【目的】

紫外线是皮肤常见的应激源。目前紫外线照射如何对角质形成细胞进行自噬调节并不清楚。

MTOR蛋白是哺乳动物细胞中的重要信号调节因子。MTOR信号在角质形成细胞中的作用仍不清楚。

研究表明海藻糖通过诱导哺乳动物细胞自噬发挥有益作用。然而，无论是海藻糖或其他糖类是否可以诱导角质形成细胞自噬仍未知。自噬诱导剂海藻糖是否具有紫外线保护效应仍未知。

【方法】

对原代角质形成细胞给予不同剂量UVB照射，在不同的时间点分析自噬流和自噬体形成。UVB照射后，使用雷帕霉素孵育分析自噬抑制是否可以恢复。

人角质形成细胞系HaCaT细胞和原代角质形成细胞给予MTOR抑制剂后，分析自噬调控和MTOR信号。分析MTOR抑制剂处理对UVB和UVA暴露后的DNA损伤信号、内质网应激、JNK和凋亡。

对HaCaT细胞给予不同浓度的海藻糖处理，孵育至不同的时间点，观测自噬标志性分子事件的变化。进而，分析自噬关键调控信号。给予葡萄糖、蔗糖、麦芽糖、棉子糖和山梨醇处理分析自噬调控变化。分析海藻糖处理对UVB损伤的HaCaT细胞的LDH释放、克隆形成以及凋亡活性。

【结果】

UVB照射抑制人角质形成细胞自噬流，但MTOR信号下调，且雷帕霉素不能恢复自噬抑制。

角质形成细胞MTOR信号对MTOR抑制剂雷帕霉素、依维莫司、Torin 1和pp242敏感，但MTOR下游信号的调控状态不同。仅有雷帕霉素可以诱导自噬。MTOR信号对UVB不敏感，但对UVA敏感。雷帕霉素、依维莫司或pp242处理不影响UVB所诱导产生的系列细胞事件，包括BIP和PERK下调，组蛋白H2A和JNK激活以及caspase 3和PARP剪切。

海藻糖处理增加HaCaT细胞LC3-I向LC3-II转换，AO染色阳性囊泡和GFP-LC3点状聚集。说明具有可以诱导自噬。海藻糖处理并没有影响MTOR信号，这表明海藻糖诱导的自噬通过非MTOR依赖性途径。在蔗糖或棉子糖处理的HaCaT细胞中也可以观察到非MTOR依赖性自噬诱导，但在葡萄糖、麦芽糖和山梨醇处理的HaCaT细胞中则不能观测到这种诱导，表明自噬诱导能力不是糖类的共性。虽然海藻糖处理对细胞增殖具有抑制作用，但它对暴露于UVB的细胞具有提高细胞活力和抑制异常克隆形成的作用，并且对UVB诱导的凋亡没有抑制作用。

【结论】

UVB以非MTOR依赖性方式抑制角质形成细胞自噬。

抑制角质形成细胞MTOR信号可以不伴随自噬诱导，在UVB触发的细胞反应中MTOR通路不发挥核心作用。

海藻糖、蔗糖和麦芽糖是角质形成细胞的非MTOR依赖性自噬诱导剂。海藻糖在UVB损伤的角质形成细胞中展现了良好的细胞保护效应。

Ultraviolet is onecommon stressor of skin.Autophagy is a self-digestive pathway to maintain cellular homeostasis. It is unclearhow autophagy is regulated by ultraviolet exposure in epidermal keratinocytes. Here, we found that UVB radiation inhibits basal autophagy flux in human keratinocytes. Moreover, mechanistic target of rapamycin (MTOR) signaling is contradictorily decreased, suggesting the MTOR-independent autophagy inhibition. Our study demonstrates that UVB radiation inhibits autophagy response in keratinocytes. This study provided a linkage of autophagy and skin disorders associated with ultraviolet.

The mechanistic target of rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes (HEKs) treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors rapamycin, everolimus, Torin 1 and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by rapamycin, everolimus or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the down-regulation of BIP and PERK, activation of Histone H2A and JNK and cleavage of caspase 3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB-triggered cellular response.

Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals. Autophagy is a process in which double-membrane autophagosomes are formed; this process mediates the sequestration, lysosomal delivery and degradation of proteins and organelles. Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells. However, whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown. Here, we found that trehalose treatment increased the LC3-I to LC3-II conversion, acridine orange-stained vacuoles and GFP-LC3B (LC3B protein tagged with green fluorescent protein) puncta in the HaCaT human keratinocyte cell line, indicating autophagy induction. MTOR-independent autophagy induction was also observed in HaCaT cells treated with sucrose or raffinose but not in glucose, maltose or sorbitol treated HaCaT cells, indicating that autophagy induction was not a general property of saccharides. Finally, although trehalose treatment had an inhibitory effect on cell proliferation, it had the cytoprotective effect and inhibition on abnormal cell proliferation in cells exposed to UVB radiation. Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes.