核因子E2通路激活在表皮渗透屏障损伤修复中的作用

Oxidative stress is involved in the pathogenesis of diverse skin diseases accompanied by compromised epidermal permeability barrier function such as psoriasis, atopic dermatitis and acne. Studies have demonstrated that both the content of oxidative products and the extent of epidermal permeability barrier abnormality correlate positively with the severity of those skin disorders．Administrations of antioxidants can improve both skin disorders and epidermal permeability barrier function. Approaches of either administering antioxidants or enhancing epidermal permeability barrier function could be helpful in preventing and treating dermatoses accompanied by defective epidermal permeability barrier function．Nrf2 pathway plays important roles in the cellular defense against oxidative stress, but its function in the skin barrier homeostasis is still largely unknown. We found in our preliminary study that after acute skin barrier injury with tape stripping, the Nrf2-responsive antioxidant enzymes expression was highly increased, which could be decreased by using moisturizer to help restore the barrier function. But in a chronic barrier injury model, Nrf2 and the phaseⅡantioxidant enzymes respond in a totally different way. We then speculate that Nrf2 pathway is involved directly in the process of the skin barrier injury and recovery, which helps in the barrier homeostasis maintenance. In this study, we will use SKH-1 hairless mice to creat acute and chronic skin injury model, by tape stripping and applying oxazolone respectively. The expression pattern of Nrf2 and its responsive enzymes will be observed before and after the skin barrier injury and also compared with the barrier-restored group at different timepoint. We will further use RNA interference to achieve Nrf2 knockdown in KC, then subsequently compare the expression of Nrf2, its responsive enzymes and differentiation marker. The corneocyte stress test will also be performed. The results of this study will help to elucidate the possible function of Nrf2 pathway in the skin barrier function. Nrf2 may serve as a novel molecular target for developing new therapies for inflammatory skin disease, which currently does not have a cure.

氧化应激与痤疮，银屑病，特应性皮炎等屏障受损的皮肤病密切相关。Nrf2通路是细胞内对抗氧化应激反应的重要保护机制，但其诱导的氧化应激反应在皮肤屏障功能中的作用尚不明确。我们在前期的研究中发现急性屏障损伤后，Nrf2通路下游的抗氧化基因HO-1,slpi,Sprr表达显著增加，外用保湿剂恢复屏障可明显抑制反应性Nrf2激活，而慢性屏障损伤Nrf2激活特点与急性期不同。本研究拟在1.急慢性屏障损伤的小鼠模型上进行比较，检测ROS水平，观察在屏障损伤的不同时相，Nrf2通路的反应特点；2.将角质形成细胞Nrf2敲低，检测不同分化程度角质形成细胞分化标记和细胞间连接蛋白，进行应力对抗实验。从体内和体外两个层面论证Nrf2诱导的氧化应激反应在屏障中的作用。研究结果将通过Nrf2将氧化应激与渗透屏障联系起来，为氧化应激反应在炎症性皮肤病的发病机理和抗氧化在皮肤病治疗中的作用提供新的思路。

研究背景：完整的皮肤屏障功能是维持皮肤稳态，防御各种外来及内在损伤的基础。恢复正常的屏障功能是皮肤病治疗中非常重要的部分。目前仅已知氧化应激参与皮肤屏障的损伤过程，抗氧化治疗对某些屏障损伤性皮肤病治疗有效，其具体机理所知甚少。Nrf2通路是人体内泛在的细胞内对抗氧化应激反应的重要的防御机制，本研究探索了Nrf2抗氧化通路在皮肤屏障损伤修复中的作用及其机理。. 主要研究内容：研究包括细胞实验和动物在体实验两部分。（1）细胞实验：培养人类原代角质形成细胞(NHEK)，分别研究Nrf2通路在细胞增殖分化不同阶段、Nrf2激活和敲低状态下不同时间点其下游Ⅱ相细胞保护蛋白HO-1、NQO-1蛋白及表皮增殖（K5/K14）和分化标记物K1/K10、兜甲蛋白Loricrin、丝聚合蛋白Filaggrin、内披蛋白Involucrin)表达情况以推测其对屏障功能的影响。（2）动物实验：将SKH-1分别在屏障破坏和Nrf2刺激不同时间点取材，检测皮肤标本Nrf2及其下游Ⅱ相细胞保护蛋白以及表皮增殖分化标记物的表达，检测经皮水分丢失量(Transepidermal Water Loss，TEWL)、皮肤组织切片H＆E染色、以推测急性屏障损伤后Nrf2通路的活化在屏障修复中的短期作用，及其过度活化对屏障功能的长期影响。.主要研究结果：1.急性皮肤屏障破坏可以激活Nrf2抗氧化通路，Nrf2通路活化参与了屏障早期修复的启动机制，通过上调Sprr2d和Slpi刺激表皮增生和分化，促进屏障修复修复的过程。Nrf2抗氧化通路在急性皮肤屏障破坏后第七天，随着皮肤屏障的修复，逐渐恢复至正常水平。2. Nrf2激活剂SFN可以在细胞培养及动物实验水平活化Nrf2抗氧化通路。SFN处理短期内（处理8h-24h）角质形成细胞增殖和分化标记物蛋白表达下降，可能与ROS的产生以及ERK等细胞信号通路的激活有关。而SFN持续作用于皮肤（处理7d-14d）皮肤增殖和分化标记物蛋白的表达明显上调，导致表皮过度增厚和鳞屑。 .研究意义：以上结果说明Nrf2抗氧化通路为快速反应通路，在屏障损伤的早期快速反应，促进屏障功能恢复，但其时效性非常重要，如果其激活因素持续存在导致Nrf2慢性过度活化，会影响表皮分化的稳态，从而影响屏障的功能。