Platonin減少glutamate誘導神經膠質星狀細胞CTX TNA2死亡作用之探討

The Effects of Platonin on Reducing Glutamate-Induced Death in CTX TNA2 Astrocytes

連芛珮(Wei-Pei Lian)

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(2012年)

https://doi.org/10.6831/TMU.2012.00183

摘要

根據2010年行政院衛生署統計，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)是意外傷害最主要的死因。遭受創傷性腦損傷後，神經傳導物質glutamate會大量釋放造成興奮性毒性(excitotoxicity)，產生神經發炎反應(neuroinflammation)和細胞死亡，臨床上創傷性腦損傷病患的預後不佳，須長期治療與觀察，將造成經濟上極大的負擔，因此開發治療創傷性腦損傷藥物是非常重要的。Platonin為臨床上用於治療灼傷、過敏性結膜炎、腸炎、藥物過敏與自體免疫疾病的藥物，且具有抗氧化、抗發炎與降低顱內壓的作用，但platonin對於腦創傷之作用仍然未知。本研究以glutamate刺激神經膠質星狀細胞CTX TNA2模擬創傷性腦損傷現象，首先利用MTT assay分析glutamate對於CTX TNA2細胞之細胞毒性，結果顯示細胞存活率具有濃度依存性下降，利用流式細胞儀搭配annexin V/PI及acridine orange分別偵測細胞凋亡(apoptosis)及細胞自噬(autophagy)比例，發現glutamate可誘導神經膠質星狀細胞進行apoptosis (58.16 ± 8.30 %)及autophagy (20.38 ± 1.12 %)，於24小時達最高百分比。將細胞處理platonin 100 nM，經由annexin V/PI及acridine orange之結果指出platonin降低glutamate所誘導CTX TNA2細胞的apoptosis (59.20±2.73 %下降至7.87 ± 0.73 %)與autophagy (22.36 ± 1.22 %下降至10.19 ± 1.26 %)的百分比。利用HEt、DCFH-DA和DHR123染劑偵測細胞內活性氧化物(reactive oxygen species, ROS)變化，發現platonin具有抑制ROS產生的效果。透過西方墨點法觀察到platonin減少IκBα的分解與COX-2的表現。綜合上述實驗結果證明platonin具有神經保護作用，可能藉由抑制ROS產生降低IκBα的分解與COX-2的表現減少glutamate誘導細胞產生apoptosis與autophagy。期望可提供臨床上開發治療創傷性腦損傷藥物的新方向，透過有效的減少神經系統的損傷，以增加創傷性腦損傷病人的存活率。

關鍵字

創傷性腦損傷 ； 神經膠質星狀細胞 ； 細胞自噬 ； 細胞凋亡

並列摘要

According to the statistics made by Department of Health in 2010, accident would rank sixth among ten leading cause of death in Taiwan. Traumatic brain injury (TBI) is the major cause of accident death. After TBI, excessive glutamate leads to the excitotoxicity of cell. TBI has poor prognosis in clinical, so the patients need long-term treatment and medical observation, which causes economic burden. Therefore, the development of drugs for TBI is very important. In clinical, platonin is used to treat with burns, allergic conjunctivitis, enteritis, drug allergies and autoimmune diseases. In addition, platonin has anti-oxidantive, anti-inflammatory and intracranial pressure downregulation effects. However, the effeciency of platonin on TBI is still unclear. In this study, glutamate is used to induce the cytotoxicity of CTX TNA2 astrocytes to mimic the phenomenon of TBI. First, using MTT assay, we demonstrated that glutamate significantly decreased the cell viability of CTX TNA2 cells in a dose-dependent manner. Furthermore, glutamate induced autophagy and apoptosis in CTX TNA2 cells with acridine orange stain and annenxin V/PI stain using flow cytometry, respectively. The percentages of apoptosis (58.16 ± 8.30 %) and autophagy (20.38 ± 1.12 %) reached to the peak at 24 hour time point. When treated with 100 nM platonin, CTX TNA2 cells attenuated glutamate-induced apoptosis (59.20±2.73 % dropped to 7.87 ± 0.73 %) and autophagy (22.36 ± 1.22 % dropped to 10.19 ± 1.26 %). In addition, we demonstrated that platonin inhibits reactive oxygen species (ROS) generation using HEt, DCFH-DA, and DHR 123 staining. Western blot analysis showed that platonin attenuated IκBα degradation and COX-2 expression through Western blot. These finding indicated that the regulation of ROS, IκBα, and COX-2 participated in the neuroprotective of platonin in CTX TNA2 astrocyte cells.We finally conclude that inhibition of ROS generation and IκBα degradation may participate in the neuroprotective of platonin. This study will provide a potential strategy for the development of a new drug for TBI by reducing the damage on nervous system and increasing the survival rate of TBI patients.

並列關鍵字

Platonin ； Traumatic brain injury ； Astrocyte ； Glutamate ； Autophagy ； Apoptosis

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