Abstract：
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.
摘要：
本研究旨在探讨NAcM-OPT的保护作用,cullinneddylation1（DCN1）缺陷的小分子抑制剂，H2O2诱导的角质形成细胞氧化损伤。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双荧光自噬指示系统用于评估自噬通量的变化。使用蛋白质印迹法测量自噬相关蛋白LC3和Beclin1的表达。用自噬激活剂雷帕霉素处理角质形成细胞，并将HaCaT细胞上清液添加到PIG1细胞（永生化人黑素细胞）中，然后通过qRT-PCR评估酪氨酸酶（TYR）的表达。NAcM-OPT增加细胞活力和细胞增殖。此外，该分子通过在H2O2诱导的氧化应激下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减轻线粒体损伤，线粒体恢复功能，并上调NFE2L2、HO1、NQO1和GCLM的表达。重要的是,NAcM-OPT也增加了表皮厚度，卵泡长度,和体内氧化应激下的黑色素合成。这些发现表明NAcM-OPT可能是治疗白癜风的一种有前途的小分子抗氧化药物。
