inhibiting p53 transcriptional activity. Previous study showed that the neuroprotective effect of PFT-μ is better than PFT-α in neonatal ischemic brain. In this study, we compared the neuroprotective effect of two p53 inhibitors, PFT-μ and PFT-α against TBI. We first examined whether these drugs protect against excitotoxicity in vitro. Primary rat cortical neuron/glia mixed cultures were treated with glutamate (100 mM) in the presence or absence of various concentrations of PFT-μ. Cell death was estimated by LDH assays, and protein expression was estimated by immunocytochemistry. PFT-μ was a neuroprotective against glutamate-induced neuronal death in primary rat cortical neuron/glia cultures. To further compare the effects of PFT-μ and PFT-α in vivo, adult SD rats were subjected to controlled cortical impact (CCI). PFT-α or PFT-μ (2 mg/kg, i.v.) was administered to rats at 5 or 7 hr after injury, Functional deficits after TBI were examined by bilateral tactile adhesive removal test, elevated body swing test (EBST), modified neurological severity score (mNSS) and Beam walking test. The functional deficits improvement of PFT-α is smaller than that of PFT-μ. Animals were sacrificed at 24 hr post-injury after functional evaluation and brains were removed and sectioned to measure brain contusion volume by cresyl violet staining. Post-injury administration of PFT-μ or PFT-α at 5 hr, but not 7 hr, after TBI significantly reduced contusion volume (P<0.01) , the number of Fluoro-Jade C (FJC) positive degenerating neurons , and Annexin V and p53 positive neurons at 24 hr These data suggest that post-trauma administration of PFT-μ improves functional outcomes, prevent neuronal cell apoptosis by inhibiting p53 after experimental TBI to exert neuroprotective effect following TBI. The neuroprotective effect of PFT-μ was significantly better than that of PFT-α.