| 摘要: | 行政院衛生福利部 2015 年統計，事故傷害位居十大死因的第六位，其中事故傷害死亡最主要的原因為創傷性腦損傷 (Traumatic brain injury, TBI)，患者又以青少年居多，且花費於 TBI 治療金額更是可觀。目前於臨床上的治療藥物大多僅是針對 TBI 發生後產生症狀或後遺症做治療，所以如何發展出有效治療 TBI 的藥物並降低醫療支出，為當前急需解決的難題。當 TBI 發生時，glutamate 會被大量釋放並導致氧化壓力及發炎反應的產生，進而造成神經細胞的損傷。如此影響了病人的預後及死亡率的增加，且目前無有效的治療藥物。platonin 為目前臨床核准使用於治療燒燙傷、切割傷、潰瘍及風濕性關節炎引起急性炎症的藥物。過去也有文獻指出 platonin 具有抗氧化、抗發炎及免疫調節的功能；然而 platonin 在 TBI 上的治療角色尚未被釐清。
本實驗室過去以 glutamate 處理大鼠星狀細胞 (CTX TNA2) 建立 TBI 細胞模式並且利用 TBI 0310 Impactor 撞擊 Spraque-Dawley rat 大腦皮質建立 TBI 動物模式，探討細胞傷害之途徑及評估platonin 治療效果。於細胞模式中發現到 glutamate 能誘發活性氧化物(Reactive oxygen species, ROS) 產生，進而活化 glycogen synthase kinase-3β(GSK-3β)，造成下游轉錄因子 Nuclear factor
erythroid 2-related factor 2 (Nrf2) 累積在細胞核中的量減少並引發 CTX TNA2 細胞的死亡。延續上述發現，本研究續以 MTT assay 分析發現 platonin 可提升細胞存活率，並由西方墨漬法分析發現glutamate 會增加 GSK-3β Tyr216 位置磷酸化、降低 GSK-3β Ser9 位置磷酸化程度，增加 Fyn 在細胞核中的累積，降低 Nrf2 在細胞核中的累積量，並降低 heme oxygenase-1 (HO-1)表現量。而若同時處理 glutamate 及 platonin 時，則可逆轉上述各種現象。綜合以上實驗結果，platonin 具有保護星狀細胞之效果，其機轉可能是透過抑制 ROS 調控之 GSK-3β 活化、降低細胞核中 Fyn 的數量使 Nrf2 得以累積於細胞核內進而增加 HO-1 表現量，降低 glutamate 誘導細胞之傷害。此外，利用西方墨漬法觀察 TBI 動物組織亦發現到，TBI 發生後的腦部組織中， GSK-3β Ser9 位置磷酸化及pro form of poly ADP ribose polymerase (pro-PARP) 表現量降低。若給予 platonin 則可抑制GSK-3β 活化及細胞凋亡。未來，本實驗室將持續探討在 TBI 動物模式中，給予 platonin 對大鼠腦部組織及記憶障礙之改善，為其用於治療 TBI 之可能性提供更進一步的證據。
本研究期望透過釐清 platonin 在星狀細胞及動物模式中的治療模式，繼而開發其臨床應用之可能性，提供治療 TBI 的新契機。
According to the Ministry of Health and Welfare investigation in 2015, accident ranked sixth leading cause of death in Taiwan. Moreover, traumatic brain injury (TBI) is the main reason of accidental death. Most patients are adolescents and the treatment of TBI cost a large financial problem. Currently, most medication treatments are only designized for symptoms or sequelae. It is important to develop an effective drug and reduce medical expenses for TBI therapy. When TBI happened, large amount of glutamate was released. These released glutamate enhanced oxidative stress and inflammation generation, resulting in nerve cells damage. These physiological character caused the poor prognosis and increased patients’ mortality. Until now, there is no effective treatment for TBI. Platonin is the clinical drug approved for treatment of burns, cuts, ulcers and acute inflammation caused by rheumatoid arthritis. previous study has shown that platonin exhibits the functions in antioxidant, anti-inflammatory and immunomodulatory. However, the role of platonin in TBI therapy has not been clarified.
In our previous study, glutamate-treated astrocyte (CTX TNA2) was established as a TBI cell model to mimic glutamate-induced cell damage. The TBI 0310 Impactor-hit Spraque-Dawley rat cerebral cortex was used to explore platonin therapeutic mechanisms. In vitro, we found that glutamate could trigger reactive oxygen species (ROS) generation, followed by activating glycogen synthase kinase-3β (GSK-3β). Activated GSK-3β enhanced downstream transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) accumulation in nucleus resulting in CTX TNA2 cell death. To continue these findings, the present study began with a MTT assay that platonin attenuated glutamate-induced CTX TNA2 cells death. The results of immunoblotting assay were also found that glutamate increased the phosphorylation levels of GSK-3β on Tyr216 position, reducing phosphorylation levels of GSK-3β on Ser9 position, increasing nuclear Fyn accumulation, reducing amount of Nrf2 in the nucleus and reducing HO-1 levels. Glutamate co-treated with platonin could reverse the above phenomenon. We suggest that platonin has a protective effect on glutamate-induced CTX TNA2 cell death through reduced GSK-3β activation, decreased Fyn and increased Nrf2 accumulation in the nucleus. Moreover, we also found reduced pro form of poly ADP ribose polymerase (pro-PARP) and phosphorylation levels of GSK-3β on Ser9 position in TBI rat brain tissues. platonin could reverse TBI-induced GSK-3β activation and apoptosis in vivo. Our findings will be used to explore the improvement of the memory disorder by platonin treatment.
This study clarifies the efficacy of platonin in TBI therapy through cells and animal models. Our findings will be used to develop the possibility of clinical applications and provide new opportunities for the treatment of TBI. |
