细菌衍生黑素对长波紫外线诱导人皮肤成纤维细胞凋亡和坏死的保护

Evaluating the photoprotection of bacteria-derived melanin against UVA-induced apoptosis and necrosis in cultured fibroblasts from both normal and DNA repair-deficient patient

目的:探讨细菌衍生黑素(b-melanin)对长波紫外线(UVA)诱导人成纤维细胞凋亡和坏死产生的光保护作用,为今后将其用作皮肤光保护剂提供依据。方法:正常人成纤维细胞(NL-FB)和着色性干皮病患者成纤维细胞(XP-FB)经UVA照射12h后,以四甲基偶氮唑蓝(MTT)法检测细胞存活率,Hoechst33258染色法观察早期凋亡细胞核形态学变化,二氯荧光素二酯(DCFH-DA)标记法测定细胞内活性氧基(ROS)水平。结果:UVA照射诱导细胞的半数致死剂量,XP-FB大约为30J/cm2,而NL-FB>40J/cm2。为了观察不同浓度(0,25,50,100,200,400,和800μg/mL)的b-melanin是否对细胞存在光保护作用,给予半数致死剂量(30J/cm2)UVA照射后,经100～400μg/mLb-melanin处理的XP-FB的细胞存活率均较未处理组明显增高(P<0.01),而NL-FB的细胞存活率变化不明显。细胞内ROS测定结果显示100、400μg/mL的b-melanin能明显清除UVA诱导产生的ROS。100μg/mL b-melanin即能阻止非致死剂量(16J/cm2)UVA照射诱导的早期凋亡细胞核改变。结论:b-melanin能对UVA诱导人成纤维细胞凋亡和坏死提供有效的光保护,这一作用很可能关系到b-melanin对ROS的清除。本研究还首次提出核酸切除修复机制缺陷的XP-FB是一敏感的可用于测试UVA光损伤作用的体外细胞模型。

Objective： To evaluate the photoprotection of bacteria-derived melanin （b-melanin） against UVA-induced apoptosis and necrosis in cultured fibroblasts from both normal subject and DNA repair-deficient patient, enabling topical application of b-melanin as a broad-spectrum sun blocker in the clinical setting. Methods： Human fibroblasts obtained from a healthy donor （NL-FB） and a well-diagnosed patient with xeroderma pigmentosum （XP-FB） were exposed to the different doses of UVA irradiation. Twelve hours after irradiation, the viabilities of the treated cells were measured by MTT assay, the morphology of apoptotic nuclei was detected using Hoechst33258 staining, and the level of intracellular reactive oxygen species （ROS） was determined by DCFH-DA labeling. ResultsL The lethal dose 50% （LD50） of UVA irradiation was about 30 J/cm^2 for XP-FB and was 〉 40 J/cm^2 for NL-FB, indicating that the cell death of XP-FB was more liable to be induced by UVA than that of NL-FB. To define the photoprotection against 30 J/cm^2 UVA irradiation with different doses of b-melanin ranged from 25 to 800 μg/mL, the data showed that the survival rates of the XP-FB treated with varying doses （100-400 μg/mL） were significantly increased in comparison with untreated control （P〈 0.01）, and the rates of NL-FB showed and no obvious change. 400 μg/ mL b-melanin dramatically suppressed intracellular ROS close to the basal level and 100 μg/mL b-melanin was sufficient to block induction of apoptosis by a non-lethal dose （16 J/cm^2） of UVA. Conclusions： b-melanin affords efficacious protection against UVA radiation in cultured human fibroblasts in vitro, which may be due to its ability to scavenge intracellular ROS. These findings provide evidence for the first time that XP-FB, a DNA excision repair-deficient cell, may be used as a sensitive model for evaluation of UVA-induced photodamage in vitro.