奈米氧化鋅（ZnONPs）因其美白和強效吸收紫外線的特性，被廣泛應用於化妝品和防曬乳中。然而，關於其經皮滲透及隨後引起的皮膚毒性（特別是在紫外線輻射等環境壓力下）的安全疑慮，仍是一個關鍵且尚未解決的爭議。本研究旨在闡明重複經皮暴露於奈米氧化鋅結合 UVR 誘發發炎性皮膚疾病的潛在角色與詳細分子機制；具體而言，本研究探討了涉及 NLRP3 發炎體（inflammasome）、自體吞噬流（autophagic flux）以及胞外體（exosome）介導之細胞間通訊的複雜調節網絡，同時建立了一個基於細胞的替代測試框架，以減少動物實驗。
本研究製備了氨基化奈米氧化鋅（NH2-ZnONPs），並對其尺寸、形貌和表面電荷進行了分析。在動物試驗中，SKH-1 無毛鼠分別進行急性或重複暴露，結合單次劑量的 UVR 照射（150 mJ/cm²）以及隨後局部塗抹的奈米氧化鋅（2 mg/cm²）。研究過程中監測了經皮水分流失（TEWL）、表皮厚度及組織病理學變化。在細胞研究中，則利用人類角質形成細胞（HaCaT）和樹突狀細胞（THP-1）進行單獨或共同培養，以根據皮膚過敏不良結局路徑（AOP）框架來識別分子起始事件。
動物實驗結果顯示，同時暴露於 UVR 和 奈米氧化鋅會顯著破壞皮膚屏障，表現為 TEWL 水平升高和明顯的表皮增厚。從機制上看，奈米氧化鋅的細胞內吞作用引發了顯著的細胞壓力。在發炎體機制方面，共同暴露啟動了經典與非經典的 NLRP3 發炎體活化，進而驅動下游 caspase-1 的蛋白水解，並導致細胞焦亡(pyroptosis)。與此同時，細胞內奈米氧化鋅的累積導致了嚴重的自體吞噬功能障礙，其特徵為溶體膜通透化，從而阻斷了末端自體吞噬的降解並困住自噬體。在這種交織的壓力下，受損細胞加速向細胞外微環境釋放具有生物活性的胞外體。這些壓力誘導的胞外體作為關鍵的細胞間信使，將致病性信號貨物傳遞給未受影響的接受細胞，進一步擴大 NLRP3 發炎體的活化，並加劇嚴重的細胞發炎。動物實驗中局部塗抹紫檀芪（Pterostilbene, PT）能透過減少活性氧物質的生成以及降低線粒體損傷水平，進而抑制 NLRP3 發炎體活化以及細胞焦亡。除了其抗氧化作用外，紫檀芪還能恢復正常的自噬流並減少載有 NLRP3 炎性細胞焦亡的胞外體釋放，從而改善自噬異常。
此外，體外替代方法也模擬了奈米氧化鋅導致過敏性發炎的關鍵事件。利用整合的 AOP 測試分析，本研究觀察到暴露奈米氧化鋅導致致敏的細胞事件，包括 Keap1/Nrf2 調節的角質形成細胞過度活化，以及隨後的樹突狀細胞成熟，從而在不依賴傳統動物數據的情況下提供了預測準確性。
總結而言，這些研究結果表明，奈米氧化鋅與 UVR 協同誘發發炎性皮膚損傷。該過程形成了一個病理循環，其中自體吞噬障礙與 NLRP3 發炎體過度活化發生交互作用並透過胞外體的傳播顯著放大發炎反應。藉由繪製這些複雜的路徑並驗證穩健、非動物的 AOP 替代測試模型，本研究提供了深入的機制見解，並為管理奈米材料引起的皮膚毒性建立了具前景且可驗證的治療與監管指標。

Zinc oxide nanoparticles (ZnONPs) are extensively utilized in cosmetics and sunscreens due to their whitening and strong UV light absorption properties. However, safety concerns regarding their dermal penetration and subsequent skin toxicity—particularly under environmental stress like ultraviolet radiation (UVR)—remain a critical and unresolved debate. This study aims to elucidate the potential roles and detailed molecular mechanisms of repeated dermal exposure to ZnONPs combined with UVR inducing inflammatory skin diseases. Specifically, it investigates the complex regulatory network involving the NLRP3 inflammasome, autophagic flux, and exosome-mediated intercellular communication, while simultaneously establishing a cell-based alternative testing framework to minimize animal testing.
NH2-ZnONPs were prepared and analyzed for size, morphology, and surface charge. For in vivo assessments, SKH-1 hairless mice were subjected to acute or repeated treatment regimens, combining a single dose of UVR irradiation (150 mJ/cm2) followed by topical applications of ZnONPs (2 mg/cm2). Transepidermal water loss (TEWL), epidermal thickness, and histopathological alterations were dynamically monitored. In vitro, human keratinocytes (HaCaT) and dendritic cell surrogates (THP-1 monocytic cell line) were utilized in single or co-culture systems to identify molecular initiating events based on the skin sensitization Adverse Outcome Pathway (AOP) framework. The in vivo findings demonstrated that concurrent exposure to UVR and ZnONPs significantly compromised the skin barrier, manifested by elevated TEWL levels and pronounced epidermal hyperproliferation. Mechanistically, ZnONPs internalization triggered prominent cellular stress. In terms of inflammasome dynamics, the combination treatment significantly licensed canonical and alternative NLRP3 inflammasome activation, driving downstream caspase-1 proteolytic cleavage and pyroptosis cell death. Concurrently, intracellular accumulation of ZnONPs caused severe autophagy dysfunction, which was characterized by blocking terminal autophagic cargo degradation and trapping autophagosomes. Under this cross-linked stress, compromised cells accelerated the release of bioactive exosomes into the extracellular microenvironment. These stress-induced exosomes acted as critical intercellular messengers, transferring pathogenic signaling cargo to naive recipient cells, which further propagated the NLRP3 inflammasome activation cascade and exacerbated severe cutaneous inflammation. Topical administration of pterostilbene (PT) attenuates NLRP3 inflammasome activation and subsequent pyroptosis by reducing total and mitochondrial reactive oxygen species (mtROS). Beyond its antioxidant properties, PT restores impaired autophagic flux and inhibits the secretion of NLRP3-laden exosomes, thereby rescuing autophagy dysfunction.
Furthermore, the in vitro alternative methodologies successfully mirrored these inflammatory milestones. Utilizing the integrated AOP-based testing assays, the study successfully captured the key cellular events, including Keap1/Nrf2-regulated keratinocyte hyperactivation and subsequent THP-1 dendritic cell maturation, thereby providing sensitization prediction without relying on traditional in vivo data.
Collectively, these findings demonstrate that ZnONPs and UVR synergistically drive inflammatory skin injury via a pathological loop where autophagy blockade cross-talks with NLRP3 inflammasome hyperactivation, a process prominently amplified by exosome dissemination. By mapping these intricate pathways and validating robust, non-animal AOP-based alternative testing models, this study offers profound mechanistic insights and establishes promising, verifiable therapeutic and regulatory targets for managing nanomaterial-induced skin toxicity.

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