細胞外囊泡（extracellular vesicles， EVs）是細胞間訊息傳遞的重要媒介，其中外泌體主要源自內體系統，經由內體膜向內出芽形成腔內囊泡，進一步形成多泡體（multivesicular ibodies， MVBs）。MVB 後續可與溶酶體融合並進行降解，亦可與細胞膜融合而將外泌體釋放至細胞外。此過程涉及 ESCRT 相關貨物分選、Rab GTPase 介導的囊泡運輸、細胞膜錨定及膜融合等多項調控機制。已有研究指出，細胞內氧化壓力的增加可能影響內體成熟、溶酶體功能及 EV 釋放，但降低細胞內氧化水平是否直接與抑制或促進外泌體生成有影響，目前仍然未知。 因此，本研究以 A375 人類黑色素瘤細胞及 WJMSC 臍帶間質幹細胞為研究模型，探討不同抗氧化處理所造成的細胞內氧化狀態改變，是否會影響外泌體生合成、MVB 命運及 EV 釋放。研究中分析細胞內及小型細胞外囊泡( small extracellular vesicles ， sEVs) 中之外泌體與 MVB 相關蛋白，包括 CD63、CD81、TSG101、Syn-1、Rab27a、Rab7、WDR4 及 LAMP1，並搭配奈米粒子追蹤分析（nanoparticle tracking analysis， NTA），評估不同處理對 sEVs 生成與釋放的影響。 研究結果顯示，細胞內氧化水平與外泌體生成之間並非單一線性之關係，而具有細胞類型及抗氧化物種類依賴性。在 A375 細胞中，抗氧化處理可造成多種外泌體或 MVB 相關蛋白表現改變，顯示腫瘤細胞可能隨細胞內氧化狀態變化，重新調節內體貨物分選機制、MVB 生成、囊泡運輸及外泌體釋放路徑。相較之下，在 WJMSC 細胞中，Compound A 與 Compound Compound .ß 雖皆可降低細胞內氧化水平，卻對 sEV 釋放產生不同影響。Compound A 處理後，儘管部分細胞內 MVB 或溶酶體相關蛋白表現下降，單次NTA檢測中sEV 數量增加，顯示其可能促進 MVB 與細胞膜 融合或提高囊泡釋放效率，而非單純增加 MVB 相關蛋白表現。另一方面，Compound ß 處理後雖使部分細胞內囊泡運輸相關蛋白上升，但 外泌體標誌蛋白表現及 單次NTA檢驗 結果均顯示 sEV 釋放下降，；細胞內 Rab7 與 LAMP1 顯著增加，同時 Rab27a 與 WDR4 亦顯著增加，顯示分泌與溶酶體相關路徑可能同時受到調控，因此現有結果尚不足以將 MVB 命運簡化為單一降解方向。推測此處理可能改變 MVB 的命運，使其較傾向進入溶酶體降解途徑，而非與細胞膜融合並釋放至細胞外。 綜合而言，本研究顯示，抗氧化物對外泌體生成及 sEV 釋放的影響，無法僅以降低細胞內氧化水平作為單一且唯一解釋，而應同時考量其對內體成熟、MVB 生成、貨物分選、囊泡運輸、溶酶體功能及膜融合效率的整體調控。不同細胞來源對氧化還原狀態變化具有不同反應，顯示腫瘤細胞與間質幹細胞可能採取不同的內體—溶酶體路徑調控策略。本研究支持細胞內氧化狀態參與外泌體生合成調控的觀點，並進一步指出，不同抗氧化物可能透過改變 MVB 命運選擇與 sEV 釋放效率，影響細胞間訊息傳遞。;Extracellular vesicles (EVs) serve as pivotal conduits for intercellular communication. Among distinct EV subpopulations， exosomes predominantly originate from the endosomal pathway via inward budding of the endosomal membrane， resulting in the generation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). Subsequently， MVBs face alternative physiological fates: undergoing lysosomal fusion for degradation， or docking and fusing with the plasma membrane to liberate exosomes into the extracellular milieu. This delicate machinery is governed by an intricate regulatory network encompassing ESCRT-dependent cargo sorting， Rab GTPase-mediated vesicular trafficking， and specialized membrane tethering and fusion events. Although elevated intracellular oxidative stress has been implicated in modulating endosomal maturation， lysosomal clearance， and EV secretion， whether attenuating intracellular redox status directly promotes or suppresses exosome biogenesis remains poorly defined. To elucidate whether variations in intracellular redox states exert a direct influence on exosome biogenesis， MVB fate determination， and sEV release， we employed human melanoma A375 cells and Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) subjected to distinct antioxidant interventions. Exosome- and MVB-associated molecular signatures—including CD63， CD81， TSG101， Syntenin-1 (Syn-1)， Rab27a， Rab7， WDR4， and LAMP1—were systematically profiled in both cellular lysates and isolated small extracellular vesicles (sEVs)， accompanied by nanoparticle tracking analysis (NTA) to quantify physical secretion kinetics. Our findings reveal a non-linear relationship between intracellular oxidative levels and exosome biogenesis， characterized by pronounced cell-type specificity and antioxidant dependency. In A375 cells， antioxidant treatment dynamically recalibrated the expressionsignatures of exosomal and MVB-associated proteins， indicating that malignant cells reconfigure cargo sorting， MVB generation， and exocytotic pathways in response to redox fluctuations. Conversely， in WJMSCs， Compound A and Compound ß triggered divergent sEV secretion profiles despite achieving comparable efficacy in scavenging intracellular reactive species. Notably， Compound A administration selectively enhanced sEV release kinetics—as reflected by elevated particle counts—despite the downregulation of specific intracellular MVB/lysosomal proteins， suggesting that Compound A facilitates plasma membrane fusion efficiency rather than merely driving vesicle synthesis. In stark contrast， while Compound ß upregulated specific trafficking-related intracellular machinery， both molecular markers and NTA metrics converged on a net reduction in sEV secretion. Crucially， the marked upregulation of Rab7 and LAMP1， alongside elevated Rab27a and WDR4 levels， implies a concurrent activation of both secretory and degradative machinery. Rather than adhering to a simplified exocytotic route， MVBs under Compound $\beta$ treatment are preferentially shunted toward lysosomal degradation over plasma membrane exocytosis. Collectively， this study demonstrates that antioxidant-mediated modulation of exosome biogenesis extends beyond a generic reduction in oxidative load. Instead， it reflects an integrated recalibration of endosomal maturation， cargo sorting， vesicular trafficking， and membrane fusion kinetics. By demonstrating that distinct cell types and antioxidant moieties differentially govern MVB fate selection， these findings establish intracellular redox status as a crucial molecular rheostat in extracellular vesicle biology.
