利用斑馬魚模式探討蔴薏水萃物對於新黴素誘導耳損傷的保護作用

胺基醣苷類抗生素（如新黴素）是臨床上造成耳毒性與永久性毛細胞損傷的主要原因之一，其機制與誘導活性氧 (ROS) 累積引發的氧化壓力及細胞凋亡有關。然而，目前尚無 FDA 核准的預防性耳保護藥物，使具備抗氧化與抗發炎潛力的植物天然物成為極具潛力的研究方向。本研究旨利用毛細胞具表達綠色螢光的基因轉殖斑馬魚Tg (pvalb3b::TagGFP)，評估蔴薏 (Corchorus capsularis L., CCL) 水萃取物對於新黴素所誘導的毛細胞損傷是否具有保護效果，並深入探討其潛在的抗氧化與抗發炎機制。研究首先透過 DPPH 與 ABTS 分析蔴薏水萃物之自由基清除能力、定量總多酚及總類黃酮含量。接著以0.5mg/ml到10mg/ml濃度的CCL水萃物處理一小時，評估急毒性。毛細胞保護作用部分，預先以不同濃度蔴薏水萃物處理一小時後，隨後以12.5 µM新黴素共處理30分鐘，並計算前、側線毛細胞數進行評估，隨後透過ROS檢測氧化壓力，並透過FM4-64、TUNEL染色評估細胞膜完整性和機械傳導功能與細胞凋亡情形、Neutral red染色探討損傷造成的免疫細胞聚集，最後進行行為學分析與 RT-PCR 檢測抗氧化相關基因的表達。結果顯示蔴薏具優異抗氧化活性與自由基清除能力，並確立1 mg/ml為最佳毛細胞保護濃度，經蔴薏水萃物預處理後，毛細胞存活率顯著提升，其機械傳導功能得以維持，並降低ROS累積與抑制細胞凋亡，並減緩由損傷引發的巨噬細胞聚集，最終改善游動行為異常。此外，可恢復新黴素處理後下降的抗氧化基因(nrf2、keap1及下游酵素)表現。綜合以上結果，證實蔴薏水萃物對於新黴素誘導耳毒性具有抗氧化、抗凋亡及免疫調節等多重保護作用，未來可望成為天然耳保護藥物後選物。

Aminoglycoside antibiotics, such as neomycin, are a primary cause of ototoxicity and permanent hair cell damage, a mechanism attributed to oxidative stress from reactive oxygen species (ROS) accumulation and subsequent apoptosis. Currently, no FDA-approved prophylactic otoprotectants are available, making natural products with antioxidant and anti-inflammatory potential promising candidates for investigation. This study aimed to evaluate the protective effects of an aqueous extract of Corchorus capsularis L. (CCL) against neomycin-induced hair cell damage in a transgenic zebrafish model, Tg(pvalb3b::TagGFP), and to investigate its underlying antioxidant and immunomodulatory mechanisms. Initially, the free-radical scavenging activity and the total phenolic and flavonoid contents of the CCL extract were determined using DPPH and ABTS assays. Following an acute toxicity assessment, the otoprotective effects were evaluated by pretreating zebrafish with various concentrations of CCL for one hour, followed by co-treatment with 12.5 µM neomycin for 30 minutes. Hair cell survival was quantified in the anterior and posterior lateral lines. The underlying mechanisms were explored by assessing oxidative stress (ROS detection), cell membrane integrity and mechanotransduction (MET) function (FM4-64 staining), apoptosis (TUNEL assay), and immune cell aggregation (Neutral red staining). Finally, behavioral analysis and RT-PCR for antioxidant-related gene expression were performed.Results showed that the CCL extract possessed excellent antioxidant activity, with 1 mg/ml identified as the optimal protective concentration. Pretreatment with CCL significantly increased hair cell survival, maintained MET function, reduced ROS accumulation, and inhibited apoptosis. It also attenuated damage-induced macrophage aggregation and ameliorated abnormal swimming behaviors. Furthermore, CCL treatment restored the expression of antioxidant genes (nrf2, keap1, and their downstream enzymes) that were downregulated by neomycin. In conclusion, the aqueous extract of Corchorus capsularis exerts multi-target protective effects against neomycin-induced ototoxicity through antioxidant, anti-apoptotic, and immunomodulatory mechanisms, suggesting its potential as a candidate for developing natural otoprotective drugs.