HSP27经P53/AMPK通路介导自噬在皮肤光损伤中的机制研究

There are many inflammatory and neoplastic skin diseases associated with photodamage in clinic.The pathogenesis of photodamage is of great importance for the prevention and treatment of such skin diseases. The study group has found the differential protein: heat shock protein 27 by comparing the human skin tissue between exposed and non-exposed parts at different ages; we have proved that HSP27 plays a protective role in skin photodamage through anti-apoptosis at the cellular and animal models.Some research groups also found that autophagy might be involved in the pathogenesis of UV-induced photodamaged skin diseases. However, whether there is an association between HSP27 and autophagy and they co-regulate cell survival are still needed to be solved in further study. In this project, human skin fibroblasts (HSF) will be used as the research object, and UVB will be used to induce acute and chronic photodamage models. We are going to elucidate the protective effects of HSP27 and autophagy in photodamage, and to determine whether HSP27 play an anti-apoptotic effect through regulating autophagy by PI3K-AKT-mTOR pathway and P53/AMPK pathway. This research will study the possible molecular mechanism of HSP27 and autophagy in dermatitis caused by photodamage, in order to provide new ideas for investigating the intrinsic link between UV-related skin diseases and tumors, and also lay a theoretical foundation for the prevention and treatment of such skin diseases.

临床中有许多炎症性、肿瘤性皮肤病与光损伤相关，明确光损伤的发病机制，对预防和治疗此类皮肤病具有重要的临床意义。前期课题组通过比较人体不同年龄段、曝光与非曝光部位皮肤组织寻找到差异蛋白：热休克蛋白27；并在细胞水平和动物模型中证明在皮肤光损伤中HSP27通过抗凋亡起到保护作用；同时国内外研究发现，细胞自噬也可能通过调控凋亡参与到光损伤皮肤病的发病过程中。但HSP27与自噬之间是否存在关联、是否共同调控细胞的存活尚需进一步研究。本项目以皮肤成纤维细胞为研究对象，用UVB诱导急、慢性光损伤模型，阐明HSP27及自噬在光损伤中的作用，通过干预HSP27探究其是否通过P53/AMPK、PI3K-AKT-mTOR途径诱导或是抑制自噬，旨在揭示HSP27与自噬在光损伤中共同影响细胞凋亡、存活，从而发挥保护作用的可能机制。为探究UV相关皮肤病及肿瘤的内在联系提供新思路，为防治此类皮肤病奠定理论基础。

光损伤是导致一类炎症性皮肤病、皮肤良恶性肿瘤等疾病的主要原因。随着紫外线辐照危害的加重，皮肤光损伤需要得到更多的重视。在光老化部分，课题组成功培养原代人皮肤成纤维细胞(HDFs)并构建了UVA和UVB诱导的细胞光老化模型。在UVA刺激下，HDFs的p53和p-HSP27的表达增加，LC3II/LC3I的表达下降，MMP-1和MMP-3的表达增加，而p62没有明显变化，说明在UVA下HDFs的应激水平升高，细胞自噬水平减弱，并在降解阶段停滞。在mTOR的抑制剂雷帕霉素的处理下，UVA照射组的HDFs衰老程度的增加被逆转，细胞自噬通量都显著增加，p53和p-HSP27的表达都显著降低，p62水平仍然没有变化。这表明雷帕霉素可能能够对抗UVA诱导的细胞应激水平的增加，进一步证实了UVA照射诱导自噬过程中的降解障碍。随后，敲除HSP27的HDFs照射UVA后LC3B的表达受到抑制，p62的表达增加, Collagen Ⅰ mRNA水平降低。在临床研究中，课题组发现光老化皮炎患者皮肤组织HSP27、LC3B和Collagen I表达水平较低，而p62表达呈弥漫性片状阳性灶；真皮胶原蛋白部分紊乱松散，胶原纤维明显减少。这些结果与体外研究结果一致，光老化皮肤表现出比正常皮肤更低的自噬和胶原纤维水平。在急性光损伤部分，成功培养原代人皮肤角质形成细胞并构建了UVB诱导的急性光损伤体外、体内模型。UVB照射下，HSP27基因敲除的角质形成细胞和HDFs增殖能力受到显著抑制，出现了p62的堆积和LC3B的减少，ROS水平增加，CAT、SOD1、SOD2的产生降低，显著减弱了细胞的抗氧化状态。在体内实验中，同样发现UVB光照后诱导了HSP27的表达，HSP27敲除后照射UVB对皮肤的损害最为显著。此外，急性光损伤小鼠模型中皮肤组织的P62，LC3B和抗氧化指标CAT, SOD1, SOD2的检测结果与体外实验结果一致。在本研究中，我们发现HSP27可能通过调节机体自噬水平及抗氧化能力起到光保护作用，为缓解光损伤提供新思路。