探討咖啡酸苯乙酯及芹菜素透過調節鐵死亡路徑對第三型小腦脊髓運動失調症的神經保護作用

第三型小腦脊髓運動失調症為體染色體顯性遺傳的神經退化性疾病，其致病基因 ATXN3 發生 CAG 三核苷酸異常擴增，轉譯出 Poly Q 突變 Ataxin-3 蛋白，造成錯誤折疊與不可溶聚集體堆積，進而引發嚴重的氧化壓力與粒線體損傷。許多研究發現，突變蛋白引發的氧化壓力會誘發鐵質代謝紊亂與抗氧化系統瓦解，促使細胞內游離亞鐵離子過度累積並參與芬頓反應，導致嚴重的脂質過氧化，最終引發神經細胞鐵死亡，加速病程惡化。本研究旨在探討天然類黃酮化合物咖啡酸苯乙酯與芹菜素透過調節鐵死亡路徑對 SCA3 的神經保護作用。利用穩定表達全長倍增 MJD 蛋白之人類神經腫瘤母細胞株模式，並藉由活細胞螢光染色、流式細胞儀、西方墨點法及酵素免疫分析法進行鐵死亡機制探討。結果顯示，SK-N-SH-MJD78 細胞具有顯著的二價亞鐵離子累積、自噬酸性囊泡與脂質過氧化物堆積，並伴隨發炎物質 (TNF-α、IL-1β) 分泌量增加，以及抗氧化防禦系統中 SLC7A11 (xCT)、SLC3A2、GPX4 蛋白表現與還原型穀胱甘肽含量的急劇下降，最終導致粒線體膜損傷。以 CAPE 與 API 處理 48 小時後，能活化鐵輸出蛋白 FPN1 並調控鐵自噬蛋白 NCOA4 、 FTH1 以重塑正常鐵代謝，顯著減少細胞內亞鐵離子的堆積。且有效增強 SLC7A11 (xCT)-GSH-GPX4 軸抗氧化路徑的活性，提升 GSH 與 GPX4 蛋白的表現量，進而緩解氧化壓力、阻斷脂質過氧化的自由基連鎖反應、降低臨床毒性指標 MDA 與 4-HNE 的生成。最後，經 CAPE 與 API 處理皆能顯著修復並改善因鐵死亡造成的粒線體膜電位障礙。綜上所述，本論文顯示 CAPE 與 API 具備增強內源性抗氧化防禦、重塑鐵質平衡並阻斷脂質過氧化的神經保護策略，能有效減緩 MJD78 細胞走向鐵死亡，展現出延緩 SCA3 臨床病程進展與治療之關鍵潛力。

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by an abnormal expansion of CAG trinucleotide repeats within the ATXN3 gene. This mutation leads to the translation of a polyglutamine (polyQ)-expanded mutant Ataxin-3 protein, which forms misfolding and insoluble aggregates, subsequently inducing severe oxidative stress and mitochondrial damage. Emerging evidence indicates that mutant protein-induced oxidative stress triggers iron dyshomeostasis and the collapse of the antioxidant system. This cascade promotes the aberrant accumulation of intracellular labile iron pool and fuels the Fenton reaction, resulting in severe lipid peroxidation and ultimately executing ferroptosis neuronal death, thereby accelerating disease progression. This study was to investigate the neuroprotective effects of two natural flavonoids, caffeic acid phenethyl ester (CAPE) and apigenin (API), against SCA3 by modulating the ferroptosis pathway. Utilizing a human neuroblastoma cell line stably expressing full-length expanded MJD protein, we elucidated the underlying ferroptosis mechanisms through fluorescence staining, flow cytometry, Western blot, and ELISA. Our results demonstrated that SK-N-SH-MJD78 cells exhibited significant accumulation of Fe²⁺, acidic vesicular organelles (AVOs), and lipid peroxides. This was accompanied by an elevated secretion of inflammatory cytokines (TNF-α、IL-1β), alongside a precipitous decline in reduced glutathione (GSH) content and the expression of key antioxidant defense proteins, including SLC7A11 (xCT), SLC3A2, and GPX4, which ultimately culminated in the collapse and dysfunction of mitochondrial membrane potential. Remarkably, treatment with CAPE or API successfully remodeled iron metabolism by activating the iron exporter FPN1 and regulating the ferritinophagy-related proteins NCOA4 and FTH1, thereby significantly reducing intracellular iron accumulation. Furthermore, these treatments robustly enhanced the activity of the SLC7A11 (xCT)-GSH-GPX4 antioxidant axis, upregulating GSH and GPX4 protein expression. Consequently, this intervention alleviated oxidative stress, intercepted the free radical chain reactions of lipid peroxidation, and diminished the production of MDA and 4-HNE. Finally, MitoTracker revealed that CAPE or API significantly restored and ameliorated the mitochondrial membrane potential impairments induced by ferroptosis. In conclusion, this study demonstrates that CAPE and API exert potent neuroprotective strategies by fortifying antioxidant defenses, restoring iron homeostasis, and neutralizing lipid peroxidation. These actions effectively mitigate ferroptosis cell death in MJD78 cells, highlighting their pivotal therapeutic potential in delaying the clinical progression of SCA3.