利用微小毛发模型验证α-倒捻子素在毛囊中的抗氧化特性的研究

doi:10.3877/cma.j.issn.2095-1221.2024.02.003

目的

探究药物α-倒捻子素（αMG）在毛囊组织中的抗氧化能力，推测其保护毛发免受自由基伤害、减缓毛发色素脱失的潜力。

方法

基于固-液培养方法建立人头皮毛囊微小毛发模型，经2%过氧化氢（H₂O₂）预刺激后，加入浓度分别为10 μmol/L、40 μmol/L的αMG处理孵育。检测经H₂O₂诱导及αMG的处理后，毛发中活性氧（ROS）的含量与黑色素颗粒产生的位置与数量，以及毛发中的细胞凋亡水平。组间比较采用单因素方差分析，多组间两两比较采用LSD-t检验。

结果

40 μmol/L αMG降低了毛发中的ROS水平，H₂O₂组、H₂O₂ + 40 μmol/ L αMG组的ROS水平分别为空白对照组的（1.44 ± 0.05）、（1.07 ± 0.09）倍。因强氧化剂导致的毛干部黑色素颗粒增加的程度受到抑制，通过抗氧化的特性抑制毛囊黑色素颗粒的分化与迁移，培养7 天异位黑色素颗粒数量从H₂O₂组的（11.3 ± 2.8）个下降至H₂O₂ + 40 μmol/L αMG组的（4.1 ± 3.2）个。αMG还表现出抑制毛发的细胞凋亡的作用，细胞凋亡荧光染色强度相较对照组从H₂O₂组的（1.79 ± 0.13）倍下降至H₂O₂ + 40 μmol/L αMG组（0.83 ± 0.34）倍，其抗氧化水平与强抗氧化剂N-乙酰半胱氨酸相当。

结论

微小毛发模型可作为毛发黑色素细胞分化迁移及色素改变的研究模型，验证药物αMG的抗氧化作用可以减少H₂O₂诱导的毛囊中ROS积累和细胞凋亡，抑制毛囊在应激状态（如强氧化剂作用）下的黑色素颗粒异位分泌。

关键词: α-倒捻子素, 色素脱失, 抗氧化, 毛囊黑色素细胞

Objective

To explore the antioxidant capacity of the drug α-mangostin (αMG) in hair follicle tissue, and speculate on its potential to protect hair from free radical damage and slow down hair pigmentation loss.

Methods

A micro-hair model of human scalp hair follicles was established based on the solid-liquid culture method. After pre-stimulation with 2% hydrogen peroxide (H₂O₂) , αMG with concentrations of 10 μmol/L and 40 μmol/L was added for incubation. After H₂O₂ induction and αMG treatment, the content of reactive oxygen species (ROS) in the hair, the location and number of melanin granules produced, and the level of cell apoptosis in the hair were detected. One-way analysis of variance was used for comparison between groups, the LSD-t test was used for pairwise comparison among multiple groups.

Results

40 μmol/L+αMG significantly decreased the ROS levels in hair. ROS levels in the H₂O₂ group and H₂O₂+40 μmol/ L αMG group were (1.44 ± 0.05) and (1.07 ± 0.09) times that of the blank control group, respectively. The increased in the production of melanin particles in the hair shaft caused by strong oxidants is inhibited, which in turn hinders the differentiation and migration of hair follicle melanin particles due to its antioxidant properties. The number of ectopic melanin particles in the H₂O₂ group increased from (11.3 ± 2.8) to (4.1 ± 3.2) in the H₂O₂+40 μmol/ L αMG group after 7 days of culture. αMG also demonstrates the inhibition of hair cell apoptosis. The fluorescence staining intensity of apoptosis decreased from (1.79 ± 0.13) in the H₂O₂ group to (0.83 ± 0.34) in the H₂O₂+40 μmol/ L αMG group when compared to the control group. Its antioxidant efficacy is comparable to that of the potent antioxidant N-acetylcysteine.

Conclusion

The micro-hair model can be used as a research model for hair melanocyte differentiation, migration and pigment changes. It verifies that the antioxidant effect of the drug αMG can significantly reduce the accumulation of ROS and cell apoptosis in hair follicles induced by H₂O₂, inhibit the ectopic secretion of melanin granules in hair follicles under stress conditions, such as the action of strong oxidants.

Key words: α-Mangostin, Hair Greying, Antioxidant, Melanocytes of the hair follicles

图1 毛囊的分离与培养、微小模型的建立注：a图为从组织中获取的毛囊单位；b图为在显微镜下将附着脂肪组织去除；c图为分离干净的单个毛囊单位垂直种植于Williams's E半固体培养基中

图2 氧化刺激下毛囊微小模型的ROS水平及黑色素颗粒数量的检测注：荧光显微镜下箭头指示处显示深色黑色素颗粒，绿色免疫荧光强度显示毛囊微环境中释放的ROS水平（×40）。在氧化刺激作用下毛囊微环境中释放大量ROS，与空白对照组相比较，^aP < 0.05；与H₂O₂组相比较，^bP < 0.05；ns为差异无统计学意义。高水平ROS会导致毛囊隆突区内的毛囊黑色素干细胞耗竭，黑色素颗粒增多，第1、3、7天时，与空白对照组相比较，^aP < 0.05；与H₂O₂组相比较，^bP < 0.05，ns为差异无统计学意义

图3 荧光显微镜下观察单个毛囊中细胞凋亡水平（×40）注：αMG可以通过调控毛囊微小模型中细胞凋亡程序，延缓毛发变白的进程；Tunel绿色荧光强度显示单个毛囊中细胞凋亡水平，DAPI蓝色荧光显示细胞核位置

图4 氧化刺激下毛囊微小模型的免疫荧光表征细胞凋亡水平注：与空白对照组相比较，^aP < 0.05；与H₂O₂组比较，^bP < 0.05，ns为差异无统计学意义

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Using a micro hair model to verify the antioxidant properties of α-mangostin in hair follicles

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Using a micro hair model to verify the antioxidant properties of α-mangostin in hair follicles