This study aimed to investigate the protective effect of NAcM‐OPT, a small molecule inhibitor of defective in cullin neddylation 1 (DCN1), on H2O2‐induced oxidative damage in keratinocytes. Immortalized human keratinocytes (HaCaT cells) were treated with NAcM‐OPT and exposed to oxidative stress. CCK‐8 assays were used to measure cell viability. The mGFP‐RFP‐LC3 dual fluorescent autophagy indicator system was utilized to evaluate changes in autophagic flux. Western blotting was used to measure the expression of the autophagy‐related proteins LC3 and Beclin 1. Keratinocytes were treated with the autophagy activator rapamycin, and HaCaT cell supernatant was added to PIG1 cells (immortalized human melanocytes), followed by evaluation of tyrosinase (TYR) expression via qRT‐PCR. NAcM‐OPT increased cell viability and cell proliferation. Furthermore, this molecule promoted autophagic flux through increased expression of autophagy‐related proteins under H2O2‐induced oxidative stress. Additionally, rapamycin increased the mRNA levels of TYR in PIG1 cells. Moreover, NAcM‐OPT alleviated mitochondrial damage, restored mitochondrial function, and upregulated the expression of NFE2L2, HO1, NQO1, and GCLM. Importantly, NAcM‐OPT also increased epidermal thickness, follicle length, and melanin synthesis under oxidative stress in vivo. These findings suggest that NAcM‐OPT may be a promising small molecule antioxidant drug for the treatment of vitiligo.

中文翻译：

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NAcM-OPT 通过促进自噬保护角质形成细胞免受 H2O2 诱导的细胞损伤


本研究旨在探讨 NAcM-OPT（一种 cullin neddylation 1 (DCN1) 缺陷的小分子抑制剂）对 H2O2 诱导的角质形成细胞氧化损伤的保护作用。用 NAcM-OPT 处理永生化人角质形成细胞（HaCaT 细胞）并暴露于氧化应激。 CCK-8 测定用于测量细胞活力。 mGFP-RFP-LC3双荧光自噬指示剂系统用于评估自噬通量的变化。采用Western blotting检测自噬相关蛋白LC3和Beclin 1的表达。用自噬激活剂雷帕霉素处理角质形成细胞，将HaCaT细胞上清液添加到PIG1细胞（永生化人黑素细胞）中，然后评估酪氨酸酶（TYR） ) 通过 qRT-PCR 表达。 NAcM-OPT 增加细胞活力和细胞增殖。此外，该分子在 H2O2 诱导的氧化应激下通过增加自噬相关蛋白的表达来促进自噬通量。此外，雷帕霉素增加了 PIG1 细胞中 TYR 的 mRNA 水平。此外，NAcM-OPT 减轻了线粒体损伤，恢复了线粒体功能，并上调了 NFE2L2、HO1、NQO1 和 GCLM 的表达。重要的是，NAcM-OPT 还增加了体内氧化应激下的表皮厚度、毛囊长度和黑色素合成。这些发现表明 NAcM-OPT 可能是一种有前途的小分子抗氧化药物，用于治疗白癜风。