利用包覆槲皮素與迷迭香酸之磷脂微脂粒負載載脂蛋白抑制β-amyloid誘發SK-N-MC細胞的絲裂原活化蛋白酶磷酸化及沉積

Using quercetin and rosmarinic acid encapsulated into phosphatidic acid liposomes grafted with apolipoprotein E for inhibition of SK-N-MC cells against β-amyloid induced phosphorylation of mitogen-activated protein kinases and deposit

鄭士珏(SHIH-CHUEH CHENG)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/碩士(2016年)

摘要

本研究將phosphatidic (PA)組成的微脂粒(liposomes)包覆槲皮素(quercetin, QU)和迷迭香酸(rosmarinic acid, RA)做為藥物載體，以載脂蛋白(apolipoprotein, ApoE)接枝於載體表面，並探討PA的標靶治療對蛋白質磷酸化的影響。本實驗利用β-Amyloid (Aβ)誘發細胞毒性於人類神經胚胎腫瘤細胞株SK-N-MC cells的蛋白質磷酸化，建立一個體外模型來模擬阿茲海默症(Alzheimer’s disease, AD)蛋白質磷酸化的情形。在AD體外模型建立，實驗結果顯示Aβ誘發SK-N-MC細胞的JNK和ERK及p38蛋白磷酸化(p-JNK:0.98; p-ERK:0.89；p-p38:0.89)明顯高於控制組(p-JNK: 0.29; p-ERK:0.39；p-p38:0.20)，而經過含有PA作為標靶且分別包覆槲皮素與迷迭香酸的微脂粒治療的研究結果顯示能有效抑制JNK、ERK、p38蛋白磷酸化的表現量(p-JNK: 0.42; p-ERK:0.45；p-p38:0.43)，減緩細胞凋亡，達到能使細胞存活的效果。由免疫螢光染色證實以10 μM的Aβ處置SK-N-MC細胞24 h後，因為caspase-3的表現量明顯增加，達到細胞凋亡的效果。
此外，於動物實驗，直接將Aβ注射在Wistar大鼠的海馬迴CA1區域建立體內模型，使大腦海馬迴神經細胞受到傷害，經過2個星期後確實能看到Aβ沉積以及斑塊的形成，經藥物治療後顯示，經由quercetin和rosmarinic acid包覆於PA-liposomes表面修飾ApoE的治療，能有效提升血腦屏障(blood-brain barrier, BBB)的穿透率，抑制Aβ誘發的神經病變並減少斑塊的沉積形成。故未來可應用於阿茲海默症的臨床治療。

關鍵字

阿茲海默症

並列摘要

Liposomes containing phosphatidic acid (PA) and with a modified ApoE peptide encapsulate quercetin (QU) and rosmarinic acid (RA) as drug carrier to explore PA on protein phosphorylation targeting treatment. In this study, we used β-Amyloid (Aβ) to induce phosphorylation of protein in SK-N-MC cells for simulating Alzheimer’s disease (AD) in vitro model. In AD vitro model, the experimental results show that Aβ-induced SK-N-MC cells, JNK and ERK and p38 protein phosphorylation (p-JNK: 0.98; p-ERK: 0.89; p-p38: 0.89) was significantly higher than the control group (p-JNK: 0.29; p-ERK: 0.39; p-p38: 0.20). And after containing PA as a target liposomes therapy shown in effect to inhibit JNK, ERK, p38 protein phosphorylation performance (p-JNK: 0.42; p -ERK: 0.45; p-p38: 0.43), slow down cells apoptosis, to make the cell survival effect. Immunofluorescence staining was confirmed by a 10 μM of Aβ disposal of SK-N-MC cells for 24 h, because of the amount of caspase-3 expression was significantly increased, reaching apoptotic effect. Furthermore, Aβ1-42 injected directly into the hippocampus CA1 region of Wistar rats to established in vivo model, after two weeks the formation of Aβ deposition can and plaques, surface ApoE is efficient in transporting PA-liposomes across the BBB and inhibiting the degeneration of SK-N-MC cells with Aβ-induced neurotoxicity. Therefore, the future can be used in clinical treatment of Alzheimer's disease.

並列關鍵字

Alzheimer's disease ； apolipoprotein E ； beta-amyloid ； blood-brain barrier ； phosphorylation ； quercetin ； rosmarinic acid ； MAPK pathway

參考文獻

[1] M.Goedert, M.G. Spillantini, “A century of Alzheimer's disease,” Science, 314, 777–781, 2006.

[2] D. Brambilla, B. Le Droumaguet, J. Nicolas, S. H. Hashemi, L. P. Wu, S. M. Moghimi, P. Couvreur, K. Andrieux, “Nanotechnologies for Alzheimer's disease: diagnosis, therapy, and safety issues,” Nanomedicine, 7, 521–540, 2011.

[3] M. Basso, J. Yang, L. Warren, M. G. MacAvoy, P. Varma, R. A. Bronen, C. H. van Dyck, “Volumetry of amygdala and hippocampus and memory performance in Alzheimer's disease,” Psychiatr. Res., 146, 251–261, 2006.

[4] T. S. Anekonda, P.H. Reddy, “Can herbs provide a new generation of drugs for treating Alzheimer's disease?,” Brain Res. Rev., 50, 361–376, 2005.

[5] H. Hampel, R. Frank, K. Broich, S. J. Teipel, R. G. Katz, J. Hardy, “Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives,” Nat. Rev. Drug Discov., 9, 560–574, 2010.