Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22850
標題: 石蓮花粉劑對缺血腦組織保護機轉之探討
Houseleek (Graptopetalum paraguayense)-mediated brain protection against cerebral ischemia
作者: 王偉迪
Wang, Wei-Ti
關鍵字: houseleek (Graptopetalum paraguayense);石蓮花;cerebral ischemia;anti-inflammation;neutrophil;macrophage;腦缺血;抗發炎;嗜中性白血球;巨噬細胞
出版社: 生命科學系所
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摘要: 
氧化壓力與發炎反應常造成缺血腦組織的二次傷害。本論文之研究目的即在探討具有抗氧化、抗發炎功效的石蓮花 (Graptopetalum paraguayense) 粉劑,對缺血腦組織是否具保護性,及參與的作用機轉。研究中首先以將大鼠雙側總頸動脈結紮 ( common carotid artery occlusion , CCAO ) 及右側中大腦動脈結紮 ( middle cerebral artery occlusion , MCAO ) 的方式使腦部缺血 90 分鐘,隨後將結紮之血管放開,使血液回流 24小時,建立一個可逆式的缺血/再灌流 ( ischemia/reperfusion ) 動物模式。此外,亦將細胞培養在缺糖、缺氧及缺血清 ( glucose- , oxygen- and serum-deprivation , GOSD ) 的環境下,建立一個模擬缺血的體外研究模式。而後分別利用這兩個研究模式,評估石蓮花對缺血腦組織及運動功能是否具保護性;石蓮花的最佳給藥方式;石蓮花對不同缺血腦細胞之保護及訊息傳遞路徑;及石蓮花對缺血鼠脾臟嗜中性白血球與巨噬細胞活性之急性及長期性調控。研究結果顯示,缺血早期 ( 缺血 15 分鐘 ) 石蓮花之腦部注射,或長期 ( 8週 ) 餵食石蓮花,均能有效降低缺血腦組織及運動功能受損的程度,但缺血晚期 ( 再灌流後 15 分鐘 ) 的石蓮花腦部注射,則不具此保護性。另將不同濃度 ( 1, 2.5, 5, 10 mg/ml ) 之石蓮花直接外加至經 GOSD 處理之腦細胞中,發現 GOSD 神經細胞及星芒狀細胞之存活明顯受到石蓮花之保護,但石蓮花對 GOSD 微膠細胞不具保護性。石蓮花明顯提升 GOSD 星芒狀細胞中 ERK、AKT 之磷酸化 ( 活化 ),但 GOSD 神經細胞中之 ERK、AKT之磷酸化現象卻明顯受到抑制,同時石蓮花對 caspase-3 之活性亦具抑制性。另外,石蓮花在體外可直接抑制缺血鼠脾臟嗜中性白血球與巨噬細胞釋放 NO 之能力,及提升其吞噬能力,反應出石蓮花可調控缺血腦組織中嗜中性白血球與巨噬細胞活性的可能性。但長期餵食石蓮花對缺血鼠體內脾臟嗜中性白血球及巨噬細胞釋放 NO 之能力卻不具抑制性,但可明顯提升細胞之吞噬能力,反應出石蓮花維護缺血鼠先天性免疫力的潛能。整體而言,石蓮花的確具有抗腦缺血之功效。不論是缺血早期的腦室注射或是缺血前長期餵食石蓮花,均對缺血腦組織具保護性,兩種方式的保護效益相近,但後者不具侵入性,是較易被接受的療法,且兼具提升先天性免疫力的保健功效。石蓮花之保護機轉可能是透過對缺血神經細胞及星狀細胞之保護,進而修復受損之運動功能;及抑制缺血腦組織中嗜中性白血球與巨噬細胞釋放 NO 之能力及提升兩者吞噬能力所致。石蓮花粉劑明顯兼具治療缺血性腦中風,及提升中風病患先天性免疫力之潛能,未來對抗中風藥物的篩選,石蓮花顯然是極佳的選擇。

Oxidative stress and inflammation have been regarded as important triggers lead to the secondary injury of ischemic brain. Houseleek (Graptopetalum paraguayense) has been known to posses anti-oxidative and anti-inflammatory activities. Study goal of the thesis was therefore to evaluate the protective effect of houseleek on cerebral ischemia and the underlying mechanisms involved. In the study, Spraque-Dawley (S.D.) rats were subjected to 90 min of bilateral common carotid artery occlusion (CCAO) plus unilateral middle cerebral artery occlusion (MCAO) followed by reperfusion for 24h to develop an animal model with reversible focal ischemia. The primary neuron, astrocyte and microglia were also isolated and exposed to glucose-oxygen-serum deprivation (GOSD) condition to mimic the cerebral ischemic condition in vitro. Briefly, the acute (intracerebroventricular injection) and chronic (8-week long feeding) effects of houseleek on ischemia/reperfusion (I/R)-induced brain infarction and the dysfunction of motor activity and on the activities (nitrite oxide or NO release and phagocytosis) of splenic neutrophils and macrophages were evaluated in ischemic animals with or without houseleek treatment. The protective effect of houseleek on GOSD neuron, astrocyte and microglia, and its regulation of AKT, ERK or caspase-3 activity of GOSD neuron and astrocyte were also assessed through the in vitro ischemia platform. The results showed that I/R-caused brain infarction was reduced and motor dysfunction was restored by houseleek given at the early stage of ischemia (at 15min after MCAO/CCAO) or by long-term (8 weeks) oral administration with houseleek. The same protection was absent when houseleek was given at the late stage of ischemia (at 15min after reperfusion). In vitro, cell viability of neurons and astrocytes (but not microglia) were protected by houseleek from GOSD-induced damage. Houseleek appeared to protect GOSD astrocytes via the activation (phosphorylation) of AKT and ERK. Houseleek however, inhibited the phosphorylation of AKT and ERK and was able to suppress caspase-3 activity of GOSD neurons. Thus, houseleek might protect GOSD neurons via the inhibition of caspase-3 activity. Treatment with houseleek in vitro, can also inhibit NO release from and elevated the phagocytic activities of splenic neutrophils and macrophages isolated from ischemic rats. Long-term feeding with houseleek on the other hand, did not suppress NO release but still enhanced the phagocytic activities of both immune cells in ischemic rats. In overall, early brain injection or long-term pre-feeding with houseleek can both protect ischemic brain, probably by increasing the viabilities of neurons and astrocytes and inhibiting the pro-inflammatory activities of brain infiltrating neutrophils and macrophages. Brain injection with houseleek can protect ischemic brain in a rapid way whereas long-term pre-feeding can provide same protection with less body intrusion. Additionally, houseleek given in both ways can elevate phagocytic activities that appeared to be beneficial to injury brain and innate immunity of ischemic rats. Above results clearly indicate a therapeutic value of houseleek in the control of cerebral ischemia as well as in the protection of innate immunity of ischemic patients.
URI: http://hdl.handle.net/11455/22850
其他識別: U0005-0702201016440900
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