维生素C溶液在H2O2诱导体外培养黑素细胞氧化损伤中的影响

In vitro effects of ascorbic acid on H2O2-induced oxidative injury in cultured melanocytes

目的探讨维生素C对体外培养的人黑素细胞增殖活性的影响并评估维生素C对H2O2诱导的人黑素细胞氧化损伤的影响。方法通过CCK8法取最佳浓度的维生素C溶液和半数致死浓度的H2O2溶液应用于实验。将黑素细胞分为对照组、维生素C组、H2O2组、联合处理组4组，经过48h处理后，分别用CCK8法和流式细胞仪检测4组细胞活率及凋亡率；将除维生素C组以外其他3组细胞，分别用生物化学法检测超氧化物歧化酶活力和丙二醛浓度，荧光染色法检测细胞内的活性氧。结果最佳浓度的维生素C溶液为1000μmol／L，半数致死浓度的H2O2溶液300μmol／L。对照组细胞活率、细胞凋亡率、超氧化物歧化酶活力、丙二醛浓度、活性氧荧光强度分别为（100±4．99）％、（6．90±0．87）％、（54．71±4．75）U／mgprot、（263．39±20．17）nmol／mgprot、342．16±27．36。H202溶液可以明显提高黑素细胞凋亡率[（16．47±1．07）％]、超氧化物歧化酶活性[（103．62±10．44）U／mgprot]、丙二醛[（493．70±31．36）nmol／mgprot]和细胞内活性氧（782．48±36．25）水平，降低细胞活率[（39．07±2．94）％]，而维生素C溶液可以显著降低H2O2溶液诱导的凋亡[（11．83±0．95）％]，降低超氧化物歧化酶活性[（76．73±5．20）U／mgprot]、丙二醛[（371．82±23．05）nmol／mgprot]、活性氧（475．64±52．18）的含量，同时恢复细胞的活率[（74．31±5．53）％）]。结论1000μmol／L维生素C溶液不仅能促进人黑素细胞的增殖，对H2O2诱导的黑素细胞氧化应激损伤具有保护作用。

Objective To evaluate effects of ascorbic acid on proliferative activity of cultured melanocytes in vitro, as well as on H2O2-induced oxidative injury in melanocytes. Methods The optimal concentration of ascorbic acid solution and median lethal dose of H2O2 solution were determined by CCK-8 assay for the following experiment. Cultured melanocytes were classified into the control group, ascorbic acid group, H2O2 group and combination group. During the first 24 hours, the control group and H2O2 group were treated with M254 medium, while the ascorbic acid group and combination group with ascorbic acid solution. During an additional 24-hour period, the control group and ascorbic acid group were treated with M254 medium, while the H2O2 group and combination group with H2O2 solution at the median lethal dose. After 48-hour treatment, CCK-8 assay and flow cytometry were performed to determine the survival rate and apoptosis rate of melanocytes, respectively, in the 4 groups. Biochemical methods were used to evaluate the superoxide dismutase （SOD） activity and determine the malondialdehyde （MDA） concentration, and fluorescent staining was conducted to detect the level of reactive oxygen species （ROS） in the control group, H2O2 group and combination group. Results The optimal concentration of ascorbic acid solution was 1 000 μmol/L, and the median lethal dose of H2O2 solution was 300 μmol/L. The cell survival rate, apoptosis rate, SOD activity, MDA concentration and ROS fluorescence intensity in the control group were 100% ± 4.99%, 6.90% ± 0.87%, 54.71 ± 4.75 U/mgprot, 263.39 ± 20.17 nmol/mgprot and 342.16 ± 27.36 respectively. Compared with the control group, H2O2 solution could significantly increase the cell apoptosis rate （16.47% ± 1.07%）, SOD activity （103.62 ± 10.44 U/mgprot）, MDA concentration （493.70 ± 31.36 nmol/mgprot） and intracellular ROS fluorescence intensity （782.48 ± 36.25）, but decrease the survival rate of melanocytes （39.07% ± 2.94% ）, while ascorbic acid solution markedly downregulated the H2O2- induced apoptosis （11.83% ± 0.95%）, SOD activity （76.73 ± 5.20 U/mgprot）, MDA concentration （371.82 ± 23.05 nmol/mgprot） and ROS level （475.64 ± 52.18）, but increased the eell survival rate （74.31% ± 5.53% ）. Conclusion Ascorbie acid solution at the concentration of 1 000 μmol/L can not only promote proliferative activity of melanocytes, but also protect melanocytes from H2O2-induced oxidative injury.