Nrf2-ARE信号通路在白癜风氧化应激发病中的作用和机制研究

发布时间：2018-08-13 14:16

【摘要】：背景：白癜风是一种常见的色素脱失性皮肤病，发病率呈逐年上升趋势，皮损局部黑素细胞减少或消失是其色素脱失的主要原因。由于白癜风发病的确切机制尚不清楚，目前仍缺乏有效的治疗方法。近年来的研究发现，氧化应激在诱发表皮黑素细胞破坏或功能障碍方面发挥关键作用。氧化应激可通过产生大量的氧自由基来攻击细胞，干扰其正常代谢、增殖和分化，并可能进一步诱发机体自身免疫反应，形成瀑布式效应，造成表皮黑素细胞不可逆性损伤。与其它表皮细胞相比，白癜风患者黑素细胞更易于受到氧化攻击而发生损伤，然而这其中的原因和机制并不清楚。因此，阐明白癜风患者黑素细胞易受到氧化损伤的原因和具体分子机制对于完善白癜风氧化应激发病机理、指导临床治疗具有重要的意义。 Nrf2-ARE通路是细胞对抗外源性刺激和氧化损伤的主要机制。Nrf2属于“CNC”家族的亮氨酸拉链蛋白，它可与胞核内的抗氧化反应元件（ARE）的DNA序列结合，调控下游一系列II相解毒酶和抗氧化基因的表达。在无任何刺激的状况下，Nrf2与胞浆伴侣蛋白Keap1相结合，处于相对抑制的状态，并被铆钉在胞浆中。当处于氧化应激条件时，Nrf2将与Keap1解偶联并转移入核，与核内的抗氧化反应元件结合，启动一系列II相解毒酶和抗氧化基因的表达。这些基因主要包括：血红素氧合酶-1（HO-1）、NADP(H)醌氧化还原酶1（NQO1）、谷氨酸-半胱氨酸连接酶催化亚基（GCLC）和谷氨酸-半胱氨酸连接酶调节亚基（GCLM）。Nrf2-ARE信号通路及其下游基因已被证实在多种细胞、组织和器官抗氧化损伤中发挥重要作用。由此，我们提出如下的假说：第一，Nrf2-ARE通路在保护黑素细胞抵抗氧化损伤中发挥重要作用；第二，白癜风患者黑素细胞中Nrf2-ARE通路存在异常，导致其对氧化应激更为敏感。目的： 1.研究Nrf2-ARE通路及其下游分子在人黑素细胞中的表达情况，明确该通路在黑素细胞抗氧化应激中的作用； 2.分析Nrf2-ARE通路在正常人和白癜风患者黑素细胞中的表达及功能差异； 3.探讨Nrf2-ARE通路及其下游分子在白癜风患者黑素细胞中表达及功能差异的分子机制。方法： 1.首先利用不同浓度的H_2O_2处理人黑素细胞PIG1，MTS实验检测细胞活性，摸索建立体外黑素细胞氧化应激模型的最佳条件； 2.在PIG1中通过转染Nrf2干涉片段及过表达质粒下调或者上调Nrf2的表达，氧化应激下MTS和流式细胞术检测细胞活性和凋亡情况。 3. H_2O_2预处理后，Real-time PCR检测Nrf2下游主要的4个抗氧化基因（HO-1,NQO-1, GCLC和GCLM）表达水平，免疫细胞化学及Real-time PCR检测原代黑素细胞中Nrf2及HO-1的表达情况。 4.分别利用MTS实验、Real-time PCR、免疫印迹、激光共聚焦显微镜、双荧光素酶报告基因实验、流式细胞术以及酶联免疫实验检测氧化应激下正常人及白癜风患者黑素细胞活性、HO-1表达水平、Nrf2的核转位情况、转录活性、细胞内ROS和MDA水平以及SOD、GPx、CAT和GSH的含量。 5.用免疫组化法检测白癜风患者皮损处Nrf2、p-Nrf2和HO-1的表达分布模式，ELISA检测患者血清中IL-2和HO-1表达水平。 6.应用ATM干涉片段或抑制剂KU55933抑制ATM的表达后，Real Time-PCR和免疫印迹实验检测Nrf2、HO-1和p-Nrf2的表达情况。H_2O_2预处理后，MTS实验、流式细胞术以及酶联免疫实验检测氧化应激下黑素细胞活性、细胞内ROS和MDA水平，免疫组化检测白癜风患者皮损处ATM和p-ATM表达分布模式。结果： 1. H_2O_2可以浓度依赖性的方式诱导黑素细胞死亡，1.0mM H_2O_2处理24h是体外建立黑素细胞氧化应激模型的最佳条件； 2. MTS实验及流式结果证实，下调Nrf2的表达可导致氧化应激下细胞活性下降，增加细胞凋亡和坏死的比率；而上调Nrf2的表达则可提高细胞活性，显著降低细胞凋亡的比率，说明调节Nrf2-ARE通路可影响黑素细胞对氧化应激的抵抗性； 3. Real-time PCR结果表明，H_2O_2可诱导Nrf2通路下游4个主要抗氧化基因表达升高，其中HO-1变化最显著（升高了近5倍）。用HO-1抑制剂ZnPP预处理可增加过氧化氢对细胞的损伤，而用其激动剂hemin预处理可提高细胞抗氧化能力，进一步研究显示，HO-1的表达水平与Nrf2的表达呈正相关，说明HO-1可通过Nrf2通路保护黑素细胞避免氧化损伤； 4. MTS实验结果显示，在未使用H_2O_2处理时，正常人和白癜风患者黑素细胞活性无显著差异，但在H_2O_2作用下，白癜风患者黑素细胞活性较正常人相比显著下降，说明白癜风患者黑素细胞对氧化应激的敏感性更高； 5.激光共聚焦显示，Nrf2在正常人黑素细胞（PIG1）中主要位于胞核，而在白癜风患者黑素细胞（PIG3V）中则主要位于胞浆。在正常状态下，PIG1细胞胞核中Nrf2的含量要高于PIG3V中，而胞浆中的含量则明显低于PIG3V中；免疫印迹实验结果显示，H_2O_2刺激后两组黑素细胞胞核中Nrf2的含量均有所增加，但PIG3V细胞胞核中Nrf2的增加量显著低于PIG1中；双荧光素酶报告基因实验显示，H_2O_2可在两组黑素细胞中以时间依赖性的方式诱导Nrf2转录活性升高，而在PIG3V中Nrf2转录活性的升高幅度显著低于PIG1中；进一步研究显示，在氧化应激下，白癜风患者黑素细胞中HO-1表达增高幅度显著低于正常人黑素细胞中，说明PIG3V中Nrf2存在激活障碍导致HO-1表达水平降低； 6.在PIG3V中上调Nrf2的表达可显著减少氧化应激下细胞死亡率，说明激活Nrf2可降低PIG3V对氧化应激的敏感性； 7.与PIG1相比，PIG3V细胞中ROS和MDA水平升高、SOD和GPx活性及总GSH和GSH含量降低，但是CAT活性在两组细胞中无显著差异，说明PIG3V细胞中氧化-抗氧化失衡； 8.白癜风患者皮损处表皮细胞中Nrf2、p-Nrf2和HO-1表达分布异常，细胞核内Nrf2、p-Nrf2和HO-1含量减少，氧化应激并未引起细胞核内Nrf2、p-Nrf2和HO-1核转位增加； 9.与健康对照相比，白癜风患者血清中HO-1水平降低，而IL-2水平升高，同时发现血清HO-1水平与IL-2水平呈显著负相关； 10.免疫组化结果表明，白癜风患者皮损处表皮细胞中ATM表达降低，而p-ATM表达升高；在PIG1中下调ATM的表达可显著抑制Nrf2、HO-1和p-Nrf2的表达，降低氧化应激下黑素细胞活性，提高细胞内ROS和MDA的水平，而对黑素合成及酪氨酸酶活性则无影响，提示ATM可通过作用于Nrf2通路影响细胞氧化应激水平，ATM表达量降低可能是白癜风患者黑素细胞中Nrf2通路存在激活障碍的原因。结论：通过本课题的研究，我们首次明确了Nrf2-ARE信号通路在黑素细胞抗氧化应激中的重要作用，发现Nrf2激活障碍是白癜风患者黑素细胞易于发生氧化损伤的原因，初步阐明了Nrf2存在激活障碍的分子机制，为进一步认识白癜风中黑素细胞的损伤机制奠定了基础。同时，本研究不仅丰富和完善了白癜风氧化应激发病机制，而且为临床治疗提供了新靶点。
[Abstract]:Background:
Vitiligo is a common depigmented skin disease. The incidence of vitiligo is increasing year by year. The decrease or disappearance of melanocytes is the main cause of depigmentation. Oxidative stress can attack cells by producing large amounts of oxygen free radicals, interfere with their normal metabolism, proliferation and differentiation, and may further induce the body's autoimmune response, forming a cascade effect, causing irreversible damage to epidermal melanocytes. Melanocytes in vitiligo patients are more susceptible to oxidative damage, but the reasons and mechanisms are not clear. Therefore, it is important to clarify the causes and specific molecular mechanisms of melanocytes in vitiligo patients susceptible to oxidative damage for improving the pathogenesis of oxidative stress and guiding clinical treatment.
Nrf2-ARE pathway is the main mechanism of cell resistance to exogenous stimuli and oxidative damage. Nrf2 belongs to the "CNC" family of leucine zipper proteins. It binds to the DNA sequence of the antioxidant response element (ARE) in the nucleus and regulates the expression of a series of downstream phase II detoxifying enzymes and antioxidant genes. When exposed to oxidative stress, Nrf2 uncouples with Keap1 and transfers to the nucleus, binds to antioxidant elements in the nucleus, and initiates the expression of a series of phase II detoxifying enzymes and antioxidant genes, including heme oxygenase-1 (HO-1). 1), NADP (H) quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase regulatory subunit (GCLM). The Nrf2-ARE signaling pathway and its downstream genes have been demonstrated to play an important role in antioxidant damage in a variety of cells, tissues and organs. Nrf2-ARE pathway plays an important role in protecting melanocytes against oxidative damage. Secondly, the abnormal Nrf2-ARE pathway in melanocytes of vitiligo patients makes them more sensitive to oxidative stress.
Objective:
1. To study the expression of Nrf2-ARE pathway and its downstream molecules in human melanocytes, and to clarify the role of Nrf2-ARE pathway in antioxidant stress of melanocytes.
2. To analyze the expression and function of Nrf2-ARE pathway in melanocytes of normal subjects and vitiligo patients.
3. To investigate the expression and functional differences of Nrf2-ARE pathway and its downstream molecules in melanocytes of vitiligo patients.
Method:
1. Firstly, human melanocyte PIG1 was treated with different concentrations of H_2O_2. MTS assay was used to detect the cell activity and explore the best conditions for establishing melanocyte oxidative stress model in vitro.
2. The expression of Nrf2 was down-regulated or up-regulated by transfection of Nrf2 interference fragment and over-expression plasmid in PIG1, and the cell viability and apoptosis were detected by MTS and flow cytometry under oxidative stress.
3. After H_2O_2 pretreatment, the expression levels of four major antioxidant genes (HO-1, NQO-1, GCLC and GCLM) downstream of Nrf2 were detected by Real-time PCR. The expression of Nrf2 and HO-1 in primary melanocytes was detected by immunocytochemistry and Real-time PCR.
4. MTS assay, Real-time PCR, immunoblotting, confocal laser microscopy, double luciferase reporter gene assay, flow cytometry and enzyme-linked immunoassay were used to detect melanocyte activity, HO-1 expression level, Nrf2 nuclear translocation, transcriptional activity, intracellular ROS and MDA levels in normal persons and vitiligo patients under oxidative stress, respectively. The contents of SOD, GPx, CAT and GSH.
5. The expression patterns of Nrf2, p-Nrf2 and HO-1 in the lesions of vitiligo patients were detected by immunohistochemistry, and the expression levels of IL-2 and HO-1 in serum were detected by ELISA.
6. The expression of Nrf2, HO-1 and p-Nrf2 was detected by Real Time-PCR and Western blotting after ATM interference fragment or inhibitor KU55933 was used to inhibit the expression of ATM. After pretreatment with H_2O_2, MTS, flow cytometry and enzyme-linked immunosorbent assay were used to detect the activity of melanocytes under oxidative stress, the levels of ROS and MDA in cells, and immunohistochemistry was used to detect vitiligo. The distribution pattern of ATM and p-ATM in skin lesions.
Result:
1. H_2O_2 can induce melanocyte death in a concentration-dependent manner. 24 h treatment with 1.0 mH_2O_2 is the best condition for establishing melanocyte oxidative stress model in vitro.
2. MTS assay and flow cytometry showed that down-regulation of Nrf2 expression could decrease cell viability and increase the rate of apoptosis and necrosis under oxidative stress, and up-regulation of Nrf2 expression could increase cell viability and significantly decrease the rate of apoptosis, suggesting that regulation of Nrf2-ARE pathway could affect the resistance of melanocytes to oxidative stress.
3. Real-time PCR results showed that H_2O_2 could induce the expression of four major antioxidant genes downstream of Nrf2 pathway to increase, and HO-1 had the most significant change (nearly five times increased). Pretreatment with ZnPP, an inhibitor of HO-1, could increase the damage of hydrogen peroxide to cells, while hemin, an agonist, could enhance the antioxidant capacity of cells. Further studies showed that HO-1 inhibitor could increase the antioxidant capacity of cells. The expression level of HO-1 was positively correlated with that of Nrf2, indicating that HO-1 could protect melanocytes from oxidative damage through Nrf2 pathway.
4. MTS results showed that there was no significant difference in melanocyte activity between normal and vitiligo patients without H_2O_2 treatment, but the activity of melanocyte in vitiligo patients was significantly lower than that in normal people under H_2O_2 treatment, indicating that melanocytes in vitiligo patients were more sensitive to oxidative stress.
5. Laser confocal focusing showed that Nrf2 was mainly located in the nucleus of normal human melanocytes (PIG1) and mainly in the cytoplasm of vitiligo melanocytes (PIG3V). Under normal conditions, the content of Nrf2 in the nucleus of PIG1 cells was higher than that of PIG3V, but the content of Nrf2 in the cytoplasm was lower than that of PIG3V. Immunoblotting results showed that H_2O_2 was mainly located in the cytoplasm of vitiligo melanocytes (PIG3V). After stimulation, the Nrf2 content in the nucleus of both groups increased, but the Nrf2 content in the nucleus of PIG3V cells was significantly lower than that in PIG1 cells; the double luciferase reporter gene experiment showed that H_2O_2 could induce Nrf2 transcriptional activity to increase in a time-dependent manner in both groups of melanocytes, while the Nrf2 transcriptional activity increased in PIG3V cells. Further studies showed that under oxidative stress, the increase of HO-1 expression in melanocytes of vitiligo patients was significantly lower than that in normal melanocytes, suggesting that the activation of Nrf2 in PIG3V may lead to the decrease of HO-1 expression.
6. Up-regulation of Nrf2 expression in PIG3V can significantly reduce cell mortality under oxidative stress, indicating that activation of Nrf2 can reduce the sensitivity of PIG3V to oxidative stress.
7. Compared with PIG1, ROS and MDA levels in PIG3V cells increased, SOD and GPx activities and total GSH and GSH contents decreased, but CAT activity was not significantly different between the two groups of cells, indicating the imbalance of oxidation-antioxidation in PIG3V cells.
8. The expression and distribution of Nrf2, p-Nrf2 and HO-1 were abnormal in epidermal cells of vitiligo patients. The contents of Nrf2, p-Nrf2 and HO-1 in nucleus were decreased. Oxidative stress did not induce the increase of Nrf2, p-Nrf2 and HO-1 translocation in nucleus.
9. Compared with healthy controls, the serum HO-1 level was lower, while the serum IL-2 level was higher in vitiligo patients, and the serum HO-1 level was negatively correlated with the serum IL-2 level.
10. Immunohistochemical results showed that ATM expression was decreased and p-ATM expression was increased in the epidermal cells of vitiligo patients; down-regulation of ATM expression in PIG1 significantly inhibited the expression of Nrf2, HO-1 and p-Nrf2, decreased the activity of melanocytes under oxidative stress, and increased the levels of ROS and MDA, but not in melanogenesis and tyrosinase activity. The results suggest that ATM can affect the level of oxidative stress by acting on Nrf2 pathway, and the decrease of ATM expression may be the reason for the inactivation of Nrf2 pathway in melanocytes of vitiligo patients.
Conclusion:
Through this study, we first clarified the important role of Nrf2-ARE signaling pathway in melanocyte antioxidant stress, and found that the inactivation of Nrf2 is the cause of melanocyte prone to oxidative damage in vitiligo patients, and preliminarily elucidated the molecular mechanism of Nrf2 activation disorder, in order to further understand melanocyte in vitiligo. At the same time, this study not only enriched and improved the mechanism of oxidative stress in vitiligo, but also provided a new target for clinical treatment.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R758.41

【共引文献】