3,3'-二吲哚甲烷对过氧化氢诱导人角质形成细胞氧化应激的预防作用及其机制

目的:探究3,3'-二吲哚甲烷(DIM)对过氧化氢(H₂O₂)诱导人角质形成细胞(HaCaT)氧化应激作用的预防效应及可能机制。方法:体外培养HaCaT细胞,用H₂O₂建立氧化应激模型。采用CCK-8法检测不同浓度(1~20 μmol/L)DIM对HaCaT细胞生长的抑制作用;流式细胞术检测DIM作用前后细胞内活性氧自由基(ROS)含量的变化;Western blot检测不同浓度DIM(0、5、10 μmol/L)对HaCaT氧化应激相关蛋白核因子(NF-κB)和丝裂原活化蛋白激酶(MAPKs)磷酸化表达水平的影响。结果:成功建立了HaCaT氧化应激模型。CCK-8法研究结果显示1~10 μmol/L DIM对HaCaT细胞无明显毒性作用(P>0.05);流式细胞术检测结果表明10 μmol/L DIM预处理可有效预防由H₂O₂诱导的HaCaT内ROS产生(P<0.05);Western blot检测结果显示,随着DIM浓度的增加,HaCaT中p38-MAPK、JNK和NF-κB磷酸化蛋白表达水平均逐渐降低(P均<0.05)。结论:DIM预处理可减弱HaCaT中由H₂O₂引起的氧化应激,其机制可能是通过抑制ROS的产生及对NF-κB、MAPK家族等氧化应激相关蛋白表达的调控作用来实现的,提示DIM可作为预防或改善氧化应激型皮肤细胞损伤的一种有效药物。

Preventive effect and mechanisms of 3,3'-diindolylmethane on oxidative stress induced by hydrogen peroxide in HaCaT cells

OBJECTIVE:To observe the effects of 3, 3'-diindolylmethane (DIM) on oxidative stress induced by hydrogen peroxide (H₂O₂) in HaCaT cells. METHODS:HaCaT cells were treated with different concentrations of H₂O₂ to establish the models of oxidative stress. CCK-8 assay was performed to evaluate the inhibitive effects of DIM (1-20 μmol/L) on cells proliferation. The intracellular reactive oxygen species (ROS) levels were measured with flow cytometry. Furthermore, the effects of DIM (0, 5 and 10 μmol/L) on H₂O₂-induced the phosphorylation level of nuclear factor κB (p-NF-κB), the expression of mitogen activated protein kinase (MAPKs) were detected with Western blot. RESULTS: The cell model of oxidative stress was successfully established. The results of CCK-8 assay showed that in the doses of 1-10 μmol/L, DIM did not have obvious influence on cell viability (P>0.05). Flow cytometry results indicated that pre-treatment with DIM (10 μmol/L) could inhibit the level of intracellular ROS (P<0.05). With increasing concentration of DIM, the levels of p-p38-MAPK, p-JNK and p-NF-κB were significantly depressed. CONCLUSION: DIM could protect HaCaT cells from H₂O₂-induced oxidative stress via suppressing production of ROS levels and down-regulating the expression of NF-κB and members of MAPKs. DIM might be used as an effective drug to treat or reduce oxidative stress-mediated injury in skin cells.