評估小孢子靈芝免疫調節蛋白對谷氨酸興奮性毒性之神經保護作用及其對大鼠創傷性腦損傷造成的認知功能障礙之改善效果

腦部損傷可分為慢性腦部損傷，例如：阿茲海默症，及本論文著正的急性腦損傷，例如：創傷性腦損傷。創傷性腦損傷為外力重擊、車禍或跌倒等等所造成，創傷性腦損傷可能會造成記憶缺失、大腦中的膠細胞增生、發炎反應或神經退行性疾病，嚴重者可能導致死亡。損傷的神經元會誘發氧化壓力的生成，進而導致發炎反應或神經傳導物興奮性毒性，如常見於神經退化性疾病或急性腦損傷中的谷氨酸興奮性毒性。谷氨酸是哺乳類動物中樞神經系統中主要的神經傳導物質之一，它與神經發育及記憶學習有關，而過度刺激谷氨酸受體會造成興奮性毒性，並促使自由基產生並形成氧化壓力，最後導致神經元功能障礙或死亡。發育完成的中樞神經系統對神經受損的修復能力降低，因為成熟的神經細胞無法複製再生替換損傷的神經細胞，也難以重建被破壞的神經網路連結。目前治療創傷性腦損傷的藥物或手術皆用於預防創傷性腦損傷的二次傷害，像是清除自由基、神經傳導物質的拮抗劑或是維持大腦血液流壓力等等。靈芝為廣泛使用的藥用大型真菌，經代謝後可產生許多生物活性氧化物，如多醣體、三萜類及免疫調節蛋白。小孢子靈芝免疫調節蛋白 (Ganoderma Microsporum immnuomodulatory proteins, GMI) 是從靈芝中萃取出來的一種真菌免疫蛋白，根據最近的文獻指出GMI可以抑制A549細胞中細胞內的活性氧物種以及與TNFα相關的細胞侵入和遷移。因此，本研究欲分為兩部分分別探討GMI是否可經由抑制活性氧物種的生成，進而保護神經元對抗谷氨酸興奮性毒性與幫助損傷神經元修復，以及藉由降低創傷性腦損傷造成的發炎反應及調控相關蛋白質，最終改善創傷性腦損傷造成的空間記憶障礙。本研究將採用神經受損常發生的谷氨酸興奮性毒性及神經纖維斷裂兩種體外測試平台及體內創傷性腦損傷模型，評估GMI於保護神經元上的功效。於谷氨酸興奮性毒性測試平台中，實驗分成三組: 1) 對照組：神經細胞受谷氨酸 (0.5、1及5 μM) 刺激10分鐘後，加入培養液24小時；2) 預防組：神經細胞預先加入GMI (0.1、0.5、1 μg/mL) 24小時後，使用谷氨酸 (0.5、1及5 μM) 刺激10分鐘，再放回原濃度GMI再培養24小時；3) 治療組：神經細胞受谷氨酸 (0.5、1及5 μM) 刺激10分鐘後，加入GMI (0.1、0.5、1 μg/mL) 24小時。於體外神經纖維斷裂測試平台中，實驗分成兩組：1) 預防組：神經細胞預先加入GMI (0、0.01、0.1、0.5、1、5 μg/mL) 24小時後，使用1mL的Gilson-pipette tip斜刮細胞，接著再放置24小時培養; 2) 治療組：神經細胞使用1mL的Gilson-pipette tip斜刮細胞後，加入GMI (0、0.01、0.1、0.5、1、5 μg/mL) 24小時。於體內創傷性腦損傷模型中，實驗分成四組：1) 口服預防組：腦損傷前服用GMI (0.33、3.3、33 μg/kg) 一個月，並且於損傷後再服用原劑量一個月；2)口服治療組：腦損傷後服用GMI (0.33、3.3、33 μg/kg) 一個月；3) 單次注射組：腦損傷後注射一劑GMI (0.33、3.3 μg/kg)；4) 多次注射組：腦損傷後連續注射四天GMI (0.33、3.3 μg/kg)。於谷氨酸興奮性毒性測試結果中，神經元的存活率隨著谷氨酸的濃度增加而降低。但是經過GMI處理過後，GMI可抑制細胞內ROS和NO的產生，進而減少谷氨酸誘導的神經元興奮性毒性，尤其是預先處理GMI組別的效果較好。此外，GMI不會對神經細胞造成毒性。在GMI對於神經纖維斷裂的保護作用結果中，處理GMI的組別皆可使斷裂的神經纖維再生，但是在預先處理GMI的組別效果較好。於創傷性腦損傷模型中，我們發現GMI可以改善創傷性腦損傷造成的空間記憶障礙、加快損傷部位的修復及調節了小膠質細胞、BDNF和星狀膠質細胞的增生。綜合以上結果，GMI確實可以保護谷氨酸誘導的興奮性毒性、幫助損傷神經纖維再生及改善創傷性腦損傷造成的認知功能障礙。

關鍵字

創傷性腦損傷；興奮性毒性；小孢子靈芝免疫調節蛋白

並列摘要

Neuronal injury and degeneration are responsible for various neurological diseases. Adult mammalian central nerve system (CNS) as a difficulty in repairing injuries because it lacks the regenerative power to replace damaged neuronal cells and reconstruct dendritic connections. Neuronal injury release some inflammatory mediators and oxidative stress. Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system, which is important for neural development, synaptic plasticity, and learning and memory. Excessive stimulation of glutamate receptors causes excitotoxicity, a phenomenon implicated in both acute and chronic neurodegenerative diseases. Some studies suggests that oxidative stress may play an important role in the common pathway for neurotoxicity in a variety of neurodegenerative diseases. Traumatic brain injury (TBI) occurs 20 admissions per 100,000 people in studies, and results in deaths and neurodegeneration disease. Many external causes lead to TBI, such as falls, traffic accidents, struck by/against and assaults. TBI induce astrogliosis, activation of microglia, oxidative stress and increase of inflammation-related protein. The treatment of traumatic brain injury are used to prevent traumatic brain injury secondary damage, such as free radical scavenging, antagonists of neurotransmitters or brain blood flow to maintain pressure. Ganoderma is a widely used medicinal macrofungus that creates a diverse set of bioactive. Ganoderma Microsperum immnuomodulatory proteins (GMI) is a immunomodulatory proteins of Ganoderma Microperum. Recent studies demonstrated that GMI is able to inhibit intracellular ROS generation and TNFα-mediated migration and invasion in A549 cells. The aim of this study is to investigate whether the GMI can inhibit glutamate-induced oxidative stress to protect neurons and repair neuronal injury in vitro. The another aim is to examine whether GMI could improve damaged effects of TBI in vivo. To distribute glutamate excitotoxicity in vitro model, we therefore separated the in vitro cultured neurons into three groups: 1) control group: incubate the neurons with glutamate for 10 min and recover in vehicle condition for additional 24 h; 2) prevention group: pre-incubate the neurons with GMI for 24 h followed by 10 min glutamate treatment, and recover in the presence of GMI for additional 24 h; 3) therapy group: treat glutamate for 10 min and recover in the presence of GMI for additional 24 h. In wound healing in vitro model, we separated into two groups: 1) prevention group: pre-incubate the neurons with GMI for 24 h before scratch the wound and recover in the presence of GMI for additional 24 h; 2) therapy group: incubate the neurons with GMI for 24 h after scratch the wound. In TBI model, GMI was provided to the rats orally (0.33, 3.3 and 33 μg/kg B.W.) or by injection (0.33, 3.3 μg/kg B.W.) before, concurrently or after TBI. The results show that GMI does not cause neuronal cytotoxicity. Glutamate treatment caused a decrease in neuronal viability in a dose dependent manner. GMI is able to protect neurons from the glutamate-induced excitotoxicity by reducing ROS production and NO production, especially in pretreatment condition. We found that advanced application of GMI before or right at trauma injury dramatically accelerated the wound healing comparing with other conditions. Meanwhile, GMI modulated the microglia and astrocytes proliferation in response to TBI. However, BDNF expression was not significantly influenced in oral gavage groups but was increased in injection groups. In summary, GMI is able to protect neurons from the glutamate-induced excitotoxicity and free radical species in in vitro model. GMI can improve cognitive dysfunction, and regulates oxidative/inflammatory response against TBI.

並列關鍵字

trauma brain injury ； excitotoxicity ； Ganoderma Microsperum immnuomodulatory proteins

參考文獻

1 Pearson-Leary, J., Osborne, D. M. & McNay, E. C. Role of Glia in Stress-Induced Enhancement and Impairment of Memory. Front Integr Neurosci 9, 63, doi:10.3389/fnint.2015.00063 (2015).

2 Sajja, V. S., Hlavac, N. & VandeVord, P. J. Role of Glia in Memory Deficits Following Traumatic Brain Injury: Biomarkers of Glia Dysfunction. Front Integr Neurosci 10, 7, doi:10.3389/fnint.2016.00007 (2016).

3 Bradl, M. & Lassmann, H. Oligodendrocytes: biology and pathology. Acta Neuropathol 119, 37-53, doi:10.1007/s00401-009-0601-5 (2010).

4 Bregman, B. S. et al. Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature 378, 498-501, doi:10.1038/378498a0 (1995).

5 Horner, P. J. & Gage, F. H. Regenerating the damaged central nervous system. Nature 407, 963-970, doi:10.1038/35039559 (2000).

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評估小孢子靈芝免疫調節蛋白對谷氨酸興奮性毒性之神經保護作用及其對大鼠創傷性腦損傷造成的認知功能障礙之改善效果

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