自噬在维持黑素细胞氧化应激平衡及白癜风发病中的作用机制研究

Autophagy is an evolutionarily conserved process by which cells are able to degrade and recycle their own structural components. It is critical for the removal of damaged proteins and subcellular organelles, the maintenance of cell metabolism during starvation, cellular renovation during development and differentiation as well as for anti-bacterial and anti-viral defence. UV exposure of cells and oxidant stress leads to oxidation of cellular lipids, which can then modify and crosslink proteins. This is harmful to cells, thus it is important to study how cells cope with it. In response to oxidative stress by both UVA irradiation and exposure to oxidized lipids, cells activate the Nrf2-driven antioxidant response which provides cellular antioxidants and detoxifying enzymes. At the same time, proteins modified by oxidizing stressors are degraded by the proteasome and autophagosomal/lysosomal pathways. (Macro) autophagy degrades cargo that is too large to enter the proteasome. We have previously shown that autophagy plays an important role in the anti-oxidant response of epidermal keratinocytes. Oxidative stressors induced massive accumulation of high molecular weight protein aggregates containing the autophagy adaptor protein p62/SQSTM1 in autophagy-deficient keratinocytes. Strikingly, even in the absence of exogenous stress, the expression of Nrf2 dependent genes was elevated in autophagy-deficient keratinocytes. Furthermore, we showed that autophagy-deficient keratinocytes contained significantly elevated levels of reactive oxidized phospholipids. Thus we demonstrated that autophagy is crucial for both the degradation of proteins and lipids modified by environmental UV-stress and for limiting Nrf2 activity in keratinocytes. In our latest study, we further demonstrated that autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of melanocytes. However, it remains unclear whether autophagy plays a role in the anti-oxidant response of melanocytes. In this study we plan to investigate the role of autophagy in MCs in vitro and in vivo with a trangenic-mouse strain which is deficient in autophagy specifically in melanocytes by crossing mice carrying a floxed ATG7 gene (ATG7 f/f) with mice expressing the Cre recombinase under the control of the tyrosinase promoter. This led to an efficient suppression of autophagy in MCs of ATG7 f/f Tyr::Cre mice. We intend to study the pigmentation and melanocyte homeostasis in transgenic mice and primary melanocyte cultures derived from these mice beyond oxidative stress including UVA and H2O2. In the meantime, we plan to explore the condition of autophgay in serums and melanocytes primarily isolated from perilesional areas of patients with non-segmental symmetrical vitiligo and normal controls. We expect this study would shed light on the mechanisms of pigment disorders including vitiligo.

白癜风是一种常见的色素脱失性皮肤病。发病机制不明，缺乏特异、高效的治疗手段。近年研究显示氧化应激损伤在白癜风病人的黑素细胞破坏中发挥始动作用。自噬是广泛存在于真核细胞内的溶酶体依赖性降解途径，保护细胞免受氧化刺激损伤，是清除破损蛋白和细胞器的重要降解机制。我们在前期研究中发现，自噬对于保护皮肤角质形成细胞的氧化应激自稳状态及维持黑素细胞的正常增殖和衰老起重要调控作用。然而自噬在黑素细胞氧化应激反应中所起的作用及其与白癜风发病是否相关尚不明确。本课题以黑素细胞缺乏自噬功能的靶向基因敲除小鼠为模型，通过UVA，过氧化氢等氧化应激原刺激，观察小鼠表型及其黑素细胞增殖，凋亡水平，色素生成，ROS水平及Nrf2抗氧化通路的变化，探索自噬在维持黑素细胞氧化应激平衡中所起的作用；并以白癜风患者为研究对象，研究自噬在白癜风患者皮损处黑素细胞及血清中的表达，探索白癜风新的发病机制，为临床寻找新的治疗靶点。

细胞自噬是广泛存在于真核细胞内的一种溶酶体依赖性的降解途径。在饥饿，感染或应激等条件下，它调节细胞内蛋白和细胞器的降解，从而为细胞提供能量和营养。有研究表明自噬参与抗氧化应激反应，保护细胞免受氧化损伤。然而，自噬在黑素细胞抗氧化应激反应中的作用尚不清楚。为了研究自噬在人表皮黑素细胞中的作用，我们首先采用Cre-loxP技术建立了特异性在黑素细胞上敲除自噬关键基因ATG7导致自噬功能缺失的小鼠模型，观察其表现型。与对照组相比，自噬缺乏黑素细胞的小鼠毛发中的黑色素含量降低了10-15%。体外培养小鼠黑素细胞时，自噬功能缺失的黑素细胞进入生长停滞期，并导致活性氧（ROS）积累和p62的堆积。此外，核因子红系2相关因子2（Nrf2）依赖的氧化应激通路被过度激活，表明自噬参与了黑素细胞中氧化还原稳态。CXCL10、1、2和12在自噬缺陷黑素细胞中的表达显著增加，表明自噬缺陷黑素细胞在体外的衰老表型伴随着分子转录水平的变化，主要是衰老相关分泌表型（SASP）的上调。其次，我们发现进展期白癜风患者体内存在着高表达的CXCL9，CXCL10及其受体CXCR3，血清CXCL10水平随治疗有效而下降。最后，我们在正常人表皮黑素细胞中敲除/过度表达ATG7从而抑制/增强自噬。我们发现ATG7依赖性自噬通过调节黑素生成而影响黑素细胞的黑素含量。抑制ATG7依赖性自噬抑制黑素细胞的增殖并促进氧化应激诱导的凋亡，而ATG7依赖性自噬的增强保护黑素细胞免受氧化应激诱导的细胞凋亡。同时，ATG7依赖性自噬的缺乏导致氧化应激下黑素细胞的过早衰老。值得注意的是，ATG7依赖性自噬可以通过调节ROS产生、Nrf2抗氧化途径和黑素细胞中几种抗氧化酶的活性来改变氧化应激稳态。综上所述，本课题证实，ATG7依赖性自噬对于维持黑素细胞氧化还原稳态是必不可少的，特别是在氧化应激下对黑素细胞的生物学功能，如黑素生成、增殖、凋亡和衰老至关重要。进展期白癜风CXCL10/CXCR3轴介导T细胞募集到皮肤中。血清CXCL10水平可能作为监测进展期白癜风疾病活动性和指导疗效的生物学标志。