- 學位論文
二十碳五烯酸經由PGC-1α與p38 MAPK路徑刺激粒線體生合成
Eicosapentaenoic acid (EPA) stimulates Mitochondrial Biogenesis through PGC-1α and p38 MAPK pathways
摘要
過氧化小體增生活化受體γ共同活化子-1α (peroxisome proliferator- activated receptor γ coactivator -1α, PGC-1α)是調節粒線體生合成的重要調節物。我們實驗室先前已發現二十碳五烯酸(eicosapentaenoic acid, EPA)可增加PGC-1α與TFAM (mitochondrial transcription factor A)基因表現與粒線體功能。本次實驗探討EPA是否經由p38裂質素活化蛋白激酶(p38 mitogen-activated protein kinase, p38 MAPK)路徑調控PGC-1α基因表現，進而影響粒線體相關基因表現。結果發現給予25 μM的EPA處理後，會顯著增加p38 MAPK蛋白質磷酸化，但不改變p38蛋白質表現，且使用共軛焦顯微鏡觀察到磷酸化之p38會轉位進入細胞核中，然而隨著EPA處理時間增加，核內之轉錄因子CREB (cAMP response element binding protein)與ATF2 (activating transcription factor 2)蛋白質磷酸化亦有增加傾向。使用報導基因分析發現，給予25 μM的EPA 24小時處理會增加PGC-1α促進子轉錄活性。使用同步定量聚合酶反應分析發現，EPA處理會增加粒線體DNA (mitochondrial DNA, mtDNA)之套數，且投予48小時之EPA會促使mtDNA轉譯的蛋白質ND6 (NADH dehydrogenase 6)表現傾向增加，但不影響核轉譯之粒線體複合體蛋白質ATP synthase與succinate ubiquinol oxidoreductase蛋白質表現。而給予EPA不同濃度與時間處理，低於100 μM才不會影響C6神經膠瘤細胞的細胞型態與產生細胞毒性。本次實驗支持予C6神經膠瘤細胞EPA處理會刺激PGC-1α轉錄且影響粒線體功能是透過p38 MAPK路徑。
並列摘要
Peroxisome proliferator- activated receptor γ coactivator -1α (PGC-1α) is a key regulator of mitochondrial biogenesis. Our previous studies have been reported that eicosapentaenoic acid (EPA, n-3 C20:5) could increase pgc-1α and TFAM gene expression and mitochondrial function. To determine whether EPA stimulated pgc-1α gene transcription and resulted in elevated mitochondrial function were through the activation of the p38 mitogen-activated protein kinase (MAPK) pathway, we investigated the role of p38 MAPK in EPA-induced transcription of pgc-1α and mitochondrial gene. We showed increased phosphorylation level of p38 MAPK in C6 glioma cells that treated with 25 μM EPA and no changed levels of total-p38 (T-p38) determined by immunobloting. Following the treatment of 25 μM EPA increased phosphorylation level of CREB and ATF2. After treatment with 25 μM EPA for 24 hr, the pgc-1α promoter transcriptional activity was increased by reporter assay. The raised mitochondrial DNA (mtDNA) copy number was also determined in the same treated cells by real-time PCR analysis. EPA treatment could raise the expression of mtDNA encoded mitochondrial protein-ND6, but not nuclear encoded mitochondrial protein-ATP synthase and succinate ubiquinol oxidoreductase. However, there was no morphology change in cells treated with both of 25 μM and 50 μM EPA for 48 hr. These studies supported that EPA stimulated pgc-1α transcription and further resulted to augmente mitochondrial function in C6 glioma cell were mediated through the activation of the p38 MAPK pathway.