Network pharmacological mechanisms of Vernonia anthelmintica (L.) in the treatment of vitiligo: Isorhamnetin induction of melanogenesis via up-regulation of melanin-biosynthetic genes

Abstract

Background: Vitiligo is a long-term skin disease characterized by the loss of pigment in the skin. The current therapeutic approaches are limited. Although the anti-vitiligo mechanisms of Vernonia anthelmintica (L.) remain ambiguous, the herb has been broadly used in Uyghur hospitals to treat vitiligo. The overall objective of the present study aims to identify the potential lead compounds from Vernonia anthelmintica (L.) in the treatment of vitiligo via an oral route as well as the melanogenic mechanisms in the systematic approaches in silico of admetSAR and substructure-drug-target network-based inference (SDTNBI).

Results: The results showed that the top 5 active compounds with a relatively higher bioavailability that interacted with 23 therapeutic targets were identified in Vernonia anthelmintica (L.) using admetSAR and SDTNBI methods. Among these compounds, Isorhamnetin and Kaempferide, which are methyl-flavonoids, performed 1st and 2nd. Isorhamnetin and Kaempferide significantly increased the expression of melanin-biosynthetic genes (MC1R, MITF, TYR, TYRP1 and DCT) and the tyrosinase activity in B16F10 cells. Isorhamnetin and Kaempferide significantly increased the mRNA-expression of melanin-biosynthetic genes (MC1R, MITF, TYR, TYRP1 and DCT), the protein level of MITF and the tyrosinase activity. Based on the SDTNBI method and experimental verification, Isorhamnetin and Kaempferide effectively increased melanogenesis by targeting the MC1R-MITF signaling pathway, MAPK signaling pathway, PPAR signaling pathway (PPARA, PPARD, PPARG), arachidonic acid metabolism pathway (ALOX12, ALOX15, CBR1) and serotonergic synapses (ALOX12, ALOX15) in the treatment of vitiligo from a network perspective.

Conclusion: We identified the melanogenic activity of the methyl-flavonoids Isorhamnetin and Kaempferide, which were successfully predicted in a network pharmacological analysis of Vernonia anthelmintica (L.) by admetSAR and SDTNBI methods.

Keywords: AdmetSAR; Isorhamnetin; Kaempferide; Melanogenesis; Substructure-drug-target network-based inference; Vernonia anthelmintica (L.); Vitiligo.

Fig. 1

Diagram of the pathway-based SDTNBI and admetSAR approaches developed to identify the network pharmacological mechanisms of Vernonia anthelmintica (L.) for the treatment of vitiligo

Fig. 2

Compound-Target-Pathway interactions. By evaluating the absorption-associated properties of compounds, the 37 potential active compounds with 72 targets were used to construct the CTI network (a). The Potential Active Compound-Target and Pathway association network (b). By evaluating the metabolism-associated properties of compounds, the top 5 active compounds with 23 targets were used to construct the CTI network (c). Isorhamnetin and Kaempferide with 17 targets were used to construct the CTI network (d)

Fig. 3

Effects on the cell viability of B16F10 melanoma cells following treatment with Isorhamnetin and Kaempferide. The structure of Isorhamnetin (a). Effects on the cell viability of B16F10 melanoma cells following treatment with Isorhamnetin (b). The structure of Kaempferide (c). Effects on the cell viability of B16F10 melanoma cells following treatment with Kaempferide (d). The B16F10 melanoma cells were incubated with medium that contained various concentrations (8–32 μM) for 24 h. Cell viability was determined using the MTT assay and is expressed as the means ± standard errors of at least 3 independent experiments performed in triplicate. ^* P < 0.05 vs. the control

Fig. 4

Effects of Isorhamnetin and Kaempferide on melanin production in B16F10 melanoma cells. Appearance of the recovered cell pellets in test tubes by Isorhamnetin (a). The intracellular melanin contents in Isorhamnetin-treated B16F10 melanoma cells for 24 h (b). Extracellular melanin contents in Isorhamnetin-treated B16F10 melanoma cells for 24 h (c). Appearance of the recovered cell pellets in test tubes by Kaempferide (d). Intracellular melanin contents in Kaempferide-treated B16F10 melanoma cells for 24 h (e). Extracellular melanin contents in Kaempferide-treated B16F10 melanoma cells for 24 h (f). Data shown represent the means ± standard error of at least 3 independent experiments performed in triplicate. ^* P < 0.05 vs. the control, ^# P < 0.05 vs. 8-MOP (100 μM)

Fig. 5

Effects of Isorhamnetin and Kaempferide on tyrosinase activity and melanin-biosynthetic genes in B16F10 melanoma cells. Effects of Isorhamnetin on tyrosinase activity in B16F10 melanoma cells (a). Quantified RT-PCR (QPCR) results in Isorhamnetin treatment via relative gene expression ratios to GAPDH (b). Effects of Kaempferide on tyrosinase activity in B16F10 melanoma cells (c). Quantified RT-PCR (QPCR) results in Kaempferide treatment via relative gene expression ratios to GAPDH (d). Data shown are the means ± standard errors of at least 3 independent experiments performed in triplicate. ^* P < 0.05 vs. the control, ^# P < 0.05 vs. 8-MOP (100 μM)

Fig. 6

Effects of Isorhamnetin and Kaempferide on the protein level of MITF in B16F10 melanoma cells. Representative Western blot of MITF in Isorhamnetin-treated B16F10 melanoma cells for 24 h (a). Relative quantitative analysis of MITF in Isorhamnetin-treated B16F10 melanoma cells for 24 h (b). Representative Western blot of MITF in Kaempferide-treated B16F10 melanoma cells for 24 h (c). Relative quantitative analysis of MITF in Kaempferide-treated B16F10 melanoma cells for 24 h (d). Data shown are the means ± standard error of at least 3 independent experiments performed in triplicate. ^*P < 0.05 vs. the control, ^#P < 0.05 vs. 8-MOP (100 μM)

Fig. 7

The melanogenic pathway of Isorhamnetin and Kaempferide from Vernonia anthelmintica (L.) for the treatment of vitiligo. The blue nodes represent the targets predicted by SDTNBI. The red nodes are closely related to melanin-biosynthetic genes and vitiligo pathogenesis