电子自旋共振捕捉技术测定与黑素结合的羟基氯喹对羟自由基的清除

The measurement of hydroxyl free radical scavenging activity of melanin-binding hydroxyl chloroquine by an electron spin resonance spectroscopy

目的:观察与黑素结合的羟基氯喹(HCQ)对Fenton反应瞬时产生羟自由基([·OH])的清除能力,以阐述抗疟药物的抗炎和抗皮肤光敏感性作用的分子机制。方法:利用Fenton反应瞬时产生[·O H],将不同浓度的H4CQ和细菌衍生黑素结合后加入Fenton反应体系,用5,5-二甲基-1-吡咯啉-N-氧化物(DMPO)捕捉[·OH],用电子自旋共振(ESR)仪记录DMPO-OH特征波谱,通过测量反应体系中自由基的减少量算出该样品对[·OH]的清除率。结果:细菌衍生黑素在20～100mg/L浓度范围对自由基的清除呈剂量依赖性,在100mg/L浓度时对[·OH]的清除率达92.5%;HCQ对[·OH]的清除呈非剂量依赖性,50μmol/L HCQ呈现出最大的清除率(43.72%);10μmol/LHCQ与黑素结合后对[·OH]清除能力与单纯HCQ组相比显著增加(P<0.05),提高与黑素结合的HCQ浓度并不能增加对自由基的清除率。结论:一定浓度的HCQ与黑素结合可增加皮肤细胞对活性氧基的瞬时清除能力。

Objective： To determine the scavenging effects of melanin-binding hydroxyl chloroquine （HCQ） on transient hydroxyl free radical generated via the Fenton reaction, it is helpful in better understanding the mechanisms of antimalarials be- hind anti-inflammation and anti-photosensitivity. Methods： Transient hydroxyl free radical was generated via the Fenton reaction and trapped by DMPO. DMPO （400 mmol/L）, ferrous sulfate （0.4 mmol/L）, H202 （0.1%） and varying concentrations of melanin-binding HCQ were mixed and incubated, then sucked into a quartz capillary for 30 sec and measured by electron spin resonance （ESR）. The relative amount of the radicals was estimated from the peak height of the second peak of the DMPO-OH signals in typical ESR spectra. Results： The bacteria-derived melanin （b-melanin） ranging from 20 mg/L to 100 mg/L exhibited a potent scavenging activity against hydroxyl free radical in a dose-dependent manner. 100 mg/L of b-melanin could afford a maximum scavenging activity （92.5%） against hydroxyl free radical. HCQ showed a limited and dose-independent scavenging activity toward hydroxyl radical. 50 mol/L of HCQ had the maximum scavenging rate （43.72%） against hydroxyl free radicals. The melanin-binding with 10 μmol/L HCQ, which was equivalent to a mean blood concentration of HCQ in SLE patients receiving 400 mg of HCQ daily, demonstrated an increased scavenging activity against the radicals to compare with that of HCQ alone （P〈 0.05）. High scavenging activity of melanin-binding HCQ against free radical was failure to reach by an increased concentration of HCQ. Conclusion： The melanin achieves an enhanced scavenging activity against hydroxyl free radical by binding with HCQ at its physiological concentration （10 μmol/L） and thereby protects skin cells from oxidative damages, which likely contributes to anti-photosensitivity of antimalarials in the treatment of cutaneous lupus erythematosus.