Platonin 於創傷性腦損傷之保護作用

The Protective Effects of Platonin on Traumatic Brain Injury

詹岱華(Tai-Hua Chan)

指導教授 ： 施純明

臺北醫學大學/醫學院/醫學科學研究所/碩士(2014年)

摘要

根據2013年行政院衛生福利部統計，事故傷害位居十大死因的第六位，其中創傷性腦損傷(traumatic brain injury, TBI)是意外傷害死亡最主要的原因，患者以15至24歲的青少年居多。根據美國Defense and Veterans Brain Injury Center 2013年統計，全球每年耗費於TBI治療之醫療支出高達765億美元，因此發展有效治療TBI的藥物以減少患者症狀並降低醫療成本，為當前十分重要的課題。TBI過程中，神經傳導物質glutamate會被大量釋放導致氧化壓力及發炎反應的產生，造成神經細胞損傷，終使病患癱瘓甚至死亡，其預後差且目前並無有效治療用藥。Platonin為臨床用藥，傳統上用於治療切創傷、潰瘍、燙傷及風濕性關節炎引起的急性發炎等。此外，文獻亦指出其具有抗氧化、抗發炎及免疫調節作用；然而目前並未有研究指出platonin是否能應用於TBI之治療。
本研究利用glutamate處理大鼠神經膠質星狀細胞 (CTX TNA2) 建立TBI細胞模式，並利用TBI 0310 Impactor撞擊Spraque-Dawley rat大腦皮質作為TBI動物模式，探討glutamate造成細胞傷害之途徑以及platonin之細胞保護機制。本實驗室過去已證實：glutamate能誘發活性氧化物 (reactive oxygen species, ROS)之產生，並透過活化下游glycogen synthase kinase-3β (GSK-3β)，造成CTX TNA2細胞死亡。本論文續以MTT assay分析指出：以glutamate處理CTX TNA2後，給予300 nM platonin可見細胞存活率回升23.39 %；利用流式細胞儀偵測細胞自噬(autophagy)及細胞凋亡(apoptosis)的比例，結果顯示platonin能降低glutamate引起CTX TNA2之細胞自噬(21.51 ± 0.21 % 下降至13.88 ± 1.25 %) 及細胞凋亡(32.94 ± 3.94 % 下降至12.85 ± 0.62 %)。再者，西方墨漬法分析結果指出：glutamate可提高GSK-3β Tyr216位置磷酸化(p-tyr216-GSK-3β)、降低GSK-3β Ser9位置磷酸化(p-ser9-GSK-3β)程度，並減少細胞核內nuclear factor erythroid 2-related factor 2 (Nrf2)的累積，而若共同處理platonin則可恢復其表現。此外，利用GSK-3β inhibitor (SB216763)搭配glutamate處理細胞後，可見核內Nrf2量回升，顯示GSK-3β與Nrf2具上、下游關係。綜合以上數據顯示platonin具有保護神經膠質細胞之效果，其機轉可能透過抑制GSK-3β活化而使Nrf2得以累積於細胞核內，降低glutamate誘導之細胞傷害。
此外，利用西方墨漬法觀察TBI大鼠患部組織蛋白發現：TBI組別中p-ser9-GSK-3β磷酸化程度及pro-poly ADP ribose polymerase (pro-PARP)之表現量下降。若搭配腹腔注射platonin則能使p-ser9-GSK-3β磷酸化程度與pro-PARP表現回升，顯示platonin能抑制TBI後腦細胞之GSK-3β活化及細胞凋亡發生，於創傷性腦損傷具保護效果。本論文透過釐清platonin在細胞及大鼠TBI模式中對於細胞傷害之保護機制以及臨床應用的可能性，提供臨床開發治療TBI藥物的新方向。

關鍵字

Platonin ； 創傷性腦損傷 ； 神經膠質星狀細胞 ； Glutamate ； GSK-3β ； Nrf2 ； 細胞自噬 ； 細胞凋亡

並列摘要

The statistical results indicate that accident ranks the sixth leading cause of death in Taiwan. Furthermore, traumatic brain injury (TBI), the main reason of accidental death, is the most common among adolescents aged from 15 to 24. An estimation made by Defense and Veterans Brain Injury Center in 2013 indicates that the medical care and rehabilitation costs of TBI patients are approximately US$ 76.5 billion. Therefore, it is very important to develop new drugs for TBI therapy.
After TBI, over-released glutamate often leads to the responses of oxidative stress and inflammation, which subsequently causes damage of nerve cells. In clinics, TBI patients exhibit poor prognosis and lack of efficient therapies. Platonin, a clinical drug with anti-oxidative and anti-inflammatory functions, is used to treat incised wounds, ulcers, burns and acute inflammation in rheumatoid arthritis. However, the effects of platonin on TBI are unclear.
In this study, glutamate-treated CTX TNA2 astrocytes were used as a cell model to mimic TBI. As for animal models, TBI was induced at the left brain cortex of Sprague-Dawley rats using a TBI 0310 Impactor. In our previous report, glutamate could induce ROS production, GSK-3β activation, and CTX TNA2 cell death. Using MTT assay, we demonstrated that 300 nM platonin could reduce glutamate-induced cytotoxicity significantly. In addition, we investigated that platonin was able to reduce glutamate-induced cell autophagy (from 21.51 ± 0.21 % to 13.88 ± 1.25 %) and apoptosis (from 32.94 ± 3.94 % to 12.85 ± 0.62 %) using flow cytometry. Immunoblotting assay demonstrated that the increase of p-tyr216-GSK-3β, the decrease of p-ser9-GSK-3β and the decrease of nuclear Nrf2 level could be reversed after co-treatment with platonin. Using GSK-3β inhibitor (SB216763), we also demonstrated that GSK-3β could be the upstream regulator of Nrf2 in the glutamate-mediated signaling pathway. These results suggested that platonin may protect astrocytes from glutamate-induced cytotoxicity through inactivating GSK-3β and maintaining the nuclear Nrf2 level.
Following the line, we further investigated whether platonin could induce GSK-3β inactivation and PARP cleavage in vivo. Immunoblotting assay showed that TBI could lead to decrease of p-ser9-GSK-3β and pro-PARP level. In contrast, this phenomenon could be reversed by platonin treatment. Taken together, the results suggested that platonin may increase cell survival by suppressing GSK-3β activation and Nrf2 translocation. This study provides the evidence that platonin could be a potential therapeutic agent for TBI treatment.

並列關鍵字

Platonin ； Traumatic brain injury ； Astrocyte ； Glutamate ； GSK-3β ； Nrf2 ； Autophagy ； Apoptosis

參考文獻

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