Targets Exploration of Hydroxychloroquine for Pigmentation and Cell Protection Effect in Melanocytes: The Clue for Vitiligo Treatment

Abstract

Objective: The treatment of vitiligo is often challenging to dermatologists. There is ample evidence to suggest that hydroxychloroquine (HCQ) is effective for vitiligo treatment; nonetheless, the underlying mechanism remains unknown. In the present study, we sought to uncover the molecular targets of HCQ by an integrated network-based pharmacologic and transcriptomic approach.

Methods: The potential targets of HCQ were retrieved from databases based on the crystal structure. Targets related to vitiligo were screened and intersected with potential targets of HCQ. A protein-protein interaction network of the intersected targets was generated. Interactions between the targets were verified by molecular docking. Moreover, human vitiligo immortalized melanocytes (PIG3V) were evaluated after treatment with HCQ (1μg/mL) for 24h. The total RNA of PIG3V was extracted and determined by RNA-seq transcriptomics for differential gene expression analysis. Network pharmacology was then used to identify the relationships between putative targets of HCQ and differentially expressed genes.

Results: Molecular docking analysis revealed four putative key targets (ACHE, PNMT, MC₁R, and VDR) of HCQ played important roles in vitiligo treatment. According to the transcriptomic results, the melanosomal biogenesis-related gene BLOC1S5 was upregulated 138005.020 fold after HCQ treatment. Genes related to protein repair (MSRB3) and anti-ultraviolet (UV) effect (UVSSA) were upregulated 4.253 and 2.603 fold, respectively, after HCQ treatment.

Conclusion: The expression of the BLOC1S5 gene is significantly upregulated, indicating upregulated melanosomal biogenesis after HCQ treatment. In addition, HCQ yields a protective effect on melanocytes by upregulating genes associated with damaged protein repair (MSRB3) and anti-UV effect (UVSSA). The protective effects of HCQ are mediated by binding to putative targets ACHE, PNMT, MC₁R, and VDR according to network pharmacology and docking verification.

Keywords: hydroxychloroquine; melanocyte protection; pigmentation; treatment; vitiligo.

Figure 1

The flow chat of strategy layout. The mechanisms of HCQ in treating vitiligo were predicted by network pharmacology. Hub targets were verified by molecular docking. The efficacy of HCQ on vitiligo were then observed on PIG3V cell line. Differential gene expression was analyzed and linked to HCQ targets that verified by docking.

Figure 2

Predicted targets of HCQ. (A) 3 dimensional and 2 dimensional structure of HCQ; (B) top 100 predicted targets of HCQ from databases; (C) categories of the predicted targets.

Figure 3

The PPI network conducted with 15 mutual genes of vitiligo and HCQ potential targets. (A) PPI network of the enlarged 1686 nodes; (B) the nodes and edges after the first screening; (C) the nodes and edges after the second screening.

Figure 4

Molecular docking verification. Target candidates (A) ACHE, (B) VDR, (C) MC1R, and (D) PNMT were shown interacting with HCQ molecule (represented by a green ball-and-stick model).

Figure 5

Transcriptomics analysis of PIG3V cells treated by HCQ. (A) number of up-regulated and down-regulated genes; (B) heat map of up-regulated and down- regulated genes; (C) KEGG enrichment of differential expressed genes; (D) volcano map of up-regulated and down-regulated genes; (E) GO enrichment of differential expressed genes.

Figure 6

The network between HCQ targets and differential expressed genes of PIG3V cells after the treatment of HCQ. (A) PPI network between docked HCQ targets and differential expressed genes of PIG3V showed enlarged 163 nodes (proteins) and 1019 edges (interactions); (B) main connections of the PPI network.

Figure 7

Illustration of related genes involved in pigmentation pathway. BLOC1S5, PMEL, DTNBP1 and PLDN are genes involved in genesis of the melanosome. MC1R, MITF, DKK1, RAB27A, MLPH and MYO5A encode proteins in membrane or cytoplasm which are involved in signaling pathways of skin pigmentation. PAX3 and SOX10 are transcription factors in melanocyte. RACK1 is a regulator to the signal transduction in melanocyte. MSRB3 and UVSSA encode proteins that are involved in damaged proteins/DNA repair under UV. MC1R, VDR, PNMT, and ACHE was targeted by HCQ according to network pharmacology and molecular docking, which could further up-regulate gene expression of BLOC1S5, MSRB3, and UVSSA, showing the promotion effect of melanosome genesis and melanocyte protection under UV damage.