Qin Dengke, Ren Runjian, Jia Chuanlong, Lu Yongzhou, Yang Qingjian, Chen Liang, Wu Xinyuan, Zhu Jingjing, Guo Yu, Yang Ping, Zhou Yiqun, Zhu Ningwen, Bi Bo, Liu Tianyi
Department of Plastic and Reconstructive Surgery, Hua Dong Hospital, Fu Dan University, Shanghai, China.
Shanghai Key Laboratory of Clinical Geriatric Medicine, Hua Dong Hospital, Fu Dan University, Shanghai, China.
Cell Physiol Biochem. 2018;46(5):1849-1860. doi: 10.1159/000489369. Epub 2018 Apr 25.
BACKGROUND/AIMS: Ultraviolet B (UVB) irradiation alters multiple molecular pathways in the skin, thereby inducing skin photoaging. Murine dermal fibroblasts (MDFs) were subjected to a series of 4 sub-cytotoxic UVB doses (120 mJ/cm2), resulting in changes in cell shape, DNA damage, cell cycle arrest, extracellular matrix variations, reactive oxygen species (ROS) generation, and alterations in major intracellular antioxidant and cellular autophagy levels. Rapamycin (RAPA) is a new macrolide immunosuppressive agent that is primarily used in oncology, cardiology, and transplantation medicine and has been found to extend the lifespan of genetically heterogeneous mice. Several studies have shown that RAPA may have anti-aging effects in cells and organisms. Thus, in this study, we explored the effects and mechanisms of RAPA against the photoaging process using a well-established cellular photoaging model.
We developed a stress-induced premature senescence (SIPS) model through repeated exposure of MDFs to ultraviolet B (UVB) irradiation. The cells were cultured in the absence or presence of RAPA for 48 h. Senescent phenotypes were assessed by examining cell viability, cell morphology, senescence-associated β-galactosidase (SA-β-gal) expression, cell cycle progression, intracellular ROS production, matrix metalloproteinase (MMP) synthesis and degradation, extracellular matrix (ECM) component protein expression, alterations in major intracellular antioxidant levels, and the cellular autophagy level.
Compared with the UVB group, pretreatment with RAPA (5 µM) significantly decreased the staining intensity and percentage of SA-β-gal-positive cells and preserved the elongated cell shape. Moreover, cells pretreated with RAPA showed inhibition of the reduction in the type I collagen content by blocking the UVB-induced upregulation of MMP expression. RAPA also decreased photoaging cell cycle arrest and downregulated p53 and p21 expression. RAPA application significantly attenuated irradiation-induced ROS release by modulating intracellular antioxidants and increasing the autophagy level.
Our study demonstrated that RAPA elicited oxidative damage in vitro by reducing ROS accumulation in photoaged fibroblasts. The anti-aging effect can be attributed to the maintenance of normal antioxidant and cellular autophagy levels. However, determination of the definitive mechanism requires further study.
背景/目的：紫外线B（UVB）照射可改变皮肤中的多种分子途径，从而导致皮肤光老化。将小鼠真皮成纤维细胞（MDFs）暴露于一系列4次亚细胞毒性剂量的UVB（120 mJ/cm2）下，导致细胞形态改变、DNA损伤、细胞周期停滞、细胞外基质变化、活性氧（ROS）生成以及主要细胞内抗氧化剂和细胞自噬水平的改变。雷帕霉素（RAPA）是一种新型大环内酯类免疫抑制剂，主要用于肿瘤学、心脏病学和移植医学，已被发现可延长基因异质小鼠的寿命。多项研究表明，RAPA可能在细胞和生物体中具有抗衰老作用。因此，在本研究中，我们使用成熟的细胞光老化模型探索了RAPA对光老化过程的影响及其机制。
我们通过将MDFs反复暴露于紫外线B（UVB）照射下建立了应激诱导的早衰（SIPS）模型。细胞在不存在或存在RAPA的情况下培养48小时。通过检测细胞活力、细胞形态、衰老相关β-半乳糖苷酶（SA-β-gal）表达、细胞周期进程、细胞内ROS产生、基质金属蛋白酶（MMP）合成与降解、细胞外基质（ECM）成分蛋白表达、主要细胞内抗氧化剂水平的变化以及细胞自噬水平来评估衰老表型。
与UVB组相比，用RAPA（5 μM）预处理可显著降低SA-β-gal阳性细胞的染色强度和百分比，并保持细胞的细长形状。此外，用RAPA预处理的细胞通过阻断UVB诱导的MMP表达上调，抑制了I型胶原蛋白含量的降低。RAPA还减少了光老化细胞周期停滞，并下调了p53和p21的表达。应用RAPA通过调节细胞内抗氧化剂和提高自噬水平，显著减轻了辐射诱导的ROS释放。
我们的研究表明，RAPA通过减少光老化成纤维细胞中的ROS积累在体外引发氧化损伤。其抗衰老作用可归因于维持正常的抗氧化剂和细胞自噬水平。然而，确定确切机制还需要进一步研究。
