篩選調控粒線體型態之潛力藥物及其於抗衰老之應用

Abstract

粒線體是一種動態的胞器，它會進行分裂或融合進而調控粒線體的功能以適應細胞環境的改變。然而，粒線體動力學的失衡會導致多種疾病的臨床進展，包括神經退化性疾病，代謝性疾病和癌症。近年許多研究指出，粒線體的功能會隨著年齡增加而逐漸下降，衰老的細胞常伴隨著粒線體動力學的失衡、ATP製造減少、ROS製造增加等等的粒線體功能缺失，而此與老化相關疾病的進展有相當大的關聯。然而，目前有效的抗老化粒線體藥物並不多。因此，為了找出能夠調節粒線體型態的潛力藥物，我們利用高通量螢光影像分析系統(High Content Screening system)以篩選Library of Pharmacologically Active Compounds (LOPAC)中可明顯調控粒線體型態的藥物。我們初步地利用肉眼判讀結果並以未加藥的控制組作為標準後，根據藥物改變粒線體型態的程度打分數。經由初步篩選1280個藥物後，先挑選出14個會明顯改變粒線體型態的藥物進行進一步驗證。而後，從14個藥物中挑選出以劑量依賴性的方式調節粒線體型態的3個藥物(Cortisone, TBBz, Diphenyleneiodonium chloride(DPI))進行後續的機制探討、粒線體功能測試以及探討其對老化的影響。在機制探討的方面，3個藥物皆不會影響粒線體動力學相關基因的表現量，亦不是藉由改變粒線體分裂蛋白Drp1 Ser616位點的磷酸化以及粒線體融合蛋白Opa1切割、Mfn2的表現量來調控粒線體型態。在粒線體功能測試方面，Cortisone和TBBz皆不會對糖解作用及氧化磷酸化造成影響，而DPI則是會抑制氧化磷酸化、促進糖解作用及ATP的產生，且其亦會導致氧化磷酸化相關的基因下調。此外，推測DPI可能是藉由促進粒線體分裂而減緩氧化壓力所誘導的細胞衰老。為了了解DPI是否可以成為預防老化的藥物，我們也積極測試用於長期治療的適當濃度，並且將利用小鼠胚胎成纖維細胞(MEF)作為細胞模型觀察藥物對於延緩複製性衰老的作用。

Mitochondria are a highly dynamic organelle that constantly undergoes fission and fusion, and this plays an important role in regulating mitochondrial functions. Imbalance of mitochondrial dynamics contributes to the clinical progression of a variety of diseases, including neurodegenerative diseases, metabolic diseases, and cancers. According to the previous studies, imbalance of mitochondrial dynamics and mitochondrial dysfunction are observed in senescent models, and this is extremely associated with the progression of age-related disorders. However, there are few effective anti-aging drugs targeting mitochondria nowadays. Therefore, we combined the LOPAC®1280 compound library with the High Content Screening system to screen potential drugs that can significantly modulate mitochondrial morphology. Mitochondrial morphology is observed and scored according to comparison with the untreated control group to select drugs. There are fourteen drugs selected for further validation after the preliminary screening of 1280 compounds. Among them, three drugs (Cortisone, TBBz, Diphenyleneiodonium chloride(DPI)) modulating mitochondrial dynamics in a dosage-dependent manner are selected for the subsequent experiments. Cortisone can induce fusion while TBBz and DPI can induce fission. There is no difference in the expression of mitochondrial dynamics-associated mRNA after treatment with these three drugs. They seem to regulate mitochondrial dynamics via neither phosphorylating DRP1 at Ser616, OPA1 cleavage, nor MFN2 expression. Cortisone and TBBz do not influence mitochondrial respiration, while DPI can inhibit oxidative phosphorylation (OXPHOS), promote glycolysis and ATP production, and it causes downregulation of OXPHOS-associated genes. In addition, we speculate that DPI may be able to inhibit oxidative stress-induced senescence through mitochondrial fission. Finally, in order to investigate whether DPI can act as an aging-preventing drug, the optimal concentration for long-term treatment is necessary to be determined first. In the future, we will assess the effect of DPI on ameliorating replicative senescence in MEFs.