建立薑黃素引發斑馬魚肌肉之病變模式

Abstract

細胞壞死性凋亡（Necroptosis）是一種利用絲/蘇安酸激酶和受體 (receptor-interacting serine/threonine protein kinases; RIPK)交互作用的非依賴凋亡蛋白酶(caspase)式的調節性細胞死亡。先前研究指出其功能廣泛參與於發炎反應、自體免疫、以及神經退化性疾病等。此外 heat shock protein 90 (hsp90) 蛋白曾被報導在細胞壞死性凋亡中具有重要角色，包含可以穩定RIP1蛋白(receptor-interacting protein 1)的穩定性以及RIP3蛋白(receptor-interacting protein 3)的啟動等。實驗室先前的研究證明了細胞凋亡以及細胞壞死性凋亡皆會參與薑黃素(curcumin)引發斑馬魚的肌肉病變以及尾部潰爛的現象中。將受精後24小時的斑馬魚胚胎浸泡在具有薑黃素的養殖水中再依序加入necrostatin-1 (RIP1 抑制劑)、dabrafenib (RIP3 抑制劑、17-DMAG (hsp90 抑制劑) 處理三小時後，發現薑黃素引發斑馬魚的尾部潰爛的現象中可能為 rip-1、rip3依賴性的途徑，且可被其抑制劑阻斷潰爛現象發生。在此實驗中，我們透過TUNEL assay 以及切割的 caspase-3 抗體免疫染色以及西方墨點法實驗進一步確認細胞凋亡途徑參與在薑黃素引發斑馬魚的尾部潰爛現象中。我們也同時偵測了斑馬魚胚胎中RIP1的蛋白表現量在此過程中的變化以及其角色。接著我們使用不同濃度的17-DMAG 去抑制薑黃素引發斑馬魚的尾部潰爛的現象，並發現其為最有效率的抑制劑。綜合以上，我們的研究模式顯示hsp90在細胞壞死性凋亡的重要性以及證明了在薑黃素引發斑馬魚的尾部潰爛與細胞壞死性凋亡有重要的關聯性。

Necroptosis is a caspase-independent form of regulated cell death that has been depended on the activity of receptor-interacting serine/threonine protein kinases. It has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. In addition, heat shock protein 90 (hsp90) has been reported to play important roles in necroptosis, including RIP1 (receptor-interacting protein 1) stability and RIP3 (receptor-interacting protein 3) activation. Previous studies indicated that both apoptosis and necroptosis were involved in the process of curcumin-induced myopathy in zebrafish. 24 hpf zebrafish embryos were treated with curcumin and followed by various known necroptosis inhibitors (necrostatin-1, dabrafenib, 17-DMAG) for 3 hr. The data suggested that curcumin-induced myopathy might be a rip1- and rip3-dependent pathway because of its inhibition by necroptosis inhibitors. In this study, we have further confirmed that apoptosis indeed plays a role in the progress of curcumin-induced myopathy in zebrafish by performing TUNEL assay and cleaved Caspase-3 to label cells that undergoing apoptosis. We also monitored the changes in rip1 protein expression in order to study the role of necroptosis in the progress of curcumin-induced myopathy in zebrafish. Next, we investigated the effect of 17-DMAG (hsp90 inhibitor) in curcumin-induced myopathy and were able to prove that 17-DMAG is the most powerful inhibitor to block the curcumin-induced myopathy progression in zebrafish. In summary, our model has revealed the importance of hsp90 in necroptosis pathway and the relationship between curcumin-induced myopathy and necroptosis.