The mechanism of Croci stigma in the treatment of melasma based on network pharmacology and molecular docking

To investigate the molecular mechanism of Croci stigma (CS) in the treatment of melasma by network pharmacology and molecular docking.

Croci stigma (CS) is a traditional chinese medicine (TCM) separated from the Iridaceae plant Crocus sativus L. 4 Modern pharmacological studies have found that Crocus sativus L. exerts the effects of anti convulsion, anti-inflammatory, anti-tumor, free radical scavenging, learning and memory improvement. 5 And as a potential drug with anti-Alzheimer's disease, antidepressant and anti schizophrenia effects, Crocus sativus L. has been used in some clinical trials. 6 As an aglycone of crocin naturally occurring in Crocus sativus L., crocetin has been demonstrated to have functions including cardioprotection, hepatoprotection, neuroprotection, antidepressant, antiviral, anticancer, atherosclerosis, antidiabetes, and memory enhancement. 7 In an experiment with B16 melanoma cells, crocetin was found to inhibit tyrosinase activity and lower the amount of melanin, as well as decrease protein levels of tyrosinase and MITF. 8 As an emerging science, network pharmacology approach has been used to study "compound-protein/gene-disease" system, which is considered as an effective measure that can describe the complexity between biological systems, drugs and diseases from a network perspective. 9 This study systematically analyzed the potential therapeutic mechanisms of CS against melasma by constructing a network pharmacology model, and molecular docking model between potential hub targets and active compounds were established to confirm the predicted results. The detailed flow is described in Figure 1.

F I G U R E 1
The specific process of network pharmacological analysis of Croci Stigma treat melasma.

| Screening of active compounds from CS
The active compounds of CS were extracted from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://tcmsp -e.com/tcmsp.php), a unique systems pharmacology platform of Chinese herbal medicines, in which the relationships between drugs, targets and diseases were captured. 10 Only compounds with oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18 were considered as active compounds and selected. 11,12 Simultaneously, download the mol2 files of these active ingredients for the later molecular docking.

| Collecting targets of active compounds
The following five databases were used to collect the targets of active compounds from CS: TCMSP, the Comparative Toxicogenomics Database (CTD, http://ctdba se.org/), 13 STITCH (v 5.0, http://stitch. embl.de/), 14 GeneCards (v 5.11, https://www.genec ards.org/), 15 SymMap (v 2.0, http://www.symmap.org/). 16 Only target genes of Homo sapiens were adopted. When the collected targets were proteins, the UniProt (https://www.unipr ot.org/) was used to replace these proteins as genes. 17 Finally, the target genes from all the above databases were merged, and duplicates were removed, and the remaining target genes were used in the next analysis.

| Clustering of CS and melasma common targets and constructing their network
Potential target genes (their common targets, co-targets) of CS therapy for melasma were gained through Bioinformatics (http://www.bioin forma tics.com.cn/). Thereafter, a visualization network was constructed to reflect the relationship between CS, co-targets and melasma.

| Constructing protein-protein interaction (PPI) network and screening hub genes
The co-targets were submitted to STRING database (v 11.5, https:// cn.strin g-db.org/) with "Homo sapiens" being selected to construct the PPI network. 21 Then, the results were imported into Cytoscape (v 3.9.1) for network generation and analysis. 22 The analysis results were used the MCC algorithm in cytoHubba to calculate hub genes, of which the top ten were used for molecular docking. 23

| Active compounds and corresponding targets of CS
And 70 compounds of CS were gained from TCMSP, of which four compounds met the conditions of OB ≥30% and DL ≥0.18 (Table 1).
And 4507 targets related to these four compounds were collected from TCMSP, CTD, STITCH, GeneCards, SymMap.

| Potential targets of CS for melasma treatment
And 49 melasma-related targets were collected from OMIM, GenBank, GeneCards, and DrugBank. And 31 co-targets were found after the intersection of drugs and diseases ( Figure 2).

| Establishment and analysis of compoundstargets-disease network
Cytoscape was used to construct the interaction network between four CS active compounds and 31 overlapping targets with melasma ( Figure 3). The network includes 37 nodes (red for targets, yellow for compounds, blue for diseases, and green for drugs) and 88 edges.

| Constructing co-targets PPI network and screening hub targets
And 31 co-targets were submitted to STRING to construct PPI network ( Figure 4A), which included 31 targets and 81 edges. Further, cytoHubba was used to find the top ten hub targets scored by MCC ( Figure 4B, Table 2).

| GO and KEGG analysis
And 468 entries were obtained by GO enrichment (p < 0.05), of which the top ten entries with significant enrichment of BP, MF, and CC were visualized according to the p.adjust and count ( Figure 5A).
The biological functions were mainly related to intracellular receptor signaling pathway, response to steroid hormone, regulation of hormone levels, hormone-mediated signaling pathway. The cellular components were primarily concentrated in transcription regulator complex, RNA polymerase II transcription regulator complex, membrane raft and membrane microdomain. And the biological processes are mainly related to nuclear receptor activity, ligand-activated transcription factor activity, nuclear steroid receptor activity, oxidoreductase activity.
And 30 entries were obtained by KEGG enrichment (p < 0.05), of which the top 20 were visualized according to p.adjust and count ( Figure 5B), such as melanogenesis, estrogen-signaling pathway, chemical carcinogenesis-receptor activation, small cell lung cancer, and endocrine resistance. Furthermore, the melanogenesis signaling pathway most closely associated with melasma is shown in Figure 6.

| Molecular docking
Four active compounds screened from CS were individually docked to the top ten hub genes (Figure 7). The fitness of molecular docking was represented by the binding energy, and its smaller value meant better ligand binding to the receptor and a more substantial likelihood of an effect occurring. It was detected that the best docking results with hub targets is crocetin, which retained lower binding energy with hub targets except CD4 and MITF.

| DISCUSS ION
As an acquired and intractable skin disease, melasma ordinarily occurs in the face of patients who frequently exposed to ultraviolet (UV) and short wavelength visible light (VL), and whose Fitzpatrick classification are IV ~ VI, of whom more than 90% are women. 31 The pathophysiological mechanism of melasma is complex, which is re-    organelle in melanocytes. 36 When stimulated by stimuli like UV, melanocytes produce excessive concentrations of reactive oxygen radicals that not only damage cellular DNA but also undergo a complex series of reactions to trigger melanin production. 2 In this study, the following modalities were considered as possible mechanisms by which CS regulates melanogenesis ( Figure 6).
Initially, CS directly acts on key targets for melanogenesis, including TYR, TYRP1, and DCT. Melanin, including brown melanin and true melanin, are enzymatic synthesized by a series of specific enzymes in melanosomes--tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and dopa pigment tautomerism isomerase (DCT, also known as tyrosinase-related protein 2 or TYRP2). 37  (SCF), which binds to the extracellular domain of its receptor KIT and causes the activation of KIT, thereby activating MAPK-signaling pathway and catalyzing the synthesis of MITF. [41][42][43] Finally, CS acts on EDNRB to regulate melanogenesis. As a specific receptor on melanocytes, endothelin b receptor (EDNRB) can be bound by endothelin generated in endothelial cells and keratinocytes or induced by ultraviolet radiation b (UVB) to promote melanogenesis. 44,45 In addition, the role of PTGS2 in chloasma should additionally be noted, which occupies lower binding energy with all 4 active compounds in this study, especially crocetin. Prostaglandinendoperoxidase synthase 2 (PTGS2, also known as cyclooxygenase-2 or COX-2) is a coxase that catalyzes the production of prostaglandin compounds such as prostacyclin (PGI 2) and thromboxane A 2 (TXA 2) from arachidonic acid, and its core role is to coordinate chronic inflammation. 46 Kim et al. considered that COX-2 could be a candidate target for the development of antimelanogenesis agents as α-MSH induced pigmentation was closely related to COX-2 expression. 47

| CON CLUS ION
This study was based on network pharmacology to explore the po-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
No ethical approval and patient consent are required.