Influences And Mechanisms Of Benvitimod On Rosacea

Background:Rosacea is a common chronic inflammatory dermatosis affecting up to 5%population worldwide.Most rosacea patients are female between 30 to 50 years old.Some rosacea patients have psychological problems because of the ugly appearances on face,resulting in poor quality of life.The pathogenesis of rosacea is still unclear.Ultraviolet light,hot and cold irritation,alcohol,spicy food,Demodex mites,gastric helicobacter pylori infection and steroid misuse may be the triggers.Dysfunctions of facial blood vessels,dysregulation of nerve vessels,imbalance of innate immune and destroy of skin barriers are demonstrated to be vital in rosacea.Traditional treatments for rosacea were systemic medicines(Tetracycline,macrolides,nitroimidazole and tretinoin),topical agents(metronidazole,azelaic acid and ivermectin,),some lights or lasers(intense pulsed light,pulsed dye laser and carbon dioxide laser)and operation.However,the efficacies were limited with high recurrence rate.Therefore,a more reliable and targeted therapy for rosacea is urgently needed.Aryl hydrocarbon Receptor(AhR)is vitral in regulating the secretion of pro-inflammatory factors,affecting the expression of relative genes of skin barrier function and maintaining the homeostasis of skin micro-environment.In normal skin,AhR is regulated by innate ligands in the process of inflammatory reaction,immune function,oxidative reaction and photoaging,while it is regulated by outside ligands in skin tumor,autoimmune diseases and inflammatory dermatoses.It was reported that Toll-like receptor 2(TLR2)was elevated in rosacea.Activation of Aryl hydrocarbon Receptor(AhR)could alleviate the inflammation induced by TLR increasing in human mononuclear-derived dendritic cells.Benvitimod,also called tapinaraff,is an AhR agonist.Benvitimod was firstly approved by China food and drug administration in 2019 and was beneficial for psoriasis and atopic dermatitis.TLR2?KLK5 and LL-37 are increased in psoriasis,AD and rosacea,which indicates that the pathogenesis of the 3 dermatoses are similar.Therefore,we hypothesis that benvitimod maybe effctive for rosacea.Objective:To investigate the treatment effect and mechanism of benvitimod on rosacea,find new treating targets for rosacea and provide proof for new therapies.Methods:1.Bioinformatic analysis:The Mesh term“rosacea”in the GEO database was retrieved to obtain datasets of differentially expressed genes(DEGs)between rosacea patients and healthy controls.Datasets with organisms other than homo sapiens or cases irrelevant to rosacea were excluded.The DEGs were analyzed by gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis via Database for Annotation,Visualization and Integrated Discovery(DAVID),KEGG gene sets via Gene Set Enrichment Analysis(GSEA)and Protein-protein interaction(PPI)network analysis via Search Tool for the Retrieval of Interacting Genes/proteins(STRING).Then the DEGs were verified in lesions of rosacea patients.2.Mouse model:The dorsal hairs of 6-8 weeks old female Balb/c mice were shaved by an electric razor and a depilatory agent 24 h before treatment.They were randomly assigned into 6 groups,including Group A,B,C,D,E and F.The rosacea-like eruptions of Group C?D?E and F were made by injecting 40?l(320?M)LL37 intradermally on the same area of dorsal skins twice a day for 2 consecutive days.No treatment was done in Group A,and 40?l endotoxin water was applied in Group B instead.1%or 0.5%benvitimod was administrated topically on the lesions 30 minutes after the second LL-37injection once a day for 3 consecutive days in Group E and F separately.Reagent dissolving benvitimod was applied in Group D instead.24 h after the last application of benvitimod,eruptions were photographed by a digital camera and skin inflammation was assessed by redness score and redness area.Then the lesions were biopsied for hematoxylin-eosin(HE)staining and quantitative real time-polymerase chain reaction(qRT-PCR).3.Cell culture:Immortalized human keratinocyte cells(HaCaT cells)were cultured in Dulbecco's Modified Eagle's medium(DMEM)with 10%(v/v)fetal bovine serum(FBS)and 1%penicillin-streptomycinin in a humidified 5%CO₂ incubator at 37°C.Cultured HaCaT cells were seeded 24 h before treated.And they were treated with different concentrations of LL-37or benvitimod.MTS was applied to evaluate the cell viability of HaCaT cells treating with LL-37 and benvitimod,in order to select the appropriate concentrations and applying times of the reagents.The DEGs found in Part 1 were examined by qRT-PCR and Western Blot/enzyme linked immunosorbent assay(ELISA).Then HaCaT cells were transfected by lentivirus for overexpression of DEGs found in Part 1,potential downstream indexes were tested thereafter.Results:1.DEGs between rosacea patients and healthy controls:There were separately359,579 and 571 DEGs between Erythematotelangiectatic(ETR)/Papulopustular(PPR)/Phymatous(PhR)patients and healthy controls in GSE65914.A total of 215 overlapping DEGs were detected between different subtypes of rosacea patients and healthy controls as indicated with Draw Venn software.The 215 overlapping DEGs were analyzed using GO,with the result that 77 biological process(BP),13 cell component(CC)and 15molecular function(MF)GO terms were enriched in rosacea.For BP,the DEGs were predominantly enriched in inflammatory reaction,chemotaxis,cell chemotaxis and chemokine-mediated signaling pathway.For CC,the DEGs were predominantly enriched in T cell receptor complex.For MF,the DEGs were predominantly enriched in chemokine activity,CXCR3 chemokine binding and CCR receptor binding.The 215overlapping DEGs were analyzed by KEGG via DAVID.The DEGs were enriched in 10KEGG pathways(P<0.05),including cytokine-cytokine receptor interactions,chemokine signaling,TLR signaling pathways and TNF signaling pathways.Moreover,KEGG pathway enrichments were also analyzed by GSEA,with a total of 127 gene sets being up-regulated in rosacea patients.Fifty-eight of these were significantly enriched with nominal P values of<5%and false discovery rates of<25%.Six gene sets of GSEA were consistent with the results obtained from DAVID.The top 4 pathways were TLR signaling pathway,hematopoietic cell lineage,cytokine-cytokine receptor interactions and the chemokine signaling pathway.The 215 overlapping DEGs were uploaded to STRING.A total of 182 nodes and 456 edges were identified in the PPI network.Further analysis as performed with the Cytoscape MCODE app showed 8 clusters with>3 nodes.The largest cluster included 15 central nodes and 96 edges,with 13 genes being up-regulated and 2 down-regulated.The 15 central DEGs were inputted into KEGG via DAVID and 6 KEGG pathways were identified(P<0.05).The TLR signaling pathway was the principal pathway,with cytokine-cytokine receptor interactions and the chemokine signaling pathway being the next two most common pathways observed.In the TLR signaling pathway,5 candidate genes were identified and consisted of TLR2,regulated on activation,normal T-cell expressed and secreted chemokine(RANTES)(C-C motif chemokine(CCL)5),monokine induced by gamma-interferon(MIG)(CXCL9),interferon-gamma inducible protein(IP)10(CXCL10)and interferon inducible T cell alpha chemokine(I-TAC)(CXCL11).TLR2 by IHC and qRT-PCR,chemokines(CCL5,CXCL9,CXCL10 and CXCL11)by qRT-PCR were further verified in lesions of rosacea patients.2.Benvitimod ameliorated LL-37 induced rosacea-like eruptions in mice:Erythema was seen on dorsal mice skin after LL-37 injection.Both 1%and 0.5%benvitimod ameliorated LL37-induced rosacea-like eruptions in mice significantly.Redness scores and redness area decreased after treatment with 1%and 0.5%benvitimod.The dermis inflammatory cell infiltrate by HE staining in LL-37 induced rosacea-like eruptions in mice were ameliorated by 1%and 0.5%benvitimod.The results examined by Image J were in correspondence with those by HE staining(P<0.05).Gene expressions of TLR2and chemokines(CCL5,CXCL9,CXCL10,CXCL11)were elevated in LL-37 group by qRT-PCR(P<0.05).Compared with LL-37 induced model group,gene expressions of TLR2,CCL5,CXCL9,CXCL10 and CXCL11 were all decreased in 0.5%benvitimod group,while only CXCL9,CXCL10 and CXCL11 were decreased in 1%benvitimod group(P<0.05).3.Protective effect of benvitimod on LL-37 induced HaCaT cells and benvitimod down-regulated TLR2 in inhibiting expressions of chemokines:HaCaT cells were incubated with 8?M LL-37 for 24 h,and then challenged with 10?M benvitimod.TLR2and chemokines(CCL5,CXCL9,CXCL10,CXCL11)were tested by qRT-PCR after 8h,TLR2 were tested by Western Blot and chemokines(CCL5,CXCL9,CXCL10,CXCL11)were tested by ELISA after 24h.The results illustrated that TLR2 and chemokines were increased after LL-37 irritation in wild HaCaT cells and the chemokines increased higher in TLR2 overexpressed HaCaT cells.The differences were statistically significant(P<0.05).Benvitimod down-regulated TLR2 in inhibiting the expressions of chemokines(P<0.05).Conclusions:1.The bioinformatic analysis and lesions verification illustrated that TLR2,CCL5,CXCL9,CXCL10 and CXCL11 were crucial DEGs between lesions of rosacea patients and healthy controls,and they were enriched in TLR pathway.2.Benvitimod inhibited expressions of TLR2 and chemokines(CCL5,CXCL9,CXCL10and CXCL11)in LL-37 induced Balb/C mice,decreased inflammatory cell infiltration,thus ameliorated rosacea-like eruptions.3.Expressions of TLR2 and chemokines(CCL5,CXCL9,CXCL10 and CXCL11)were elevated in LL-37 irritated HaCaT cells,and the chemokines were increased higher in TLR2 overexpressed HaCaT cells.Benvitimod suppressed TLR2 and chemokines(CCL5,CXCL9,CXCL10 and CXCL11)in LL-37 irritated HaCaT cells.