奈米氧化鋅（ZnONPs，粒徑小於100奈米），為工程奈米粒子（ENPS）其中之一，目前被廣泛的使用並添加於許多消費產品當中，其中在防曬乳中為一重要成分，也因此增加人體暴露奈米氧化鋅的機會。然而，目前對於奈米氧化鋅所造成的毒性以及危害風險的了解仍不足。本研究之目的為探討在皮膚受損與發炎的情況之下，暴露奈米氧化鋅是否會透過自噬功能失調而引起巨噬細胞進一步的促發炎現象。在體外實驗中，透過trypan blue來計算細胞存活率，並透過流式細胞儀對自體吞噬作用與細胞凋亡百分比進行分析。自噬作用與發炎體相關蛋白的表現則使用西方墨點法來分析。在動物實驗中，使用SKH：HR-1無毛鼠建立UVB誘導皮膚損傷的模式。利用H&E組織切片染色觀察皮膚組織的病理變化，並量測皮膚經皮水分散失的表現情形，組織相關蛋白使用免疫組織化學染色 (IHC) 和西方墨點法分析表現量。細胞實驗方面，以不同劑量之紫外線與奈米氧化鋅暴露24小時並計算細胞存活率，最後選定以導致HaCaT細胞存活率在50%以下之紫外線 (150 mJ/cm2) 以及奈米氧化鋅 (10, 12.5 μg/ml) 劑量組別來模擬皮膚受損情形並進行後續實驗。在HaCaT細胞株合併暴露組中發現自體吞噬相關蛋白LC3-II和p62在暴露24小時後呈現增加的情形，同時NLRP3表現亦增加，透過螢光顯微鏡觀察奈米氧化鋅在短時間內 (4小時) 會經由細胞攝入並分布至溶酶體當中，並且也發現p62在不同時間點中的累積情形，同時應證了自體吞噬功能失調的結果。而THP-1細胞株暴露奈米氧化鋅後也發現到發炎體相關的蛋白NLRP3、ASC、caspase-1、cathepsin B的表現增加。在動物實驗方面，組織切片染色結果發現先誘導曬傷之合併暴露組有明顯的角質層剝落與表皮層增厚的情形，皮膚功能相關指標之經皮水分散失也在相同之合併暴露組呈現明顯上升，並且奈米氧化鋅也會因表皮層受損而更容易穿透至皮膚深層，可能導致奈米氧化鋅對皮膚的影響時間增加。同時也發現皮膚修復相關蛋白galectin-7的表現減少與IL-1β增加的現象，證明了皮膚先誘導曬傷後，奈米氧化鋅會大幅影響皮膚的受損與發炎情形。根據以上結果顯示，紫外線合併暴露奈米氧化鋅可能藉由誘發自體吞噬失調，進一步造成NLRP3 inflammasome的活化及增加，導致發炎激素的表現增加，並使皮膚受損及發炎情形更為嚴重。

SUMMARY
Zinc oxide nanoparticles (ZNONPs) have been widely formulated in sunscreens as protective agents against exposure to ultraviolet radiation. Many prior studies have concluded that NPs do not penetrate healthy skin, but damaged skin slightly enhanced metal oxide NPs penetration. Therefore, hazard evaluation of ZnONPs exposure in different situation should be investigated urgently. In this study, we explored the toxic and inflammatory effect of ZnONPs on HaCaT, THP-1 cells and animal model. Results demonstrated UVB exposure enhanced ZnONPs-induced cytotoxic, autophagy and apoptosis percentage, also the expression of LC3-II, p62, NLRP3 were enhanced in UVB and ZnONPs cotreated groups in HaCaT cells. We also found uptake of ZnONPs in lysosome and p62 accumulation as time goes on, it may mean the disruption of autophagic flux－usually caused more inflammation and cell damage . Over-expression of inflammatory proteins in THP-1 cells also showed immunotoxicity after exposure to ZnONPs. In in vivo study , after sunburn occurred, ZnONPs enhanced significantly more inflammatory responses and severe damage than cotreat groups with healthy skins and alone groups, meanwhile, skin repair was also interfered with ZnONPs. In summary, this study found that UVB exposure enhanced ZnONPs toxicity and the potential mechanism is caused by NLRP3-inflammasome activation and the damage of skin repair system.

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