Ji-Hong Li,Jia-Yu Zhang,Yuan-Xia Zou

1Department of Dermatology, Leshan Traditional Chinese Medicine Hospital, Leshan, Sichuan, 614001, China.2The First Clinical Medical College of Yunnan University of Traditional Chinese Medicine,Kunming,Yunnan,650051,China.3Department of Newborn Medicine,Hospital(T.C.M)Affiliated to Southwest Medical University,Luzhou,Sichuan,646000,China.

Abstract Objective: To explore the mechanism of Piperine of Piperis Longi Fructus in the treatment of vitiligo based on network pharmacology and transcriptome. Methods:Piperine,the active component of Piperis Longi Fructus, was screened from Traditional Chinese Medicine Systems Pharmacology,and the target network and protein interaction network of piperine-vitiligo were constructed.PPI analyzed the cross targets, GO function, and KEGG pathway analysis.In the GEO database, vitiligo gene expression profile chips GSE65127 and GSE75819 were selected for differential gene screening and gene set enrichment analysis.The potential key signal pathways of piperine in the treatment of vitiligo were identified by transcriptome microarray data verification. Results: 31 potential targets of piperine in the treatment of vitiligo were found, of which 9 targets such as ESR1, AKT1, and IGF1 were closely related to the treatment of vitiligo.Enrichment analysis showed that signal pathways such as PI3K-Akt, MAPK,and Melanogenesis were related to piperine's mechanism in treating vitiligo.The cross-validation of chip analysis results confirmed that Melanogenesis is a potential key signal pathway for piperine in vitiligo treatment.Conclusion: This study discusses the potential targets and signal pathways of piperine in the treatment of vitiligo,which helps clarify the mechanism of piperine in the treatment of vitiligo and provides a theoretical basis for the clinical treatment of vitiligo.It also provides direction for the research and development of new drugs for vitiligo.

Key words:Piperine,Vitiligo,Network pharmacology,Transcriptome,Action mechanism

Background

Vitiligo is a common depigmented skin disease,characterized by melanocyte destruction and lead to depigmentation with a global prevalence rate of 0.5-2%.There are no significant differences in sex and age [1, 2].The etiology of vitiligo is complex,involving genetic, immune, oxidative stress,psycho-neurological, and environmental factors.Controlling skin lesions and promoting the recovery of pigment in leukoplakia are still the primary targets in vitiligo treatment.To date, widely used drugs include calcineurin inhibitors, local antioxidants, vitamin D derivatives, glucocorticoids; the non-drug treatments include phototherapy and transplant therapy [3].Although these methods have achieved good results,there are still some limitations.In recent years, with the continuous breakthrough of traditional medicine research methods, traditional Chinese medicine in vitiligo treatment has attracted more attention because of its good effect[4,5].

Piperine is the main active ingredient ofPiperis Longi Fructus, which could stimulate melanocyte proliferation and dendritic formationin vitro, activate the adhesion and migration ability of unpigmented melanocytes in the outer root sheath of hair follicles,as well as enhance tyrosinase activity [6-8].Promote melanin synthesis of epidermal melanocytes.Although some progress has been made in the study of piperine in vitiligo treatment, the relationship between its targets and therapeutic approaches has not systematically and comprehensively understood[9].

Network pharmacology is widely used in drug research because of its holistic, systematic, and influential characteristics of using bioinformatics,molecular biology, and database to study the relationship between drugs, targets, pathways, and diseases.This study identified the potential targets and signal pathways of piperine in the treatment of vitiligo through biological function, pathway analysis, and transcriptome chip data analysis [10].From the perspective of network pharmacology, we should understand piperine as much as possible and reveal its mechanism in the treatment of vitiligo to provide new ideas and methods for treating vitiligo.

Materials and methods

The possible targets of piperine

In Traditional Chinese Medicine Systems Pharmacology (TCMSP,(http://ibts.hkbu.edu.hk/LSP/tcmsp.php)) database usesPiperis Longi Fructusas the keyword to search for all its active ingredients, with oral bioavailability (OB) ＞30% and drug-likeness (DL) ＞ 0.18 as screening conditions.According to the screening results and previous basic research, piperine was determined as the main active ingredient ofPiperis Longi Fructusin vitiligo treatment.The piperine's 2D chemical structure was searched in PubChem(https://pubchem.ncbi.nlm.nih.gov/) database, and its image was loaded into PharmMapper(http://www.lilab-ecust.cn/pharmmapper/) database to determine the predictive target and pharmacophore model related to piperine.The UniProt database(https://www.uniprot.org/) was used to screen the coding matching of piperine target proteins [11].The potential target of piperine was obtained by eliminating repetition and non-standard target to improve its credibility.

Acquisition of vitiligo-related targets

The vitiligo-related genes were searched in the GeneCards (https://www.genecards.org/) database and OMIM (https://www.omim.org/) database and the target genes related to vitiligo were obtained after removing duplicates.

Construction of piperine-vitiligo target network

R package VennDiagram is used to construct the piperine-vitiligo target crossover.Web-based STRING(http://string-db.org) is a database for known and predicted protein-protein interaction analysis (PPI)[12].For more accurate results, PPI's confidence score is set to 0.900, and Cytoscape (https://cytoscape.org/)software platform is used to visualize and analyze the interactive network.The results of the PPI analysis of piperine-vitiligo were imported into Cytoscape software.To improve the accuracy of potential target screening, two different algorithms, MCODE and MCLique, were used to calculate the score, and an optical network was constructed according to the node score and the relationship between proteins.Use the Cytoscape plug-in STRING to visualize potential key gene clusters.

Enrichment analysis

Metascape (https://metascape.org/) is an enrichment test based on hypergeometric distribution to calculate GO terms and KEGG pathways, which can more accurately analyze and visualize the functional profiles of genes and gene clusters [13].GO and KEGG analysis was performed in Metascape to study the biological process and signal pathway of a piperine-vitiligo cross target.

Data verification of gene expression profiling chips(GEPCs)

Vitiligo gene expression microarray GSE65127 and GSE75819 were selected from the GEO public database (https://www.ncbi.nlm.nih.gov/geo/) to exclude patients with complications and drug therapy.A total of 50 samples were extracted from the chip,including 25 skin lesions and 25 healthy control samples.The limma package of R software was used to screen differentially expressed genes.P＜0.05 and|logFC | ＞1 were used as criteria for data processing and analysis [14].Gene set enrichment analysis(GSEA) was used to analyze the potential signal pathway of GEPCs, and GSE65127 and GSE75819 were analyzed by GSEA to verify the results of KEGG analysis of the piperine-vitiligo cross target.

Results

Piperine, the active component of Piperis Longi Fructus and its target screening

By screening all the active components ofPiperis Longi Fructusin the TCMSP database, 104 active components were found.According to OB and DL's standards, 15 active components were obtained, of which three active components were directly related to piperine(Table 1).

Table 1 Screening results of active components of Piperis Longi Fructus

Piperine-vitiligo target network

Piperine is 1-2E, 4E piperonylpiperidine, which has two trans double bonds in the chain connecting the piperidine and methylenedioxyphenyl groups.258 piperine targets were screened by Pharmmapper database and UniProt database.1096 vitiligo-related target genes were identified in GeneCards and OMIM databases, and 44 potential targets were obtained by crossing piperine with vitiligo targets using R software package VennDiagram (Figure 1A).Import these potential targets into the STRING database and use Cytoscape software to draw the piperine-vitiligo target topology network (Figure 1B), Cytoscape extended applications MCODE MCLique for gene cluster analysis (Table 2).Use Cytoscape to visualize candidate gene clusters (Figure 1C, Figure 1D and Figure 1E).These potential targets include Mitogen-activated protein kinase 8 (MAPK8),Interleukin-2 (IL2), RAC-α serine/threonine-protein kinase (AKT1), estrogen receptor β (ESR2),mitogen-activated protein kinase 14 (MAPK14),estrogen receptor (ESR1).Mitogen-activated Mitogen-activated protein kinase-1 (MAPK1),insulin-like growth factor-1 (IGF1) and insulin-like growth factor-1 receptor (IGF1R).These potential targets reveal the key pharmacological targets of piperine in the treatment of vitiligo.

Table 2 Topological analysis of piperine-vitiligo target network

Figure 1 Identify cross targets and carry out network construction and topology analysis.

Enrichment analysis

44 cross targets of piperine and vitiligo were loaded into Metascape for enrichment analysis.When correctedP≤0.05(P.adjust),GO analysis showed the first 20 significantly rich biological processes (Figure 2A-C), closely related to the functional enrichment of organic cyclic compounds, lipopolysaccharides,nitrogen compounds, reactive oxygen species,apoptosis signals, and cytokines.WhenP.adjust ≤0.05, KEGG pathway analysis selected five pathways containing more than two most relevant targets to the pathology of vitiligo (Figure 2D), including FoxO signaling pathway, PI3K-Akt signaling pathway,MAPK signaling pathway,melanogenesis,and tyrosine metabolism.Based on the results of KEGG analysis(Table 3), we speculate that these pathways are potential signal pathways for piperine in vitiligo treatment.

Data verification of GEPCs

GSE75819 gene expression microarray of vitiligo from GEO and generated a heat map and volcano map by screening differentially expressed genes in order to visualize the distribution of differentially expressed genes between vitiligo and normal tissues.The results of differentially expressed genes were obtained usingP＜0.05 and | logFC | ＞1 as truncation criteria (Figure 3A-D).The marked gene set database was used to analyze the genes in the whole expression profile in GSEA, and the significantly enriched gene sets were set toP＜0.05 and FDR ＜0.25, respectively.The GSEA analysis of GSE65127 and GSE75819 and the comparison with the KEGG analysis of the cross target of piperine in the treatment of vitiligo confirmed that melanogenesis is the potential critical signal pathway of piperine in the treatment of vitiligo(Figure 3E-F).

Table 3 Signaling pathway enrichment analysis of piperine-vitiligo cross targets

Figure 2 Functional enrichment analysis of cross targets.

Discussion

As an immune-mediated depigmentation dermatosis,vitiligo seriously affects the physical and mental health and quality of life of patients.A genome-wide association study has found the risk of vitiligo caused by multiple genetic loci,including gene polymorphism,immunity, antigen presentation, melanocytes, and peptidase [15].Due to the complexity and protracted cycle of vitiligo treatment, the current treatment scheme can improve the skin lesions, but the therapeutic effect still needs improvement.In this study,through network analysis, it was found that 31 potential targets of piperine, the active component of Piperine long, may have an effective relationship with vitiligo-related genes, of which nine potential genes are near related to the mechanism of piperine in the treatment of vitiligo.MAPK1, MAPK8 and MAPK14 are members of the MAPK family.For vitiligo cells,long-term exposure to sub cytotoxic oxidative stress may lead to overstimulation of MAPK and imbalance of MAPK kinase phosphorylation [16].Transcription factor Nrf2 can regulate multiple genes' expression to encode antioxidant enzymes, detoxification factors,anti-apoptotic proteins, and drug transporters.The protective effect of Nrf2 expressed through the enzyme network protects cells from oxidative and electrophilic stress [17].IGF-1 can promote the activation of Nrf2 and inhibit the formation of ROS and the expansion of the endoplasmic reticulum in HaCaT.IGF-1 inhibits oxidative damage to HaCaT and immunosuppresses the proliferation and activation of CD8+T cells to resist decolorization induced by hydrogen peroxide[18].The deficiency of AKT1 protein can lead to stress of the endoplasmic reticulum.The destruction of the redox balance of the endoplasmic reticulum can lead to the accumulation of misfolded proteins,thus activating the unfolded protein response and then initiating the apoptosis of melanocyte death[19,20].

IL-2, as a pro-inflammatory cytokine, signals and drives T cell proliferation through IL-2 receptors,induces regulatory T cell differentiation and participates in immune regulation by activating the immune system [21].It is reported that the level of IL-2 in patients with vitiligo is increased, which is related to the duration and activity of vitiligo [22].Besides, the high level of estrogen in serum is related to the increase of skin pigmentation, and estrogen may increase pigment cells' activity.Genotypic distribution and allele frequency show that the gene polymorphism of ESR1 and ESR2 is closely related to vitiligo occurrence [23].Based on the enrichment analysis results,this study found that some potential key targets could regulate multiple signal pathways.Among the most significant pathways related to the pathology of vitiligo, AKT1, MAPK1, MAPK8, MAPK14, and IGF1 can act on more than two signal pathways simultaneously, reflecting the synergistic effect of multiple targets.

In this study, we found five signal pathways containing more than two targets, which are near related to piperine's mechanism in vitiligo treatment.According to previous studies, it is found that genes related to the Melanogenesis signal pathway play a key role in piperine in the treatment of vitiligo.Therefore,we further verified the Melanogenesis signal pathway in the GSEA analysis of vitiligo GEPCs data.

Figure 4 The major predicted melanogenesis signaling pathway

Melanogenesis is a biological process in which melanocytes synthesize melanin to resist ultraviolet radiation, oxidative stress, and other factors.Melanocytes are cells derived from the nerve ridge,mainly located in the epidermis' basal layer, and produce melanin.Destruction of melanocytes,dysfunction, or obstruction of the synthesis pathway can lead to vitiligo.Melanogenesis in human skin is regulated by multiple signal pathways and transcription factors, among which Melanogenesis signal pathway plays a crucial role in melanogenesis(Figure 4).In human melanocytes, signals from the melanocortin system reach melanogenic enzymes through cAMP/PKA pathways, such as tyrosinase(TYR), tyrosinase associated protein 1 (TYRP1), and dopa pigment tautomerase (DCT), which are regulated by Wnt and MAPK pathways [24].Melanocyte-specific markers involved in melanin synthesis include tyrosinase-related protein-(TRP-) 1(TRPmur2) and tyrosinase, which are mainly responsible for the conversion of tyrosine to melanin[25].In the process of melanogenesis,α-melanocyte-stimulating hormone (α-MSH)stimulates tyrosinase activity through the cAMP pathway and promotes tyrosinase overexpression [26].Melanocyte-specific melanocortin one receptor(MC1R) binds to α-MSH and activates adenylate cyclase,increasing the level of intracellular cAMP[27].These melanin synthesis and expression genes are regulated by downstream microphthalmos related transcription factor (MITF).It can regulate the differentiation of melanocytes and increase TYR,TRP-1, and TRP-2 [28].The downstream signal transduction of MC1R mediates cAMP and response element-binding (CREB) transcription factors.The signal transduction of MC1R is one of the main factors affecting melanogenesis.CREB can induce the expression of MITF [29].P38 MAPK phosphorylation induces melanin synthesis by increasing the expression of MITF and TYR.Phosphatidylinositol 3 kinase(PI3K) / AKT signal pathway inhibits melanogenesis by down-regulating the expression of TYR, MITF,and TRPs [30, 31].P44/42 MAPK reduces melanin synthesis through the degradation of tyrosinase protein[32].Our enrichment analysis results explored the effect of piperine on melanogenesis in vitiligo involving Melanogenesis, Tayrosine metabolism,MAPK signaling pathway, and PI3K-Akt signaling pathway signaling pathways.However, piperine's specific melanogenesis mechanism is still unclear, and its regulation of many critical links in the melanogenesis pathway, such as TYR, MC1R, MITF,TRP-1 and TRP-2, and the targeted signal pathway is the focus of our next research.These potential targets and signal pathways of melanin production can provide further theoretical support and practical guidance for piperine in vitiligo treatment.

Conclusion

This study explored the molecular mechanism of piperine in the treatment of vitiligo employing network pharmacology and transcriptome chip data verification and identified 31 potential targets and 9 key piperine targets in vitiligo treatment.The fatty pathways most related to the pathology of vitiligo were verified by GO and KEGG enrichment analysis, and the mechanism of piperine in the treatment of vitiligo was explored.The critical signal pathways are verified by GSEA analysis of transcriptome chip data.According to the enrichment analysis results, we revealed the characteristics of multi-target synergism and verified that the melanogenesis signal pathway is the critical signal pathway of piperine in vitiligo treatment.This study provides a theoretical basis for piperine in the treatment of vitiligo and provides new ideas and methods for treating vitiligo.