与黑素结合的羟氯喹对长波紫外线诱导HaCaT细胞死亡的光保护机制研究

Photoprotection of HaCaT cells against UVA radiation by melanin-binding hydroxychloroquine in vitro

目的:观察与黑素结合的羟氯喹(HCQ)对细胞内活性氧基(ROS)的清除以及对长波紫外线(UVA)诱导人角质形成细胞(HaCaT细胞)凋亡和坏死的保护。方法:自一株产黑素的嗜麦芽假单胞菌AT18的培养液中分离纯化获得细菌衍生黑素,在体外与不同浓度HCQ结合后处理培养的HaCaT细胞,随后给予半数致死剂量(30J/cm2)的UVA照射。照射12h后,以四甲基偶氮唑蓝(MTT)比色法测定细胞存活率;碘化丙啶(PI)染色结合流式细胞仪检测细胞凋亡率;采用二氯荧光素二酯(DCFH-DA)标记法测定细胞内ROS水平。结果:低浓度(10、50μmol/L)HCQ与黑素结合后能明显保护HaCaT细胞抵抗半数致死剂量(30J/cm2)UVA照射,尤其是50μmol/LHCQ+50mg/L黑素处理组细胞存活率较单纯黑素和单纯HCQ组显著增高(P<0.05);相反,用高浓度(250μmol/L)HCQ与黑素结合处理细胞,细胞存活率较单纯黑素和单纯HCQ组减低。与单纯黑素和单纯HCQ组相比,10μmol/LHCQ与黑素结合还能显著提高细胞内ROS的清除能力和抑制UVA诱导HaCaT细胞凋亡。结论:低浓度HCQ与黑素结合能协同增强黑素对细胞内ROS的清除,抑制UVA诱导的皮肤细胞凋亡,这些作用很可能关系到治疗皮肤型红斑狼疮时抗疟药的抗光敏机制。

Objective： To define the photoprotection of HaCaT cells against UVA radiation and the capacity of scavenging reactive oxygen species （ROS） by melanin-binding hydroxychloroquine （HCQ）. Methods： The bacteria-derived melanin （bmelanin）, an analog of synthetic eumelanin, was isolated from the cultivation of Pseudomonas moltophila AT18. To mimic the accumulation of HCQ in the epidermal melanin in vivo, the varying concentrations of HCQ were incubated with 50 g/L bmelanin for 3 h to allow their binding sufficiently, and then pre-treated HaCaT cells with melanin-binding HCQ and determined their photoprotection against the lethal dose 50% （30 J/cm^2） of UVA radiation. The viabilities of HaCaT cells were measured by MTT assay after 12 hours of UVA radiation, the apoptosis was detected by flow cytometry with a propidium iodide （PI） staining, and the level of intracellular ROS was monitored by DCFH-DA labeling. Results： Low concentration （10 or 50 μmol/L） of HCQ was bound with 50 mg/L b-melanin, which could afford an enhanced photoprotection of HaCaT cells against UVA radiation （30 J/cm^2）. In comparison with b-melanin or HCQ treatment, the survival rate was significantly increased in the HaCaT cells treated with 50 μmol/L HCQ-bound b-melanin （P〈 0.05）. It was surprising that high concentration （250 μmol/L） of HCQ was bound with b-melanin to demonstrate phototoxicity to some extent on UVA-exposed HaCaT cells, Furthermore, the augmentation of ROS scavenging and the inhibition of UVA-induced apoptosis were also found in HaCaT cells treated with 10 μmol/L HCQ-bound b-melanin. Conclusions： The binding of low dose （10-50 μmol/L） of HCQ with melanin dramatically increases the capacity of ROS scavenging and protects the keratinocytes from UVA-induced apoptosis, which may contribute to anti-photosensitivity of antimalarials in the treatment of cutaneous lupus erythematosus.