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  • 學位論文

桑黃菌絲體萃取成分保護PC-12細胞對抗氧糖剝奪/復氧所造成傷害的機制探討

Mechanisms of Phellinus Linteus Mycelium Extract Protects PC-12 Cells against Oxygen-Glucose Deprivation/Reoxygenation Injury

指導教授 : 詹明修 黃相碩

摘要


缺血性中風是造成成人死亡的第二大死因,並且會引起嚴重的認知及運動功能的退化、神經退化性疾病及猝死的發生。缺血性中風會造成細胞面臨缺氧缺糖的環境,使細胞因為缺氧缺糖而啟動自體吞噬 (autophagy) 、細胞凋亡 (apoptosis) 、壞死 (necrosis) 等路徑。已知在缺血性中風中,自體吞噬扮演著一個很重要的角色,但是否為保護機制或加劇腦損傷至今仍具爭議。桑黃 (Phellinus linteus) 為一種橘色的多年生蕈類,被認為具有抗氧化、抗發炎等特性,但將桑黃使用在中風疾病的研究並不多。在本研究中,以氧糖剝奪(oxygen glucose deprivation, OGD)/復氧 (OGD reoxygenation, O/R) 來模擬活體中腦部缺血再灌流 (ischemia/reperfusion)的狀況,偵測桑黃菌絲體萃取物是否保護類神經細胞株免於OGD或OGD/R的傷害。在結果中發現,低濃度的桑黃菌絲體萃取物能夠保護PC-12類神經細胞免於氧糖剝奪或氧糖剝奪/復氧所造成的傷害,細胞死亡率從70%降至5%。相較於在OGD時不給予桑黃菌絲體萃取物的組別,給予桑黃菌絲體萃取物的PC-12細胞中p62、Beclin 1及LC3 I蛋白質的表現有顯著的增加,並維持PC-12細胞中自體吞噬的進行;另外,在氧糖剝奪實驗中,低濃度的桑黃菌絲體萃取物能增加Bcl-2蛋白質的表現,且明顯的抑制pro-caspase 3的活化;桑黃菌絲體萃取物能有效抑制JNK及eIF-2α的磷酸化,避免啟動細胞走向凋亡路徑。此外,,在氧糖剝奪實驗時,桑黃菌絲體萃取物能誘導表現CHOP蛋白質。然而,結果中也發現cycloheximde (CHX) 不影響自體吞噬相關蛋白的表現,卻能抑制CHOP蛋白質的表現,在此我們認為CHOP蛋白質的表現很可能與p38的活化有關但並不影響LC3 I蛋白質的表現。綜合以上,我們認為桑黃菌絲體萃取物可維持細胞的自體吞噬、抑制促凋亡訊號及抑制細胞凋亡來保護類神經細胞免於受到氧糖剝奪及氧糖剝奪/復氧所誘導的細胞損傷。因此,未來桑黃可能提供一個新的策略來預防中風。

並列摘要


Ischemia stroke has become the second leading cause of death in adult population. It could cause sereve cognitive and motor dysfunction, neurodegenerative disease, and even acute death. Ischemia stroke could lead cells expose to the environment without oxygen and glucose, and induce autophagy, apoptosis and necrosis pathways. It has been known that autophagy plays an important role in ischemia stroke; however, whether the autophagy mitigates or exacerbates the brain injury is still controversial. Phellinus linteus, an orange Basidiomycete fungus has antioxidative, anti-inflammatory and other properties. However, reports about P. linteus application to stroke are still limited. In our study, we had established both in vitro oxygen glucose deprivation (OGD), and reoxygenation (O/R) system to mimic in vivo brain ischemia/reperfusion injury. Our results indicated that P. linteus mycelium extract (PLME) treatment was able to protect PC-12 cells from damage caused by OGD or O/R. (The ratio of cell death was decresed from 70% to 5%). Compared to control, PLME pretreated cells under OGD and O/R conditions significantly upregulated p62, Beclin-1and LC3 I protein expression to maintain autophagy in low dose. Furthermore, PLME could up-regulate apoptosis-related protein Bcl-2 expression and significantly inhibit the activation of pro-casapase 3. We also demonstraeed that PLME was capable to suppress the phosphorylations of JNK and eIF-2α protein to avoid initiatiation of apoptosis. Interestingly, only PLME-pretreated cells could significantly upregulate CHOP protein expression under OGD, however, cell treated with cycloheximde do not effect autophagy-related protein expression, but CHOP. Accrodingly, we suggest that CHOP expression may regulated by other signal pathway (such as p38) and not the dominant regulator controlling autophagy related protein expression. In conclusion, PLME could sustain autophagy, suppress pro-apoptotic pathways and inhibit apoptosis to prevent neuronal-like cells from OGD or O/R-induced cell damage. Therefore, Phellinus linteus may offer a new strategy to prevent stroke in the future.

並列關鍵字

ischemia storke autophagy apoptosis necrosis Phellinus linteus OGD O/R ischemia/reperfusion p62 Beclin 1 LC3 I JNK eIF-2α Bcl-2 pro-caspase 3 CHOP cycloheximde

參考文獻


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