目的：

皮肤是抵挡外界刺激的第一道屏障，中波紫外线（UVB）是导致皮肤光损伤的主要原因，可以引起皮肤老化、松弛、炎症甚至肿瘤等。自噬是细胞自我消化的过程，是维持细胞存活的重要生理功能。我们前期研究发现紫外线在角质形成细胞中诱导的自噬呈现剂量相关性，50 mJ/cm²中波紫外线可以抑制细胞自噬，也发现了许多天然糖类/糖醇对自噬的调控有一定作用。然而，关于这些天然物质能够否调节人原代角质形成细胞紫外线损伤模型的炎症活动，能否发挥其它生物学效应，以及这些效应是否与细胞自噬相关尚不明确。因此，我们初步研究了紫外线诱导的细胞因子分泌情况，并筛选能够影响这些细胞因子分泌的糖类/糖醇，然后对糖类/糖醇影响紫外线损伤模型的其他生物学效应的蛋白标志物、信号通路等进行近一步探索，通过自噬抑制药物干预，评估糖类/糖醇诱导的自噬能否在紫外线损伤模型的细胞因子分泌中发挥作用，以及推测在其中发挥作用的信号通路。与此同时我们也粗浅的探索了多糖/糖醇诱导自噬的作用机制。

方法：

1． 以人原代角质形成细胞（HEK）为研究对象，以50 mJ/cm² UVB为照射剂量诱导紫外线损伤模型，通过细胞因子芯片检测该模型细胞因子分泌的情况；通过 QPCR以及ELISA检测来筛选多种天然糖类/糖醇对紫外线损伤模型释放的细胞因子的作用。

2． 分别采用CCK-8、BrdU和LDH法检测筛选出糖醇（白藜芦醇）对细胞增殖活性和死亡的影响；

3. 采用Western Blot、AO染色RFP-GFP-LC3A/B串联荧光标记分析糖醇（白藜芦醇）对紫外线损伤模型自噬诱导的情况，以及自噬相关蛋白（ATG5、Beclin1和ATG9A）的表达情况；

4． 采用Western Blot检测，MAPK信号通路相关蛋白（JNK、Erk 1/2和p38）、DNA损伤信号通路相关蛋白（ATM、ATR和histone H2A.X）以及内质网应激通路相关蛋白（BiP、IRE1α和PERK）表达或磷酸化水平的影响。

5． 采用蛋白质免疫印迹法检测凋亡相关蛋白PARP和caspase-3以及周期相关蛋白cyclin A1/A2、cyclin B1和cyclin E1的表达水平，采用FITC Annexin V凋亡检测试剂盒以及PI/RNase染料处理细胞，流式细胞仪检测细胞凋亡水平和细胞周期分布。

6． 通过Western Blot以及流式细胞学，验证凋亡激活剂以及自噬抑制剂在白藜芦醇干预后的紫外线损伤模型发挥作用，继而给予凋亡激活以及自噬抑制并检测经紫外线诱导的模型分泌IL-8的情况。

7.  通过Western Blot、荧光共定位检测ATGs在Trehalose干预后细胞各个细胞器分布的情况，检测胞内定位发生变化的基因（ATG9A）与LC3 A/B的共定位；通过Western Blot、AO染色观察，敲低ATG9A后Trehalose在Hacat细胞和HEK细胞诱导自噬的变化。ELISA检测敲低ATG9A后，白藜芦醇对紫外线诱导的IL-8抑制作用的变化。

结果

1.  细胞因子芯片检测显示，50 mJ/cm²中波紫外线诱导的角质形成细胞分泌IL-8显著增高，从诱导后的12h开始升高，并在24小时后达到峰值；在若干天然糖类和糖醇中，白藜芦醇能够同时抑制紫外线诱导的IL-8的mRNA表达量和分泌量；

2.  CCK-8和BrdU实验发现白藜芦醇可以抑制人原代角质形成细胞的增殖活力，结合LDH的实验结果，100 μmol/L白藜芦醇不影响细胞死亡即无细胞毒作用，因此，选用100 μmol/L浓度进行后续实验；

3.  在紫外线诱导的HEK细胞中，白藜芦醇抑制了自噬流，抑制UVB激活的MAPK信号通路，并且降低了c-Fos蛋白的表达水平。白藜芦醇还能够抑制UVB诱导PARP和caspase-3的剪切和细胞凋亡，影响细胞周期相关蛋白cyclin B1和cyclin E1的表达以及细胞周期的进程。

4.  在紫外线诱导的HEK细胞中，自噬抑制剂LY294002、3-MA和ATG5 siRNA抑制RSV介导的IL-8蛋白质分泌，而PAC-1和AA2没有发挥类似的作用，这表明自噬可能参与调节RSV对UVB诱导的IL-8分泌的抑制作用。

5.  ATG9A通过胞内定位变化参与了Trehalose诱导的自噬活动。敲低ATG9A后，白藜芦醇对UVB诱导的IL-8分泌的抑制作用减弱。

结论

1． 中波紫外线可以诱导HEK细胞分泌的IL-8明显升高，白藜芦醇可以抑制紫外线诱导的IL-8分泌；

2.  白藜芦醇可以诱导紫外线抑制的自噬流，可以通过MAPK/c-Fos信号通路发挥对紫外线诱导的HEK细胞的调节作用；

3.  白藜芦醇可以通过调控自噬流调节紫外线诱导的IL-8的分泌；

4.  ATG9A通过胞内定位变化参与了Trehalose诱导的自噬活动，ATG9A参与白藜芦醇调节紫外线诱导的IL-8的分泌；

研究表明，白藜芦醇可以通过自噬流调节紫外线诱导的促炎因子IL-8的分泌、抑制凋亡、调节细胞周期进程发挥细胞保护效应，对于是自噬否通过MAPK/c-Fos信号通路调控IL-8的分泌尚待验证。重要自噬调节基因ATG9A参与了白藜芦醇对IL-8的调控作用，ATG9A在白藜芦醇诱导的自噬中如何发挥作用有待进一步验证。

Objective:

Skin is the first barrier to resist external stimulation. Numerous studies have demonstrated that Ultraviolet (UV) B radiation induces inflammatory mediators and DNA damage which can lead to various skin disorders involving inflammations, skin ageing and even skin cancers. Among multiple mechanisms reported to participate in resisting UVB-induced skin damage, the involvement of autophagy has been revealed. Our previous study showed 50 mJ/cm² UVB could inhibit autophagy, and some of the natural polysaccharide/ polyphenols can induce autophagy. However, it is remains unclear whether these natural substances can regulate the inflammatory activities by autophagy in UVB-damaged model, and what else biological effects they can play. Therefore, our study detected the secretion of cytokines induced by UVB, and screened the polysaccharide/ polyphenols which can affect the secretion of these cytokines. Then, we explore the influence of polyphenol the protein markers and signal pathways of some important biological effects in UVB-damaged model. Finally, the autophagy inhibitors and the apoptosis activators were used to investigate the roles of autophagy and apoptosis in the polyphenol-mediated cytokine secretion.

Method:

1. The human primary keratinocytes (HEKs) were used in this study, and the UVB-damaged model was induced by 50 mJ/cm² UVB. The secretion of cytokines was detected by cytokines array assay. The effects of several natural polysaccharide/ polyphenols on the cytokines released by UVB-damaged model were detected by QPCR and ELISA.

2. CCK-8, BrdU and LDH were used to detcet the effects of resveratrol on cell proliferation and cell death;

3. Western blot was used to measure the accumulation level of LC3-II which could analyze the autophagy flux，Ao staining, RFP-GFP-LC3A/B tandem fluorescent labeling were also used to analyze autophagy, and the expression of autophagy related proteins (ATG5, Beclin1 and atg9a) in UVB-irradiated HEks;

4. Western blot was used to measure MAPK signaling pathway related proteins (JNK, Erk 1/2 and p38), DNA damage signaling pathway related proteins (ATM, ATR and histone H2A.X) and endoplasmic reticulum stress pathway related proteins (BiP, IRE1α and PERK).

5. The levels of apoptosis related proteins PARP, caspase-3 and cell cycle related proteins cyclin A1/A2, cyclin B and cyclin E1 were measured by western blot; cell apoptosis and cell cycle distribution were measured by flow cytometry.

6. Western blot and flow cytometry were used to verify that apoptosis activator and autophagy inhibitor play a role in the UVB-irradiated HEKs after resveratrol treatment. Then apoptosis activator and autophagy inhibitor were added and IL-8 secretion was detected by Elisa.

7. Western blot and co localization fluorescence were used to detect the distribution of ATGs in various organelles after trehalose intervention, and the co localization of ATG9A and LC3 A/ B was detected; Western blot and AO staining were used to observe the autophagy flux induced by trehalose in HaCaT cells and HEK cells. The inhibitory effect of resveratrol on UV induced IL-8 was detected by ELISA.

Result:

1. UVB-induced inflammatory cytokine secretion was detected by cytokine array,  result showed that RSV inhibited the UVB-induced secretion of interleukin-8 (IL-8), and the release of IL-8 started to rise 12 h after UVB radiation and reached a peak after 24 h. Resveratrol could inhibit the expression of IL-8 mRNA and the secretion of IL-8 induced in UVB-irradiated HEKs.

2. Results of CCK-8 and BrdU showed that resveratrol inhibited the proliferation of HEK. Combined with the LDH results, 100 μ mol/l resveratrol does not affect cell death or cytotoxic effect. Therefore, 100 μ mol/l concentration is selected for the following experiment.

3. In UVB-irradiated HEKs, RSV inhibited autophagy flux, inhibited the MAPK signaling pathway activated by UVB, and reduced the expression level of c-fos. Resveratrol could also inhibit UVB induced the cleavage levels of PARP and Caspase-3 as well as cell apoptosis, RSV reduced the level of cyclin B1 and increased cyclin E1 and had an influence on the process of cell cycle.

4. ELISA data showed that LY294002, 3-MA and ATG5 siRNA reversed the RSV-mediated inhibition of IL-8 protein secretion by UVB-irradiated HEKs, while PAC-1 or AA2 did not exert similar effects, suggesting that autophagy might contribute to the inhibitory effect of RSV on UVB-induced IL-8 secretion.

5. ATG9A participates in Trehalose induced autophagy through intracellular localization. siATG9A reversed the RSV-mediated inhibition of IL-8 protein secretion by UVB-irradiated HEKs.

Conclusion:

1. UVB induced inflammatory cytokine secretion by HEKs, particularly Interleukin-8 (IL-8), RSV inhibited the UVB-induced secretion of IL-8 but increased the secretion of IL-8 itself.

2. RSV could induce the UVB-inhibited autophagy flux. MAPKs/c-Fos signaling might mediate the regulation of RSV on UVB-irradiated HEKs;

3. RSV could regulate the UVB induced IL-8 secretion by regulating autophagy flux;

4. ATG9A participates in Trehalose induced autophagy through intracellular localization, ATG9A has an influence on resveratrol regulates UV induced IL-8 secretion;

Our study indicated that resveratrol could regulate the secretion of IL-8 in UVB-irradiated HEKs. LY294002, 3-MA and ATG5 siRNA resisted RSV-inhibited IL-8 protein secreted by UVB-irradiated HEKs. However, whether autophagy can regulate IL-8 secretion through MAPK / c-fos signaling pathway remained unclear. ATG9A, an important autophagy regulatory gene, is involved in the regulation of IL-8 by RSV. Whether ATG9A plays a role in RSV induced autophagy remains to be further verified.