| Background:Vitiligo is an autoimmune disease caused by the destruction or disappearance of melanocytes in the epidermis,resulting in white patches of the skin or mucous membrane.Domestic and foreign studies have shown that CD8~+T cell-mediated immune response is the critical effector process that induce melanocyte damage,but the factors and mechanism of activating the abnormal immune response in vitiligo have not been fully clarified.Metabolic homeostasis is the basic regulator of tissue and organ immune response.Some metabolites have immunomodulatory effects independent of traditionally being substrates and intermediates in biosynthesis and energy metabolism,known as immune metabolites,which can activate or suppress immune functions by regulating inflammatory factor secretion,transcription factor activity,etc.Our previous meta-analysis on vitiligo combined with metabolic diseases revealed that vitiligo was associated with diabetes,and other studies showed that lipid-lowering drugs could promote repigmentation of leukoplakia,suggesting that glucose and lipid metabolism disorders in organisms are involved in the process of vitiligo.However,there is no systematic study on the role and mechanism of glucose and lipid metabolism disorders in the pathogenesis of vitiligo so far.In this study,the role of glucose and lipid metabolism disorder in the process of vitiligo was firstly determined by case-control study,and the mechanism of glucose and lipid metabolism disorder in regulating the process of vitiligo was further explored by combining targeted metabolomics and in vitro experiments.The results will provide a new direction and theoretical basis for the prevention and treatment of vitiligo.Objectives:1.Clarify the correlation between glucose and lipid metabolism disorder and vitiligo.2.Screening the key immune metabolites that mediate glucose and lipid metabolism disorder regulate the pathogenesis of vitiligo.3.Explore the mechanism of key immune metabolites regulating the abnormal immune response of vitiligo.Methods:1.Clarify the correlation between glucose and lipid metabolism disorder and vitiligo by case-control study.Vitiligo patients(case group)and natural population(control group)were enrolled according to the inclusion and exclusion criteria,and subjects in case group and control group were matched 1:2 according to gender and age.Subjects signed informed consent and filled in the questionnaire.The basic information,epidemiology of diseases,physical examination and other data of the subjects were collected.Peripheral blood samples were also collected from the study subjects to detect the levels of glucose and lipid metabolism index,including glucose,glycated hemoglobin A1c,total cholesterol,triglycerides,high-density lipoprotein and low-density lipoprotein.Glucose and lipid metabolism disorder is determined as hyperglycemia,high glycated hemoglobin A1c,high total cholesterol,high triglycerides,low high-density lipoprotein,high low-density lipoprotein according to medical reference range.Using univariate analysis(2×2 chi-square test,R×C Chi-square test,Mantel-Haenszel chi-square test)and multivariate analysis(binomial logistic regression,disordered multi-classification logistic regression,ordered multi-classification logistic regression)to explore the relationship between the glucose and lipid metabolism disorder and the process of vitiligo,including suffering from vitiligo,vitiligo activity,vitiligo severity and vitiligo type.2.Screening the key immune metabolites that mediate glucose and lipid metabolism disorder regulate the pathogenesis of vitiligo by targeted metabolomics.(1)Thirty patients with advanced moderate or moderate-severe vitiligo(case group)and thirty age-and sex-matched natural population(control group)were selected from the case-control study.Liquid chromatography tandem mass spectrometry was used to detect the levels of 12 energy metabolites in serum,including glucose and 11 immune metabolites(pyruvate,lactic,citric,α-ketoglutarate,succinate,fumarate,malate,oxaloacetate,itaconate,acetyl-Co A,succinyl-Co A).Unidimensional statistical analysis(Mann-Whiney U test,volcano plot),multidimensional statistical analysis(principal component analysis,orthogonal partial least squares discriminant analysis,VIP plot)and spearman rank correlation analysis were used to screen differential immune metabolites in serum between vitiligo patients and natural populations.(2)The serum succinate levels of all subjects collected in the case-control study were detected by the succinate detection kit.Mann-Whitney U test or Kruskal-Wallis H test were used to analysis the correlation between serum succinate and the process of vitiligo,including suffering from vitiligo,vitiligo activity,vitiligo severity and vitiligo type.(3)The blister fluid succinate levels of leukoplakia lesions and normal non-leukoplakia lesions in vitiligo patients were detected by succinate detection kit.The correlation between the levels of succinate in skin microenvironment and the formation of vitiligo leukoplakia was analyzed by paired t test.3.Explore the mechanism of key immune metabolites regulating the abnormal immune response of vitiligo by in vitro experiments.(1)The expression of succinate specific receptor SUCNR1 in CD8~+T cells was detected by immunofluorescence.The expression levels of marker molecules of proliferation(CFSE),activation(CD69)and killing function(IFN-γ,Granzyme B,Perforin)of CD8~+T cells were detected by flow cytometry.(2)The expression of SUCNR1 in melanocytes was detected by immunofluorescence.The expression of SUCNR1 in PIG1 cells treated with different concentrations of succinate was detected by western blot.The levels of CRT,IL-6,IL-8,HMGB1 and CXCL12 in the supernatant of PIG1 cells treated with different concentrations of succinate was detected by ELISA.The level of CRT in the supernatant of PIG1 cells pretreated with SUCNR1 si RNA or SUCNR1 antagonist prior to succinate disposition was detected by ELISA.(3)The expression of SUCNR1 in keratinocytes was detected by immunofluorescence.The expression of SUCNR1 in Ha Ca T cells treated with different concentrations of succinate was detected by western blot.The levels of IL-1β,IL-15,CXCL9,CXCL10 and CXCL16 in the supernatant of Ha Ca T cells treated with different concentrations of succinate was detected by ELISA.The level of IL-1β,CXCL9 and CXCL10 in the supernatant of Ha Ca T cells pretreated with SUCNR1 si RNA or SUCNR1antagonist prior to succinate disposition was detected by ELISA.Results:1.Clarify the correlation between glucose and lipid metabolism disorder and vitiligo by case-control study.(1)A total of 321 vitiligo patients(case group)and 642 natural population(control group)were enrolled according to the inclusion and exclusion criteria.2×2 chi-square test and binomial logistic regression analysis showed that hyperglycemia was associated with vitiligo and was a risk factor for vitiligo.The risk of vitiligo in people with hyperglycemia was 1.781 times higher than that in people with normal blood glucose(OR=1.781,95%CI=1.131～2.806,P=0.013),while other metabolic abnormalities were not significantly correlated with vitiligo(P>0.05).(2)According to the criteria of vitiligo activity,321 vitiligo patients were divided into108 stable vitiligo patients and 213 advanced vitiligo patients.2×2 chi-square test and binomial logistic regression analysis showed that hyperglycemia and high cholesterol were correlated with vitiligo activity and were risk factors for vitiligo activity.The risk of vitiligo activity progression was 2.576 times higher in patients with hyperglycemia than in those with normal blood glucose(OR=2.576,95%CI=1.027～6.460,P=0.044).The risk of vitiligo progression in patients with high cholesterol was 3.217 times higher than that in those with normal cholesterol(OR=3.217,95%CI=1.080～9.580,P=0.036),and there was no significant correlation between other metabolic abnormalities and vitiligo activity(P>0.05).(3)According to the criteria of vitiligo severity,321 vitiligo patients were divided into 119 mild vitiligo patients,157 moderate vitiligo patients,and 45 moderate-severe vitiligo patients.Mantel-Haenszel Chi-square test and ordered multiple logistic regression analysis showed that hyperglycemia was correlated with the severity of vitiligo,which was a risk factor for vitiligo severity,the risk of increasing vitiligo severity by one grade was2.707 times higher in hyperglycemic patients than in normoglycemic patients(OR=2.707,95%CI=1.073～6.828,P=0.035),and there was no significant correlation between other metabolic abnormalities and vitiligo activity(P>0.05).(4)According to the criteria of vitiligo type,321 vitiligo patients were divided into 36segmental vitiligo patients,258 non-segmental vitiligo patients,19 mixed vitiligo patients and 8 undetermined vitiligo patients.R×C-chi square test and disordered multi-classification logistic regression analysis showed that there was no significant correlation between abnormal glucose and lipid metabolism and vitiligo type(P>0.05).2.Screening the key immune metabolites that mediate glucose and lipid metabolism disorder regulate the pathogenesis of vitiligo by targeted metabolomics.(1)According to the results of targeted metabolomics,the level of glucose in vitiligo patients was higher than that in natural populations(P<0.05),the levels of immune metabolites itaconate,succinate and fumarate in vitiligo patients were higher than that in natural populations(P<0.05).The levels of itaconate,succinate and fumarate were positively correlated with glucose(P<0.05).Studies have shown that succinate could activate immune response,while itaconate and fumarate could suppress immune response,and both of them tend to increase under immune activation conditions to balance the immune response.Therefore,succinate has been selected as the potential key immune metabolite mediating the pathogenesis of hyperglycemia-regulated vitiligo to further verified.(2)Enlarge sample size detection 642 natural population and 321 cases of vitiligo patients serum level of succinic acid,the results showed that vitiligo patients serum succinate level higher than that of natural population(P<0.05),advanced vitiligo patients serum succinate level higher than that of stable vitiligo patients and the level of succinate tended to increase with increasing vitiligo severity(P<0.05).There was no significant difference in serum succinate level among different types of vitiligo patients(P>0.05).Our results suggested that succinate was correlated with suffering from vitiligo,vitiligo activity,vitiligo severity.It was further confirmed that succinate is the key immune metabolite mediating the pathogenesis of hyperglycemia-regulated vitiligo.(3)We enrolled 30 vitiligo patients and detected succinate levels in blister fluid from leukoplakia and normal non-skin lesions.The results showed that the levels of succinate in blister fluid from leukoplakia were higher than that in normal non-skin lesions(P<0.05),indicating that the level of succinate in skin microenvironment was related to the formation of vitiligo leukoplakia.It was further confirmed that succinate is the key immune metabolite mediating the pathogenesis of hyperglycemia-regulated vitiligo.3.Explore the mechanism of key immune metabolites regulating the abnormal immune response of vitiligo by in vitro experiments.(1)SUCNR1 was detected in CD8~+T cells.The CFSE positive ratio,the level of CD69,IFN-γGranzyme B and Perforin of CD8~+T cells in succinate group were not significantly different with control(P>0.05),indicating that succinate had no significant effect on the function of CD8~+T cells.(2)SUCNR1 was detected in melanocytes.The expression of SUCNR1 was up-regulated in PIG1 cells after treatment with succinate.The secretion of CRT in PIG1cells was increased after treatment with succinate(P<0.05),while the level of IL-6,IL-8,HMGB1 and CXCL12 had no significantly difference with control(P>0.05).Inhibition of SUCNR1 in PIG1 cells can inhibit succinate-induced CRT secretion(P<0.05),suggesting that succinate can bind to SUCNR1 of melanocytes and promote the secretion of CRT.(3)SUCNR1 was detected in keratinocytes.The expression of SUCNR1 was up-regulated in Ha Ca T cells after treatment with succinate.The secretion of IL-1β,CXCL9 and CXCL10 in Ha Ca T cells was increased after treatment with succinate(P<0.05),while the level of IL-15 and CXCL16 had no significantly difference with control(P>0.05).Inhibition of SUCNR1 in Ha Ca T cells can inhibit succinate-induced IL-1β,CXCL9 and CXCL10 secretion(P<0.05),suggesting that succinate can bind to SUCNR1 of keratinocytes and promote the secretion of IL-1β,CXCL9 and CXCL10.Conclusions:Our study firstly explored the role and mechanism of glucose and lipid metabolism disorder in the pathogenesis of vitiligo.The study found that hyperglycemia was correlated with vitiligo,and was a risk factor for the illness,activity and severity of vitiligo,while lipid metabolism disorder was not correlated with the process of vitiligo.Succinate is the key immune metabolite mediating the pathogenesis of hyperglycemia-regulated vitiligo.Succinate binds to the succinate receptor SUCNR1 on the surface of epidermal melanocytes and keratinocytes and promotes the secretion of proinflammatory factors CRT,IL-1β,CXCL9 and CXCL10,initiating CD8~+T cell-mediated immune responses,leading to melanocyte destruction.This study provided a new direction and theoretical basis for all levels of prevention strategies for vitiligo,provided new clues for etiological research of vitiligo,and provided new targets for treatment strategies of vitiligo. |
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