利用斑馬魚探討龍眼花水萃物對放化療造成毛細胞損傷的保護效果

以放射治療合併順鉑 (Cisplatin) 化療控制腫瘤是許多癌症的治療方式，例如頭頸癌等，雖具療效，卻常伴隨感音神經性聽力損失 (Sensorineural Hearing Loss, SNHL)。此副作用主要源於X光照射及Cis治療過程中產生過量自由基，進而導致毛細胞不可逆氧化損傷。龍眼花水萃物 (Longan Flower Water Extract, LFWE) 富含酚酸與類黃酮等天然植化素，已被證實具抗氧化能力。本研究旨在利用內耳聽斑與側線毛細胞會表達綠色螢光的基因轉殖斑馬魚Tg(pvalb3b:TagGFP) 評估LFWE對順鉑或X光所引發之聽覺毛細胞損傷的保護效果。首先以0 mg/ml到1.0 mg/ml濃度的LFWE處理斑馬魚1小時，評估其急毒性，針對Cis引發的耳毒性部分，實驗以不同濃度的LFWE前處理1小時後，再與順鉑共處理1小時，並透過計算前、後側線活毛細胞數進行評估，隨後進一步透過FM4-64、TUNEL及ROS染色探討其可能的保護機制，最後進行行為分析分析藥物處理後的斑馬魚的游動距離、速度與轉彎頻率等表現。結果顯示，即使以1.0 mg/ml的LFWE處理1小時，亦不影響斑馬魚存活率，且0.2 mg/ml 濃度具有最佳的毛細胞保護效果。染色結果，證實LFWE可有效抑制自由基累積並減少毛細胞凋亡。此外，行為分析顯示，經LFWE前處理可改善順鉑所造成的游動異常行為。 在探討X光引發之聽覺毛細胞損傷方面，由於其損傷具有延遲性，本研究首先評估了X光對毛細胞的長期影響，然後探討LFWE對X光引發之聽覺毛細胞損傷的保護效果。首先觀察並紀錄照射0 Gy到16 Gy之X光後10天內斑馬魚及其毛細胞的存活率，評估X光對毛細胞的長期影響，接著，在照射16 Gy之X光後處裡新黴素 (Neomycin) 30分鐘，並觀察紀錄之後10天內斑馬魚及其毛細胞的存活率，評估X光對毛細胞再生的影響，最後，在照射16 Gy之X光前處理1小時0.25 mg/ml到0.75 mg/ml的LFWE。結果顯示，在照射不同劑量之X光後第10天，斑馬魚存活率約降至50%，而毛細胞的存活率隨照射劑量增加與照射後間隔時間延長而下降，並且X光可能抑制毛細胞再生，使毛細胞在照射X光後無法再生回正常毛細胞的數量。進一步以LFWE保護，發現每個濃度對X光誘導的毛細胞損傷在照射X光後十天內皆無保護效果。綜合以上結果，LFWE前處理可減緩順鉑引起的耳毒性傷害，這表明未來LFWE有潛力成為順鉑誘導耳毒性之保護藥物，但LFWE對X光誘導的傷害無保護作用，可能與兩者傷害發生的時效不同有關。未來可嘗試調整處理方式，以尋求對 X 光傷害具保護效果的用藥策略。

Radiotherapy combined with cisplatin chemotherapy is a common cancer treatment strategy, especially for head and neck cancer. However, it often leads to sensorineural hearing loss (SNHL). This side effect primarily results from the excessive generation of free radicals during X-ray irradiation and cisplatin treatment, causing irreversible damage to hair cells. Longan flower water extract (LFWE), rich in natural phytochemicals such as phenolic acids and flavonoids, has been proven to possess antioxidant properties. The aim of the present study is to assess the protective effect of LFWE on cisplatin-induced or X-ray-induced sensory hair cells loss using transgenic zebrafish Tg(pvalb3b:TagGFP), in which inner ear and lateral line hair cells express green fluorescence. We firstly determine the acute toxicity, treatment of the larvae with 0 to 1.0 mg/mL of LFWE for one hour. For cisplatin-induced ototoxicity, zebrafish were pretreated with various concentrations of LFWE for one hour, followed by co-treatment with cisplatin for one hour. The number of viable hair cells in the anterior and posterior lateral lines was then counted for evaluation. To further investigate the protective mechanism of LFWE on cis-induced hair cell loss, FM4-64 staining, TUNEL assay, and ROS detection were performed. Behavioral analyses were conducted to evaluate changes in swimming distance, speed, and turning frequency after drug treatment. Results showed that even at a concentration of 1.0 mg/ml, LFWE did not affect zebrafish survival, and the 0.2 mg/ml concentration provided the most effective hair cell protection. Fluorescent staining confirmed that LFWE effectively inhibited ROS accumulation and reduced hair cell apoptosis. Behavioral tests also showed that LFWE pretreatment improved cisplatin-induced abnormal swimming behavior. Regarding X-ray-induced auditory hair cell damage the study first evaluated the long-term effects of X-ray exposure. Zebrafish and their hair cell survival rates were monitored for 10 days after exposed to 0 to 16 Gy of X-rays. Next, to assess the impact on hair cell regeneration, zebrafish were treated with 30-minute neomycin after 16 Gy irradiation, and survival were tracked for another 10 days. Finally, zebrafish were pretreated with 0.25 to 0.75 mg/ml of LFWE one hour before 16 Gy X-ray exposure. Results showed that by day 10 post-irradiation, zebrafish survival dropped to around 50%, and hair cell survival declined with higher X-ray doses and longer time intervals. X-rays appeared to inhibit hair cell regeneration, preventing a return to normal hair cell numbers. LFWE pretreatment showed no protective effect against X-ray-induced hair cell damage within 10 days post-irradiation at any concentration. In conclusion, LFWE pretreatment effectively mitigated cisplatin-induced ototoxic damage, suggesting that LFWE has the potential to become a protective agent against cisplatin-induced hearing loss. However, LFWE showed no protective effect against X-ray-induced damage, possibly due to the difference in timing and mechanism of injury. Future studies could explore modified treatment strategies to identify effective protective measures against X-ray-induced ototoxicity.