Niacinamide Down-Regulates the Expression of DKK-1 and Protects Cells from Oxidative Stress in Cultured Human Dermal Papilla Cells

Abstract

Purpose: An increasing number of people are suffering from hair loss disorders. Niacinamide has long been used as an active ingredient for anti-hair loss preparations but the exact mechanism has not been clearly elucidated yet. The effects of niacinamide were investigated in cultured human dermal papilla cells (hDPCs).

Methods: To investigate the anti-hair loss effect of niacinamide and its molecular mechanisms, Western blot analysis, ELISA, quantitative RT-PCR and immunocytochemistry were performed. To study the protective effects of niacinamide against H₂O₂-induced oxidative stress, ROS generation and cytotoxicity were evaluated by DCF-DA assay and LDH release assay, respectively. Minoxidil was used as a positive control.

Results: Niacinamide decreased the protein expression level of DKK-1 which promotes regression of hair follicles by inducing catagen. The protein expression levels of cell senescence markers, p21 (CDKN1A) and p16 (CDKN2A) which are related to cell cycle arrest, were decreased. The expression of versican was increased by niacinamide treatment in cultured hDPCs. We have found that niacinamide decreased the H₂O₂-induced intracellular ROS production in cultured hDPCs. Moreover, niacinamide decreased the protein expression levels of H₂O₂-induced p21 and p16 and diminished the secretion of H₂O₂-induced DKK-1.

Conclusion: Our data demonstrate that niacinamide could enhance hair growth by preventing oxidative stress-induced cell senescence and premature catagen entry of hair follicles.

Keywords: ROS; anti-hair loss; cell cycle; hair follicle.

Figure 1

Cell viability of niacinamide in hDPCs. (A) Chemical structure of niacinamide. (B) Cells were plated on 96-well plates with complete medium. CCK-8 assay was performed to determine cell viability after treatment with various concentrations of niacinamide (0, 0.5, 1, 2 and 4 mM) and 10 µM of minoxidil for 24 h. Data represent mean ± S.D. ^#p < 0.05 compared to non-treated control.

Figure 2

Niacinamide decreased the expression of DKK-1 and increased the expression of versican in hDPCs. (A) Cells (5.0 × 10⁵ cells/well) were seeded in 60 mm dishes. After 24 h, cells were treated with niacinamide (1, 2, and 4 mM) and 10 µM of MNX in complete medium for 24 h. Western bot analysis was performed using 40 μg of total protein. GAPDH was used as a loading control (B) The band intensity was quantitated. (C) Cells (2 × 10⁴ cells/well) were seeded in 24-well plates and cultured for 24 h. Cells were treated with niacinamide (1, 2, and 4 mM) and 10 µM of MNX in complete medium for 24 h. After treatment, culture medium from each well was analyzed using DKK-1 ELISA kit. (D) Versican expression was visualized as green by fluorescent microscopy. The scale bar represents 200 μm. Data represent mean ± SD. ^#p < 0.05 and ^##p < 0.01 compared to non-treated.

Figure 3

Effects of niacinamide on H₂O₂-induced oxidative stress. (A) Protective effect of niacinamide on H₂O₂-induced ROS generation. Niacinamide (1, 2 and 4 mM) was treated with 1 mM H₂O₂ for 30 min. After treatment, cells were stained with DCF-DA probe for 30 min and fluorescent images were obtained. The scale bar represents 200 μm. (B) ROS generation was quantitated using Celleste™ Image Analysis Software. (C) LDH release was assessed in hDPCs following H₂O₂ exposure for 24 h. (D) Protective effect of niacinamide on H₂O₂-induced LDH release. Niacinamide (1, 2 and 4 mM) was treated with 750 µM H₂O₂ for 24 h. Data represent mean ± S.D. ^#p < 0.05 and ^##p < 0.01 compared to non-treated control. *p < 0.05, **p < 0.01 compared to H₂O₂-treated control.

Figure 4

Continued.

Figure 4

Effects of niacinamide on protein and mRNA expression of cyclin dependent kinase Inhibitors in hDPCs with and without H₂O₂ treatment. (A) Cells (5.0 × 10⁵ cells/well) were seeded in 60 mm dishes. After 24 h, cells were treated with niacinamide (1, 2, and 4 mM) and 10 µM of MNX in complete medium for 24 h. Western bot analysis was performed using 40 μg of total protein. GAPDH was used as a loading control (B) The band intensity was quantitated. Data represent mean ± SD (n = 4) (C) Cells (5.0 × 10⁵ cells/well) were seeded in 60 mm dishes and cultured for 24 h, then medium was replaced with serum limitation medium. After serum limitation for 24 h, cells were treated with various concentrations of niacinamide (1, 2 and 4 mM) and 10 µM of MNX in the presence of 500 µM of H₂O₂ for 24 h. Western bot analysis was performed using 40 μg of total protein. GAPDH was used as a loading control (D) The band intensity was quantitated. Data represent mean ± SD (n = 4) (E) Cells (1.5 × 10⁵ cells/well) were seeded in 6-well plates and incubated for 24 h. After serum limitation for 24 h, niacinamide was treated at concentrations of 1, 2, and 4 mM for 24 h in the presence of H₂O₂. The mRNA expression levels of p21 (CDKN1A) and p16 (CDKN2A) were measured by RT-PCR. Data represent mean ± SD (n = 3). ^#p < 0.05 and ^##p < 0.01 compared to non-treated control. *p < 0.05 and **p < 0.01 compared to H₂O₂-treated control.

Figure 5

Niacinamide decreased H₂O₂-induced DKK-1 protein secretion in hDPCs. Cells (2 × 10⁴ cells/well) were seeded in 24-well plates and cultured for 24 h, then medium was replaced with serum limitation medium. After serum limitation for 24 h, cells were treated with various concentrations of niacinamide (1, 2 and 4 mM) and 10 µM of MNX in the presence of 500 µM of H₂O₂ for 24 h. After treatment, culture medium from each well was analyzed using DKK-1 ELISA kit. Data represent mean ± SD (n = 4). ^##p < 0.01 compared to non-treated control. **p < 0.01 and ***p < 0.001 compared to H₂O₂-treated control.