利用斑馬魚模式評估綠原酸對藥物引起聽覺毛細胞損傷之保護作用

根據實驗室先前的研究發現扁桃斑鳩菊 (Vernonia amygdalina) 對毛細胞有保護作用，之後透過成分分析發現其中的綠原酸 (Chlorogenic acid, CGA) 含量最高，因此為了確認CGA是否對毛細胞有顯著的保護作用，將單獨使用CGA純物質作為毛細胞保護藥物來進行後續實驗。CGA是一種具有廣泛生物活性的多酚化合物，廣泛存在於多種植物中，如綠咖啡豆、蘋果及茶葉。其已證實具有神經保護、抗發炎、抗氧化、抗病原體等作用。耳毒性藥物常導致毛細胞損傷並誘發聽力喪失。本研究採用基因轉殖斑馬魚Tg(pvalb3b :: TagGFP) 作為模式動物，利用其內耳及側線神經丘中特異性表達綠色螢光的毛細胞，評估CGA對耳毒性藥物誘導毛細胞損傷的保護作用。實驗設計中，7天大 (7 days post-fertilization, dpf) 斑馬魚經不同濃度CGA (25 μM、50 μM、100 μM) 預處理1小時，隨後分別暴露於12.5 μM新黴素共處理30分鐘或250 μM順鉑共處理1小時。為進一步評估毛細胞功能，本研究採用FM4-64染色來確認不同濃度的CGA和耳毒性藥物共處理後對毛細胞型態和功能的保護效果，以及TUNEL 染色來確認耳毒性藥物引起的細胞凋亡發生在毛細胞中，結果顯示，低劑量CGA(25 μM、50 μM) 有保護效果，高劑量CGA(100 μM) 顯著保護毛細胞。還有使用行為學來檢測斑馬魚側線毛細胞受損後對整體運動能力和協調性的影響。數據分析顯示，本研究利用基因轉殖斑馬魚證實綠原酸對耳毒性藥物誘導的毛細胞損傷具有顯著保護作用。隨著CGA濃度升高，毛細胞存活率提高、功能恢復及細胞凋亡減少，並使用FM4-64和TUNEL染色結果進一步確認CGA對毛細胞功能的保護及抗細胞凋亡作用。此外，行為學分析顯示高劑量CGA能顯著降低耳毒性藥物對斑馬魚側線功能及運動行為的損害，保護效果隨CGA濃度增加呈劑量效應，後續能透過更多實驗來證實CGA的保護效果後，CGA有望能成為未來聽力保護藥物的選擇之一。

Previous studies in our laboratory demonstrated that Vernonia amygdalina exerts protective effects on hair cells, with subsequent component analysis revealing chlorogenic acid (CGA) as its most abundant constituent. To confirm whether CGA alone exhibits significant protective effects on hair cells, pure CGA was used as a protective agent in subsequent experiments. CGA, a polyphenolic compound with diverse bioactivities, is widely found in plants such as green coffee beans, apples, and tea leaves, and is known for its neuroprotective, anti-inflammatory, antioxidant, and antimicrobial properties. Ototoxic drugs often cause hair cell damage, leading to hearing loss. This study utilized transgenic zebrafish (Tg(pvalb3b::TagGFP)) as a model organism, leveraging the specific expression of green fluorescent protein in hair cells of the inner ear and lateral line neuromasts, to evaluate CGA’s protective effects against ototoxin-induced hair cell damage. In the experimental design, 7-day post-fertilization (dpf) zebrafish were pretreated with CGA at concentrations of 25 μM, 50 μM, and 100 μM for 1 hour, followed by co-treatment with 12.5 μM neomycin for 30 minutes or 250 μM cisplatin for 1 hour. Hair cell morphology and function were assessed using FM4-64 staining, while TUNEL staining was employed to detect ototoxin-induced apoptosis in hair cells. Results showed that low-dose CGA (25 μM and 50 μM) provided protective effects, with high-dose CGA (100 μM) significantly protecting hair cells. Behavioral assays were conducted to evaluate the impact of lateral line hair cell damage on overall motor ability and coordination. Data analysis confirmed that CGA significantly protected against ototoxin-induced hair cell damage in transgenic zebrafish, with increasing CGA concentrations correlating with higher hair cell survival, improved functional recovery, and reduced apoptosis. FM4-64 and TUNEL staining further validated CGA’s protective and anti-apoptotic effects on hair cell function. Additionally, behavioral analysis revealed that high-dose CGA markedly reduced ototoxin-induced damage to lateral line function and motor behavior, with protective effects exhibiting a dose-dependent response. These findings suggest that CGA holds promise as a potential candidate for future hearing protection therapies, pending further experimental validation.