研究背景

白癜风是一种主要靶向黑素细胞损伤的自身免疫性疾病，由于黑素细胞进行性丢失而导致皮肤、粘膜脱色，甚至出现白发，发病机制主要和黑素细胞自身缺陷、氧化应激及自身免疫有关。其中，氧化应激被认为是白癜风发生的始动因素，很多证据表明活性氧（ROS）水平升高引起的氧化应激可从内质网应激和DNA损伤等多个方面在起始阶段促进了黑素细胞的破坏。内质网应激的特征是内质网腔内异常折叠的蛋白，进一步触发未折叠蛋白反应（Unfolded protein response, UPR），持续过度的内质网应激会促发细胞的死亡机制。证据表明来自白癜风皮损周围的黑素细胞表现出扩张的内质网结构异常，这提示白癜风黑素细胞的内质网应激水平是升高的。Sirtuin1（SIRT1）是一种烟酰胺腺嘌呤二核苷酸（NAD+）依赖的组蛋白去乙酰化酶，作为重要的代谢和应激调控因子，SIRT1在维持细胞内质网稳态和生存中起重要作用。但是否影响黑素细胞应对内质网应激以及凋亡，是否参与白癜风发病尚未明确。

目的

明确SIRT1是否可以通过调控内质网应激及黑素细胞凋亡参与白癜风发病及其具体机制。

方法

人原代黑素细胞加过氧化氢刺激后，通过quantitative real-time PCR（qRT-PCR）和Westerning-blot（WB）实验检测了内质网应激相关分子GRP78（Glucose Regulated Protein 78）的mRNA和蛋白的水平；收集白癜风患者皮损及周周组织、建立黑素瘤-调节性T细胞白癜风小鼠模型，通过免疫荧光的方法检测白癜风人、小鼠组织中黑素细胞的内质网应激水平的变化；通过单细胞数据分析了白癜风患者组织中黑素细胞的SIRT1的表达变化，并使用免疫荧光进行验证。使用三种内质网应激诱导剂衣霉素（tunicamycin, TM）、毒胡萝卜素（thapsigargin, TG）、二硫苏糖醇（dithiothreitol，DTT）诱导黑素细胞建立内质网应激的细胞模型，并经流式细胞术检测细胞凋亡。应用SIRT1激动剂（Resveratrol，SRT501）和抑制剂（EX527）预处理黑素细胞后再进行内质网应激，检测其内质网应激水平及细胞凋亡改变；黑素瘤-调节性T细胞白癜风小鼠出现脱色表型后应用SRT501和EX527分别干预，经30天后采集小鼠脱色表型的照片，通过Image J软件分析不同实验组小鼠尾部的脱色表型变化，免疫荧光验证不同组小鼠的黑素细胞及内质网应激水平变化；SIRT1激动剂、抑制剂预处理以及小干扰RNA敲降SIRT1后的黑素细胞给予内质网应激诱导后，通过WB实验检测了内质网应激相关蛋白（PERK、IRE1α、Xbp1s等）变化，同时流式细胞术方法检测了细胞凋亡的变化。

结果

氧化应激诱导后的黑素细胞的内质网应激相关分子GRP78的mRNA和蛋白水平表达升高。免疫荧光验证了白癜风患者及白癜风小鼠模型的黑素细胞中内质网应激水平是升高的；白癜风的单细胞数据分析结果显示，黑素细胞中SIRT1的表达是下降的，且通过免疫荧光验证了白癜风患者组织的黑素细胞SIRT1表达是减低的；

通过三种内质网应激诱导剂的比较，选择衣霉素作为后续实验用的诱导剂，内质网应激时，黑素细胞的凋亡是增加的。SRT501和EX527预处理黑素细胞后再进行内质网应激时，前者能减轻其内质网应激并减轻黑素细胞凋亡，后者会加重内质网应激并加重黑素细胞凋亡；SRT501和EX527分别处理白癜风小鼠后，SRT501能减轻黑素细胞的这种内质网应激，而EX527会加重黑素细胞的内质网应激水平，且加重了白癜风小鼠的脱色表型。

SIRT1激动剂和抑制剂分别预处理或小干扰RNA敲降SIRT1后的黑素细胞经衣霉素诱导，SIRT1的激动剂主要影响了内质网应激的IRE1α/Xbp1s通路，并减轻黑素细胞的凋亡，而抑制剂或SIRT1低表达后会加重内质网应激并加重黑素细胞的凋亡。

结论

SIRT1可以通过抑制IRE1α/Xbp1s 通路来减轻内质网应激诱导的黑素细胞凋亡，抑制SIRT1可以影响白癜风小鼠的黑素细胞的内质网应激水平并加重小鼠的脱色表型。

Background 

Vitiligo is an autoimmune disease targeting melanocytes, which ultimately leads to skin and mucosal depigmentation, and even the appearance of white hair, due to the loss of melanocytes. The pathophysiology of vitiligo is primarily associated with melanocyte autoimmunity, oxidative stress, and intrinsic defects of melanocytes. Among these factors, oxidative stress is considered the initiating factor in the development of vitiligo. Numerous evidences suggest that oxidative stress, resulting from elevated levels of reactive oxygen species (ROS), contributes to the destruction of melanocytes in the initial stages through various mechanisms, including endoplasmic reticulum (ER) stress and DNA damage. ER stress is characterized by abnormally folded proteins in the ER lumen, which further triggers the unfolded protein response (UPR). While excessive and continuous UPR may activate cellular death mechanisms. Evidence indicates that melanocytes from the periphery of vitiligo lesions exhibit abnormal dilated ER structures, suggesting elevated ER stress levels in vitiligo melanocytes. Sirtuin1 (SIRT1), a kind of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, plays a crucial role in maintaining ER homeostasis and cell survival as a significant metabolic and stress regulator. However, it remains unclear whether SIRT1 play role in melanocytes and the fate of ER stress and apoptosis, and whether

it is involved in the pathogenesis of vitiligo.

Objective

To determine whether SIRT1 participates in the pathogenesis of vitiligo and its specific mechanisms by regulating ER stress and melanocyte apoptosis.

Methods

After stimulating human primary melanocytes with hydrogen peroxide, we detected the mRNA and protein levels of the ER stress-related molecule GRP78 (Glucose Regulated Protein 78) using qRT-PCR and Western blot (WB) experiments. We examined changes in ER stress levels in melanocytes from vitiligo patients and melanoma-regulatory T cell vitiligo mouse model tissues using immunofluorescence. We analyzed SIRT1 expression changes in melanocytes from vitiligo patient tissues using single-cell data and verified them using immunofluorescence. We induced ER stress in melanocytes using three ER stress inducers: tunicamycin (TM), thapsigargin (TG), and dithiothreitol (DTT). Apoptosis was detected by flow cytometry. Melanocytes were pretreated with SIRT1 agonist (SRT501) and inhibitor (EX527) before inducing ER stress with TM. Changes in ER stress levels were examined. After the appearance of melanoma-regulatory T cell vitiligo mouse phenotype, mice were treated with SRT501 and EX527, respectively. After 30 days, depigmentation photos of the mice were collected, and depigmentation phenotype changes in different experimental groups were analyzed using Image J software. Change in melanocyte quantity and ER stress levels in different groups of mice were verified by immunofluorescence. Pretreated with SRT501 and EX527, or SIRT1 knockdown using small interfering RNA, and  melanocytes were subjected to ER stress induction,then, ER stress-related proteins (PERK, IRE1α, Xbp1s, etc.) were detected by WB, and apoptosis was detected by flow cytometry.

Results

1. The mRNA and protein expression levels of endoplasmic reticulum (ER) stress-related molecule GRP78 are elevated in melanocytes following oxidative stress induction. Immunofluorescence confirmed increased ER stress levels in melanocytes from both vitiligo patients and a vitiligo mouse model.Single-cell data analysis of vitiligo reveals a decrease in SIRT1 expression in melanocytes, consistent with the low expression of SIRT1 detected by immunofluorescence in vitiligo patient tissues.

2. Among three ER stress inducers compared, tunicamycin was selected as the experimental inducer due to its superior induction effect. During ER stress, melanocyte apoptosis increases, while SIRT1 expression decreases. Pretreatment of melanocytes with SIRT1 agonists and inhibitors prior to ER stress induction demonstrated that SIRT1 agonists could alleviate ER stress, whereas inhibitors exacerbated it. Similarly, in vitiligo mice treated with SIRT1 agonist and inhibitor, respectively, the agonist reduced ER stress, while the inhibitor enhanced ER stress level in melanocytes and aggravated the phenotype of vitiligo mice.

3. After pretreatment with SIRT1 agonist or inhibitor, or SIRT1 knockdown using small interfering RNA, tunicamycin-induced melanocytes showed that SIRT1 agonist affected the IRE1α/Xbp1s pathway of ER stress and reduced melanocyte apoptosis. Conversely, inhibitors or SIRT1 downregulation augmented ER stress and melanocyte apoptosis.

Conclusion 

SIRT1 can alleviate ER stress-induced melanocyte apoptosis by inhibiting the IRE1α/Xbp1s pathway. Inhibiting SIRT1 affects ER stress levels in melanocytes and exacerbates the depigmentation phenotype of vitiligo mice.