Yang Chun-Tao, Meng Fu-Hui, Chen Li, Li Xiang, Cen Lai-Jian, Wen Yu-Hua, Li Cai-Chen, Zhang Hui
Key Laboratory of Protein Modification and Degradation, School of Basic Medical Sciences, Guangzhou, China
Affiliated Cancer Hospital & Institute, Guangzhou, China
Cell Physiol Biochem. 2017;41(2):742-754. doi: 10.1159/000458734. Epub 2017 Feb 13.
BACKGROUND/AIM: Accumulation of advanced glycation end products (AGEs) is a major cause of diabetes mellitus (DM) skin complications. Methylglyoxal (MGO), a reactive dicarbonyl compound, is a crucial intermediate of AGEs generation. N-acetyl-L-cysteine (NAC), an active ingredient of some medicines, can induce endogenous GSH and hydrogen sulfide generation, and set off a condensation reaction with MGO. However, there is rare evidence to show NAC can alleviate DM-induced skin injury through inhibition of AGEs generation or toxicity. The present study aimed to observe the effects of NAC on MGO-induced inflammatory injury and investigate the roles of AGEs and its receptor (RAGE) in NAC's dermal protection in human HaCaT keratinocytes.
The cells were exposed to MGO to simulate a high MGO status in diabetic blood or tissues. The content of AGEs in serum or cell medium was measured with ELISA. The protective effects of NAC against MGO-induce injury were evaluated by administration before MGO one hour, in virtue of cell viability, mitochondrial membrane potential, inflammation reaction, nuclear factor (NF)-κB activation, matrix metalloproteinase (MMP)-9 expression, as well as cellular behavioral function.
We found the AGEs levels of patients with DM were elevated comparing with healthy volunteers. The in vitro AGEs generation was also able to be enhanced by the exposure of HaCaT cells to MGO, which reduced dose-dependently cellular viability, damaged mitochondrial function, triggered secretion of interleukin (IL)-6 and IL-8, activated NF-κB and upregulated MMP-9 expression. Furthermore, the exposure caused cellular adhesion and migration dysfunction, as well as collagen type I inhibition. Importantly, before the exposure to MGO, the preconditioning with NAC significantly attenuated MGO-induced AGEs generation, improved cellular viability and mitochondrial function, partially reversed the overexpression of proinflammatory factors and MMP-9, as well as the activation of NF-κB. Lastly, NAC blocked MGO-induced RAGE upregulation, and inhibition of RAGE with its neutralizing antibody significantly alleviated MGO-induced NF-κB activation, MMP-9 upregulation and inflammatory injury in HaCaT cells.
The present work indicates the administration of NAC can prevent MGO-induced dermal inflammatory injury through inhibition of AGEs/RAGE signal, which may provide a basal support for the treatment of diabetic skin complications with NAC-containing medicines in the future.
背景/目的：晚期糖基化终末产物（AGEs）的积累是糖尿病（DM）皮肤并发症的主要原因。甲基乙二醛（MGO）是一种活性二羰基化合物，是AGEs生成的关键中间体。N-乙酰-L-半胱氨酸（NAC）是一些药物的活性成分，可诱导内源性谷胱甘肽（GSH）和硫化氢生成，并与MGO引发缩合反应。然而，鲜有证据表明NAC可通过抑制AGEs生成或毒性来减轻DM引起的皮肤损伤。本研究旨在观察NAC对MGO诱导的炎症损伤的影响，并研究AGEs及其受体（RAGE）在NAC对人HaCaT角质形成细胞的皮肤保护作用中的作用。
将细胞暴露于MGO以模拟糖尿病血液或组织中的高MGO状态。用酶联免疫吸附测定法（ELISA）测量血清或细胞培养基中AGEs的含量。通过在MGO处理前1小时给予NAC，借助细胞活力、线粒体膜电位、炎症反应、核因子（NF）-κB激活、基质金属蛋白酶（MMP）-9表达以及细胞行为功能，评估NAC对MGO诱导损伤的保护作用。
我们发现DM患者的AGEs水平高于健康志愿者。HaCaT细胞暴露于MGO也能增强体外AGEs的生成，这会剂量依赖性地降低细胞活力、损害线粒体功能、引发白细胞介素（IL）-6和IL-8分泌、激活NF-κB并上调MMP-9表达。此外，该暴露导致细胞黏附和迁移功能障碍以及I型胶原蛋白抑制。重要的是，在暴露于MGO之前，用NAC预处理可显著减弱MGO诱导的AGEs生成，改善细胞活力和线粒体功能，部分逆转促炎因子和MMP-9的过表达以及NF-κB的激活。最后，NAC阻断MGO诱导的RAGE上调，用其阻断抗体抑制RAGE可显著减轻MGO诱导的HaCaT细胞中NF-κB激活、MMP-9上调和炎症损伤。
目前的研究表明，给予NAC可通过抑制AGEs/RAGE信号来预防MGO诱导的皮肤炎症损伤，这可能为未来用含NAC的药物治疗糖尿病皮肤并发症提供基础支持。
