The Therapeutic Effect And Mechanism Of Demethylzeylasteral In Vitiligo

Background:Vitiligo is a common autoimmune depigmenting skin disorder characterized by melanocyte destruction due to cytotoxic CD8⁺ T cells,resulting in hypo-pigmentary patches.The depigmented lesions commonly affect visible sites like the face and hands,leading to appearance anxiety,a reduction in quality of life,and growing demand for treatment.Stacks of research have shown that in genetic predisposition,inflammation factors cause the release of melanocyte-specific antigens,which are recognized by antigen-presenting cells and stimulate the development of cytotoxic CD8⁺ T cells.Further,through the induction of chemokines secreted by skin cells,melanocyte specific CD8⁺ T cells are finely regulated to migrate to the colonization site of melanocytes and then specifically kill melanocytes,eventually leading to the development of vitiligo.Among them,the overexpression of keratinocyte-derived chemokines CXCL9 and CXCL10 in the presence of inflammatory factors such as IFN-γ induces the skin migration of CXCR3⁺CD8⁺ T cells.Meanwhile,the CD8⁺ T cells release effector molecules including IFN-γ to further induce the production of chemokines to form a positive feedback chemotaxis signal loop.This is the main mechanism for the development of vitiligo,therefore targeting the critical pathogenesis process that is urgent for vitiligo treatment exploration.Demethylzeylasteral（T-96）,a bioactive phenolic demethylated pentacyclic triterpene monomer isolated from Tripterygium wilfordii Hook F（Tw HF）,possesses potent immunosuppressive,anti-inflammatory,and antiviral properties,and several studies have confirmed that it has strong immunosuppressive and anti-inflammatory pharmacological effects,and has achieved efficacy in the treatment of lupus nephritis,allogeneic kidney transplantation,and rheumatoid arthritis.In vitro studies have found that demethylzeylasteral can significantly inhibit the proliferation of isolated immune cells in mouse spleens in the presence of Con A at a concentration of 0.5 μg/m L,and the immunosuppressive effect is comparable to cyclosporine at 1.0 μg/m L.Compared with other active monomers,demethylzeylasterol has the advantage of low toxicity and is a promising natural compound to develop into a new immunosuppressant.In addition,Professor Tienan Li of China took the lead in the clinical use of TWHF tablets（20 mg each time,2 times a day）to treat patients with active vitiligo and found that the treatment of TWHF has better efficacy and safety.Still,the molecular mechanism and its monomer active ingredients in the treatment of vitiligo remain blank.Our research group has been committed to translating the knowledge of the pathogenesis of vitiligo into clinically practical treatments.Based on the high efficacy of TWHF during the treatment of vitiligo in China and the strong immunosuppression and anti-inflammatory pharmacological effects of demethylzeylasteral,we hypothesized that demethylzeylasteral suppressed the proliferation,activation,and effect function in CD8⁺ T cells of patients with vitiligo,reduce the production and release of chemokines from keratinocytes under inflammatory context,thereby protecting melanocytes from damage and playing a therapeutic role in vitiligo.Objectives:1.To explore the immunomodulatory functions of demethylzeylasteral in CD8⁺ T cells of vitiligo patients.To clarify the immunosuppression and possible mechanism of demethylzeylasteral on the proliferation,activation,migration,and effect function in CD8⁺ T cells of vitiligo patients.2.To clarify the inhibitory roles and the underlying mechanisms of demethylzeylasteral on keratinocyte production and secretion of chemokines under inflammation stress.To clarify the effect of demethylzeylasteral on CXCR3 membrane expression in CD8⁺ T cells of patients with vitiligo,and the chemotaxis ability of CXCR3⁺CD8⁺ T cells following the culture supernatants of stressed keratinocytes pretreated with demethylzeylasteral.3.To evaluate the therapeutic effect of demethylzeylasteral using our mouse model of vitiligo and provided a promising practical application for vitiligo.Methods:1.To clarify the safe dose of demethylzeylasteral in human CD8⁺ T cells.Peripheral blood of patients with vitiligo and age-sex-matched normal controls were collected,CD8⁺ T cells were isolated from PBMC,and the IC₅₀ values of CD8⁺ T cells of demethylzeylasteral in vitiligo patients and healthy controls were detected using CCK8 assay,and the concentration gradient was further reduced to screen for the safe concentration of demethylzeylasteral in CD8⁺ T cells.2.Analysis of the effect of demethylzeylasteral on the proliferation,activation,and function in human CD8⁺ T cells.Cell Trace^TM Violet-labeled CD8⁺ T cells and flow cytometry analysis were used for the proliferation.The surface frequency level of early activation marker CD69 in CD8⁺ T cells was detected using flow cytometry to identify the activation effect of demethylzeylasteral in CD8⁺ T cells.The intracellular staining flow cytometry was used to detect the effects on proinflammatory cytokine IFN-γ and cytotoxic-associated cytokines granzyme B（Gzm B）and perforin（PRF）to clarify the effect function.3.Analysis of the molecular mechanism of demethylzeylasteral in CD8 T cells.First,to identify the potential binding proteins of demethylzeylasteral in CD8 T cells,we synthesized biotinylated demethylzeylasteral(T-96^Bio)and obtained the streptavidin bead-T-96^Bio-protein complexes from CD8 T cell’s total lysates by pulldown assay.Subsequently,the binding protein profiles were combined with vitiligorelated literature to screen out binding proteins related to vitiligo CD8⁺ T cell function,further verified by computer molecular simulation methods and identifying the possible binding sites of binding proteins.Next,to clarify the binding protein function using flow cytometry.4.Analysis of the role and underlying mechanism of demethylzeylasteral in inflammatory keratinocytes.First,the safe dosage of demethylzeylasteral in keratinocytes and Ha Ca T cells was evaluated using cytotoxicity assessment CCK8 assay.The binding protein of demethylzeylasteral in the keratinocyte protein lysate under IFN-γ treatment was obtained by pull-down experiment combined with mass spectrometry,and the proteins associated with vitiligo disease were screened out in combination with the existing research literature on the pathogenesis of vitiligo,and the binding site of demethylzeylasteral and the target protein was further determined by computer molecular simulation.The effect of demethylzeylasteral on the m RNA and protein levels of binding proteins was detected by q RT-PCR,Western blotting,immunofluorescence,and the production and secretion of downstream chemokines were further detected by overexpression of upstream genes,q RT-PCR,and ELISA.5.Analysis of the effect of demethylzeylasteral on CXCR3 membrane expression in CD8⁺ T cells of patients with vitiligo,and the chemotaxis ability of CXCR3⁺CD8⁺T cells following the culture supernatants of stressed keratinocytes pretreated with demethylzeylasteral.CD8⁺ T cells were sorted from vitiligo patients,and the effect of demethylzeylasteral on the expression of chemokine receptor CXCR3 of CD8⁺ T cell membrane surface was further detected by cell flow cytometry.And the chemotactic effects of secretion supernatants on CXCR3⁺CD8⁺ T cells under different treatment conditions were detected by transwell experiments.6.Constructing a mouse model of vitiligo.Summarizing the existing vitiligo mouse models in the literature and the research objectives of this study,we generated specific CD8⁺ T cells against melanocytes in mice by injecting melanoma cells subcutaneously in C57BL/6 mice and then injected CD4 antibodies intraperitoneally to eliminate the immunomodulatory effect of Treg on CD8⁺ T cells,and then excised intradermal melanoma,so that CD8⁺ T cells attacked normal melanocytes in mice,and then pigment loss occurred.This animal model of vitiligo is like the immunological pathogenesis of human vitiligo,in that it is a killer CD8⁺ T cell that specifically attacks normal melanocytes to form white patches,which can be used for vitiligo translation studies.7.Analysis of the therapeutic role of demethylzeylasteral in a mouse model of vitiligo.Based on the construction of the mouse model of vitiligo in the early stage,different doses of demethylzeylasteral were injected intraperitoneally for treatment.The epidermis infiltration numbers of CD8⁺ T cells and the decrease of melanocytes were detected by the method of full skin fluorescence staining,and the therapeutic effect of demethylzeylasteral in this animal model was evaluated.Results:1.The safe concentration of demethylzeylasteral in human CD8⁺ T cells is 1.0μM.The 50% inhibitory concentration IC₅₀ of demethylzeylasteral in CD8 T cells was4.54 μM in vitiligo patients,and 6.32 μM in healthy controls.We further tested different concentration gradients for CD8⁺ T cells in vitiligo patients and healthy controls and showed that 1.0 μM was the safe concentration.2.Demethylzeylasteral suppressed the proliferation,activation,and function of CD8⁺ T cells.The results of Cell Trace^TM Violet-labeled CD8⁺ T cells and subsequent flow cytometry analysis showed that demethylzeylasteral suppressed CD8⁺ T cell proliferation in patients with vitiligo.The results of surface marker staining flow cytometry showed that CD69 expression exhibited a heavy decrease when pretreated with demethylzeylasteral compared with the control and the results of intracellular staining flow cytometry showed that demethylzeylasteral can significantly reduce the expression of IFN-γ,cytotoxic-associated cytokines granzyme B（Gzm B）and perforin（PRF）in CD8⁺ T cells of patients with vitiligo.At the same time,we compared these immunosuppressive effects of demethylzeylasteral with the JAK inhibitor tofacitinib（Tofa）,and the results showed that demethylzeylasteral and tofacitinib were comparable to the effect functions in CD8⁺ T cells.3.Demethylzeylasteral inhibited JAK3-STAT5 signaling in CD8⁺ T cells of patients with vitiligo.We obtained the binding proteins of demethylzeylasteral in CD8⁺ T cells using pull-down assay and mass spectrometry.The results of Gene Ontology（GO）biological processes showed demethylzeylasteral participated in immune effector processes and the regulation of cytokine presence in CD8⁺ T cells and JAK3 was one of the binding proteins.Furthermore,the computational structure prediction for docking simulation also found demethylzeylasteral docked into the JAK3 kinase domain.The pretreatment with demethylzeylasteral significantly decreased the expression of p-JAK3 and p-STAT5 in the presence of IL-2 and the inhibition effects were in a dose-dependent manner.Together,these data demonstrated that demethylzeylasteral inhibited the JAK3-STAT5 signaling in CD8⁺ T cells.4.Demethylzeylasteral decreased the expression of CXCL9/10 via Jthe AK2-STAT1 pathway in inflammatory keratinocytes.Firstly,the IC₅₀ of demethylzeylasteral was 12.02μM in NHEKs and 15.08μM in Ha Ca T cells,and the concentration of 1μM was applied for subsequent experiments based on the cell viability assay.We performed the pull-down assay and mass spectrum analysis to obtain the binding proteins of demethylzeylasteral in IFN-γstimulated keratinocytes.The bioinformatic results indicated that demethylzeylasteral might bind with JAK2and respond to IFN-γsignaling.What is more,molecular docking analysis also confirmed that demethylzeylasteral had a strong bind with JAK2.Next,we discovered that pretreatment with demethylzeylasteral inhibited the phosphorylation of JAK2 in IFN-γtreated keratinocytes by both the flow cytometry and蛋白印迹.Then,we found that demethylzeylasteral reduced the expression of m RNA,total and phosphorylation proteins of STAT1,as well as the nuclear translocation of STAT1.Subsequently,we showed that demethylzeylasteral pretreated markedly decreased the m RNA and secretion levels of CXCL9 and CXCL10 compared with the IFN-γstimulated keratinocytes via q RT-PCR and ELISA.However,the inhibition of CXCL9/10 of demethylzeylasteral was impeded when JAK2 overexpressed.At the same time,we found that demethylzeylasteral and tofacitinib have comparable inhibitory effects on JAK2-STAT1-CXCL9/CXCL10 signals.In summary,these data demonstrated that demethylzeylasteral suppressed the IFN-γinduced expression of CXCL9 and CXCL10 by targeting JAK2-STAT1 signaling.5.Demethylzeylasteral downregulated the cell surface expression of CXCR3 in CD8 T cells and blocked the chemotaxis ability of CXCR3⁺CD8⁺ T cells.We found both demethylzeylasteral and tofacitinib reduced the expression of CXCR3 on CD8⁺ T cells of patients with vitiligo using flow cytometry.The results of transwell assay in vitro the culture supernatants of pretreated with demethylzeylasteral following IFN-γinduced keratinocytes blocked the migration of CXCR3⁺CD8⁺ T cells compared with the control,and the blocking effects were comparable with tofacitinib,joint stimulation,and neutralization antibodies of CXCL9 and CXCL10.Furthermore,the blocking effect of demethylzeylasteral was significantly decreased when JAK2 was overexpressed.6.Constructing a mouse model of vitiligo.B16 tumor-bearing mice treated with anti-CD4 depletion followed by surgery to excise primary melanoma could prime vigorous,protective CD8⁺ T cells responding to melanoma to cross-react with normal melanocytes,resulting in the development of autoimmune vitiligo.We continuously observed the infiltration of CD8⁺ T cells in the tail skin epidermis after surgery using whole-mount tail epidermis staining.CD8⁺ T cell initial epidermis infiltration was observed from day 24 to day 28 characterized by mainly accumulating around the follicle portion,then activated CD8 T cells migrated to the entire scale and caused melanocyte-specific elimination.The tail depigmentation usually appeared from day35 to day 50 and after that increased gradually.7.Demethylzeylasteral ameliorated ongoing depigmentation by inhibiting the CD8⁺ T cell skin infiltration in a mouse model of vitiligo.We performed treatment on day 28 with demethylzeylasteral at two doses（low: 2.5 mg/kg and high: 5.0 mg/kg）of intraperitoneal injection once every other day until five weeks later.The results showed that demethylzeylasteral significantly reduced depigmentation compared to vehicle-treated controls,and the high dose of 5 mg/kg had a better response.5 mg/kg of both demethylzeylasteral and tofacitinib improved the loss of pigment with considerable ability.Next,we examined the tail skin to quantify the number of CD8⁺T cells and melanocytes and found that the mice treated with demethylzeylasteral and tofacitinib exhibited less CD8⁺ T cell invasion and more melanocytes remaining than the vehicle group.These results demonstrated that demethylzeylasteral might be an effective targeted treatment option for vitiligo.Conclusions:In this study,we demonstrated that demethylzeylasteral suppressed the proliferation,activation,and effect function of CD8⁺ T cells in patients with vitiligo,inhibited the JAK3-STAT5 signaling in CD8⁺ T cells,and decreased the membrane expression of CXCR3 on CD8⁺ T cells from patients with vitiligo.Additionally,demethylzeylasteral attenuated the JAK2-STAT1-CXCL9/10 pathway in IFN-γinduced keratinocytes and blocked the chemotaxis of CXCR3⁺CD8⁺ T cells.Finally,demethylzeylasteral ameliorated ongoing depigmentation in our vitiligo mouse model and decreased the infiltration of CD8⁺ T cells.Our research showed that demethylzeylasteral as a multi-target small molecule had the potential to be applied to clinical trials in vitiligo treatment or another autoimmune disease.