Astragaloside IV Enhances Melanogenesis via the AhR-Dependent AKT/GSK-3 β/ β-Catenin Pathway in Normal Human Epidermal Melanocytes

Abstract

Astragalus membranaceus root has been widely used for repigmentation treatment in vitiligo, but its mechanism is poorly understood. We sought to investigate the effect of astragaloside IV (AS-IV), a main active extract of the Astragalus membranaceus root, on melanin synthesis in normal human epidermal melanocytes (NHEMs) and to elucidate its underlying mechanisms. Melanin content, tyrosinase activity, qPCR, western blot, and immunofluorescence were employed. Specific inhibitors and small interfering RNA were used to investigate the possible pathway. AS-IV stimulated melanin synthesis and upregulated the expression of melanogenesis-related genes in a concentration-dependent manner in NHEMs. AS-IV could activate the aryl hydrocarbon receptor (AhR), and AS-IV-induced melanogenesis was inhibited in si-AhR-transfected NHEMs. In addition, we showed that AS-IV enhanced the phosphorylation of AKT and GSK-3β and nuclear translocation of β-catenin. AS-IV-induced MITF expression upregulation and melanin synthesis were decreased in the presence of β-catenin inhibitor FH353. Furthermore, AhR antagonist CH223191 inhibited the activation of AKT/GSK-3β/β-catenin signaling, whereas the expression of CYP1A1 (marker of AhR activation) was not affected by the AKT inhibitor in AS-IV-exposed NHEMs. Our findings show that AS-IV induces melanogenesis through AhR-dependent AKT/GSK-3β/β-catenin pathway activation and could be beneficial in the therapy for depigmented skin disorders.

Figure 1

Chemical structure of AS-IV.

Figure 2

Effects of AS-IV on cell viability and melanogenesis in NHEMs. NHEMs were incubated with various concentrations (0, 1, 10, and 100 μM) of AS-IV for 48 h for (a) morphology (magnification: 40x) and (b) cell viability; NHEMs were incubated with various concentrations (0, 1, 10, and 100 μM) of AS-IV or 8-MOP (100 μM) for 24 h for (c) melanin synthesis detection and (d) tyrosinase activity. Results were expressed as mean ± SD (n = 3) based on the representative experiment of at least three replicates. ^∗∗P < 0.01 and ^∗∗∗P < 0.001, versus untreated cells; ^#P < 0.05, versus AS-IV-treated (100 μM) cells according to one-way ANOVA followed by Tukey's multiple comparisons.

Figure 3

AS-IV upregulated the expression of melanogenesis-related genes in NHEMs. NHEMs were exposed to various concentrations (0, 1, 10, and 100 μM) of AS-IV or 8-MOP (100 μM) for 24 h for (a) qPCR, (b) western blot, and (c) quantitative analyses. Results were expressed as mean ± SD (n = 3) of one representative experiment of at least three replicates. ^∗P < 0.01, ^∗∗P < 0.01, and ^∗∗∗P < 0.001, versus untreated cells; ^#P < 0.05, versus AS-IV-treated (100 μM) cells according to one-way ANOVA followed by Tukey's multiple comparisons.

Figure 4

AhR signaling was essential for AS-IV-induced melanogenesis in NHEMs. NHEMs were incubated with AS-IV (100 μM) or FICZ (100 nM) for 4 h. AhR nuclear translocation was detected by (a) confocal laser scanning microscopic analysis (scale bar = 50 μm) and (b) western blot analysis and quantitative analysis. NHEMs were incubated with various concentrations (0, 1, 10, and 100 μM) of AS-IV for 12 h or 24 h for (c) qPCR and (d) western blot and quantitative analyses. (e) The effect of si-AhR transfection. si-control and si-AhR-transfected NHEMs were treated with AS-IV at 100 μM for 24 h for (f) western blot and quantitative analyses and (g) melanin content assay. Results were expressed as mean ± SD (n = 3), and images showed one representative experiment of at least three replicates. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 according to one-way ANOVA followed by Dunnett's multiple comparisons.

Figure 5

AKT/GSK-3β/β-catenin pathway was involved in AS-IV-induced melanogenesis in NHEMs. NHEMs were incubated with various concentrations (0, 1, 10, and 100 μM) of AS-IV for 24 h, and the protein expression of p-AKT, AKT, p-GSK-3β, GSK-3β, and β-catenin was analyzed by (a) western blot and (b) quantitative analyses. NHEMs were incubated with or without AS-IV (100 μM) for 24 h, and the protein expression of β-catenin in the cytoplasm and nucleus was analyzed by (c) western blot analysis and quantitative analysis. NHEMs were incubated with AS-IV (100 μM) for 24 h with or without pretreatment with FH535 (25 μM) for 3 h. Protein expression of MITF was analyzed by (d) western blot and quantitative analyses, and (e) melanin synthesis was detected. Results were expressed as mean ± SD (n = 3). ^∗∗P < 0.01, ^∗∗∗P < 0.001, and NS indicates nonsignificance according to one-way ANOVA followed by Dunnett's multiple comparisons.

Figure 6

AKT/GSK-3β/β-catenin pathway was controlled by AhR activation in response to AS-IV in NHEMs. NHEMs were incubated with AS-IV (100 μM) for 24 h with or without pretreatment with AKT inhibitor IV (10 μM) or CH223191 (10 μM) for 3 h. Protein expressions of CYP1A1, p-AKT, AKT, p-GSK-3β, GSK-3β, MITF, and nuclei β-catenin were analyzed using (a) western blot and (b) quantitative analyses; (c) melanin synthesis detection. Results were expressed as mean ± SD (n = 3). ^∗∗P < 0.01 and ^∗∗∗P < 0.001 according to one-way ANOVA followed by Dunnett's multiple comparisons.

Figure 7

A proposed mechanism of AS-IV-induced melanogenesis. Astragaloside IV activates AhR, which then induces the phosphorylation of AKT and GSK-3β to promote β-catenin nuclear translocation. β-Catenin binds the promoter of the MITF gene to increase MITF transcription and subsequently upregulates TYR, TRP-1, and TRP-2 expressions and ultimately stimulates the melanin synthesis.