Study On The Mechanism Of BMS-470539 Activation Of MC1R To Regulate The Treatment Of Vitiligo

Part Ⅰ Identification and analysis of vitiligo pathogenic genes and transcription factors based on the method of confidence analysisResearch backgroundVitiligo is a depigmentation skin disease caused by various reasons.In recent years,the incidence rate of vitiligo has increased.Once suffering from vitiligo,it can affect the patient’s personal image and have a significant impact on the patient’s life,marriage,work,and other aspects.The pathogenesis of vitiligo is unknown,and it is a disease that is relatively easy to diagnose and difficult to treat clinically.Whether it is medication,physical therapy,or surgical treatment,it is difficult to achieve a radical cure.Although vitiligo has little impact on the lives of patients,it has an impact on their physical and mental health,and even causes a decline in their quality of life.Therefore,it is particularly important to study the etiology and pathogenesis of vitiligo,find new therapeutic targets,and improve the clinical cure rate of patients.The study of the pathogenesis of diseases can begin with cell function,which begins with gene expression,including transcription and translation,which transforms the genetic information carried by the body’s genes into phenotypes.Analysis based on transcriptome data is conducive to understanding the physiology and pathology of cells and studying the pathogenesis of diseases at the cellular level.There are indeed differences in gene expression between patients with vitiligo and healthy individuals.Therefore,this study analyzed the differentially expressed genes(DEGs)of patients with vitiligo through the method of bioinformatics analysis,in order to identify the pathogenic genes of patients with vitiligo and facilitate targeted intervention and treatment.Research objectiveTo understand the molecular expression differences between vitiligo and healthy people through the method of biographical analysis,screen out the targeted genes for vitiligo pathogenesis through functional annotation of the differential genes,KEGG enrichment,transcriptional factor-DEGs regulatory network analysis,protein interaction network analysis,and further study the regulation of these genes in vitiligo,clarify the molecular pathogenesis of vitiligo,and provide new possibilities for systematic treatment.Research methodsThe relevant data were retrieved through the Gene Expression Omnibus(GEO)database,and the messenger RNA(mRNA)and transcription factors(TFs)of vitiligo and normal controls were analyzed simultaneously using the method of bioinformatics.Identify the DEGs in vitiligo and construct the TFs target gene regulatory network to screen the key genes and TFs in vitiligo.The obtained data were verified by Real-Time PCRand electronic verification.Research resultsAccording to the inclusion and exclusion criteria,the final two sets of vitiligo mRNA data sets were included in our study.The data series numbers were GSE75819 and GSE65127 respectively.Through comprehensive analysis,we identified 1863 DEGs,including 744 down-regulated DEGs and 1119 up-regulated DEGs(FDR<0.05,| Combined.ES |>1).Functional annotation results of DEGs:ubiquitin-mediated proteolysis and endoplasmic reticulum are significant enrichment pathways of DEGs.The PPI network construction and analysis results of DEGs:Preelastic(PMEL),Melan-A(MLANA),Dopachrome tautomerase(DCT),SRY-box translation factor 10(SOX10)Tyrosinase-related protein 1(TYRP1)and melanocortin 1 receptor(MC1R)are involved in the pathogenesis of vitiligo.TYRP1 and MC1R are regulated by Forkhead box J2(FOXJ2).The validation results are consistent with the analysis results through Real-TimePCRand electronic validation.Research conclusionPMEL,MLANA,DCT,SOX 10,TYRP1 and MC1R may play a role in the pathogenesis of vitiligo.The expression of MC1R is regulated by the transcription factor FOXJ2,resulting in a downregulation of MC1R expression,affecting melanocyte RNA metabolism,ribosomal proteins,and other dysfunction,leading to decreased proliferation and increased destruction of melanocytes,leading to the onset of disease.MC1R agonists may serve as a promising drug for the treatment of vitiligo,which provides a biological target for further research into the pathogenesis of vitiligo,and may provides new ideas and directions for further identifying the pathogenesis of vitiligo.Part Ⅱ:The therapeutic effect of activating MC1R by BMS-470539 on vitiligoResearch backgroundSo far,the available treatment methods for vitiligo are still limited,and the treatment strategy depends on non-specific treatment for inflammation and immune response,such as local or systemic steroids or local calcineurin inhibitors,to promote the regeneration of melanocytes,increase the production of melanin by melanocytes,and play a role in treating vitiligo.Melanocyte is a pigment-producing cell,and its key regulatory transcription factor is melanocyte-specific microphthalmos related transcription factor(m-MITF).The opioid melanocortinogen(POMC)gene and melanocortin-1 receptor(MC1R)are modulators of melanogenesis and pigmentation.POMC is a protein synthesized mainly in pituitary and other tissues including skin.In more than 30 years of research on the biology of melanocortin(MC)hormone and synthetic peptide,the activation of MCI receptor has been identified as a feasible target for the development of new anti-inflammatory therapeutic agents.The short sequence modification of natural MCs is a feasible approach to drug development.In addition,the recognition and development of new chemical entities that can selectively activate MC1 or MC3 receptors is also an important direction of drug development.Many studies have been carried out on new chemical entities that activate MC1R.BMS-470539 is a MC1R-specific agonist.When BMS-470539 is specifically combined with MC1R,it can inhibit the adhesion and migration of white blood cells,reduce the inflammatory reaction of the body,and inhibit cell apoptosis.However,the regulation of BMS-470539 activating MC1R has not been reported in the study of vitiligo disease.Therefore,this study uses vitiligo mice as animal models to explore whether BMS-470539 can improve the phenotypic changes of vitiligo after activating MC1R and through what mechanism to improve the phenotypic changes of vitiligo,thus opening up a new way to treat vitiligo diseases.Research objectiveThe animal model of vitiligo was constructed with C57BL/6 mice,and vitiligo mice were treated with MC1R agonist BMS-470539.The effects of MC1R agonist on melanogenesis in skin tissue were observed at the level of tissue,cell and molecular biology,and the relevant molecular mechanisms were analyzed,which opened up a new way for the treatment of vitiligo disease.Research methodTwenty C57BL/6 male mice,aged 5～6 weeks and weight 18～22g were selected.According to the body weight number 1-20,the mice were randomly divided into 3 groups according to the random number table.Six rats in group A were the control group,six rats in group B were the vitiligo model group,and eight rats in group C were the BMS-470539 treatment group.After treatment,observe the epidermis of mice,count the number of melanin hair follicles,and detect TGF-β,IL-6,IL-17,IL-23,TYR,MDA,CHE by ELISA.The expression of MC1R,POMC and TYR protein was detected by immunohistochemistry.The protein expressions of MC1R,POMC and TYR were detected by Western Blot.Research resultsThe number of melanin hair follicles in the model group was lower than that in the control group significantly,and the number of melanin hair follicles in the treatment group was higher than that in the model group(P<0.05).ELISA test showed that:Compared with the control group,the levels of TGF-β,IL-6,IL-17,IL-23,MDA and CHE in the model group were significantly increased.After BMS-470539 treatment,the results showed that the inflammatory cytokines TGF-β,IL-6,IL-17 and IL-23 were significantly decreased(P<0.05).The contents of CHE and MDA were also significantly decreased,while the contents of TYR were significantly increased.Both immunohistochemistry and Western blot showed that the expression of MC1R,POMC and TYR protein in the model group was significantly lower than that in the control group.After treatment with BMS-470539,the results showed that the expression of MC1R,POMC and TYR protein was significantly higher(P<0.05).Research conclusionBMS-470539 can significantly reduce the skin lesions of vitiligo mice and increase melanocytes in vitiligo mice.The possible mechanism of treatment is to regulate the immune response of various cytokines,enzymes and proteins and the effect of inflammatory reaction on melanocytes,reduce the damage of melanocytes,promote the repair of melanocytes,and play a therapeutic role.Part Ⅲ:Proteomic sequencing study on the mechanism of BMS-470539 activating MC1R in the treatment of vitiligoResearch objectiveThrough proteomic sequencing of skin tissue of vitiligo mice,the mechanism of MS-470539 activating MC1R to treat vitiligo was revealed from the protein level.Research methodAccording to the above experimental results,we selected the following three groups of skin tissue samples(normal group,model group and treatment group)for proteomic sequencing.After quality inspection,each group selects five samples for subsequent proteomic sequencing.Firstly,the whole protein of the sample is extracted,quantified,detected,digested,and the polypeptide is obtained and desalted.Finally,the download data of the original mass spectrometry file is obtained through liquid phase separation and mass spectrometry scanning.Based on the original documents obtained by mass spectrometry detection,MaxQuant software searched the database,statistically analyzed the differential proteins,and further studied their functions and involved pathways through bioinformatics analysis.Research results1 Differential proteinsCompared with the normal group,there were 122 different proteins in the model group,among which 68 proteins were up-regulated and 53 proteins were down-regulated.BMS-470539 treatment group compared with model group,there were 197 different proteins,among which 50 proteins were up-regulated and 117 proteins were down-regulated.2 Differential protein GO enrichment analysisThe functional enrichment analysis of 122 differential proteins in the model group compared with the normal group,it showed that the differential proteins were mainly involved in the biological processes such as nucleotide phosphorylation,the forward regulation of ERK1 and ERK2 cascades,the proliferation and metabolism of myoblasts,and the chaperone mediated protein folding.The differential proteins were mainly involved in the cell components such as phosphopyruvate hydratase complex,myofibrils,and the central region of the spindle β-Tubulin binding,fructose phosphate aldolase activity,various metabolic kinase activities and other molecular functions.The 197 differential proteins in the BMS-470539 treatment group were compared with those in the model group for GO bioinformatics analysis.In terms of cell composition,differential proteins were mainly concentrated in proteasome regulatory particles,proteasome auxiliary complex,translation initiation complex,secretory granules and vesicles;Molecular functions mainly focus on the activities and binding of translation initiation factors,translation regulation activities and RNA/nucleic acid binding;The biological process mainly focuses on the initial translation stage,protein synthesis stage,molecular function regulation,etc.3 Enrichment analysis of differential protein KEGGKEGG enrichment analysis of 122 different proteins in the model group compared with the control group showed that the different proteins were involved in glycolysis and glucose metabolism synthesis pathway,HIF-1 signaling pathway,metabolic pathway,etc.The 197 differential proteins in the BMS-470539 treatment group compared with the model group were analyzed for KEGG pathway,and the differential proteins were involved in the RNA transport process,endoplasmic reticulum protein processing and tight junction process.4 Protein interaction network analysisCompared with the normal group,the model group received a total of 7 genes(RPS13,LDHA,RPS17,ALDOA,SUMO3,CCT5,Pa2g4)from the core genes of the PPI network.The BMS-470539 treatment group received a total of 9 genes(EIF3M,ABCE1,RPS11,RPS18,Eif3b,PSMD7,Eif2s3,EIF5B,Eif3c)from the core genes of the PPI network.Research conclusionBMS-470539 activates MC1R to treat vitiligo in mice,which may play a therapeutic role by up-regulating or down-regulating some key genes(EIF3M、ABCE1、RPS11、RPS18、Eif3b、PSMD7、Eif2s3、EIF5B、Eif3c),promoting the proliferation of melanocytes,inhibiting their apoptosis,regulating their cell cycle.Innovation1.The topic of this study is based on the pathogenesis of the most common refractory diseases in dermatology,based on clinical problems,and is reasonable.2.The design logic of this study is clear.The design ranges from the prediction of key molecules,the clarification of experimental studies to the clarification of relevant molecular mechanisms.This study first identified the relevant key molecules in the pathogenesis of vitiligo through bioinformatics,and then determined the role of MC1R in vitiligo through the MC1R agonist of the key molecules through the experimental animal model;Finally,the molecular mechanism of MC1R agonist and its effect on melanocytes were elucidated by proteomics.3.This research method is reliable and cutting-edge.This research method involves bioinformatics analysis,experimental animal model,immune and molecular biology technology and proteomics technology.The research results of research technology and method suggest that MC1R molecule and its molecular signal pathway play a key role in the occurrence and development of vitiligo.MC1R agonist BMS-470539 can activate multiple mechanism molecules mediated by MC1R to promote the proliferation of melanocytes and inhibit their apoptosis,And then prevent the pathological formation and progression of vitiligo.Therefore,the results of this study can provide evidence for the clinical diagnosis and treatment of vitiligo.Limitations1.The sequencing sample size of this study is small,which will lead to low confidence of some results.2.This study is only from the skin tissue samples of vitiligo mice,lacking comprehensiveness;The sample size of microarray data is small,which is prone to result bias.