| Background:Leprosy is a chronic infectious disease caused by Mycobacterium leprae..leprae mainly affects the skin and nerves,causing deformity and deformity of the limbs.Leprosy diagnosis includes bacteriological examination of skin slit smear,histopathology,serum antibody detection and other methods,but some leprosy cases of atypical clinical manifestations,T-lep,pure neuritis will still be missed and misdiagnosed.Delayed diagnosis may result in irreversible nerve injury and facial deformities.Therefore,early accurate diagnosis of leprosy is very important.Classic leprosy patients are divided into five categories,presenting a spectral distribution.Studies on related molecules,metabolites and proteins that are differentially expressed in patients with different types of leprosy are conducive to the exploration of more valuable diagnostic markers as well as the exploration of their pathogenesis.Objectives:To explore the specific molecular changes of serum exosomes in patients with leprosy of different types andexplore the relevant mechanisms.Methods:1.MiRNA microarray detection and analysis were performed on serum exosomes of patients with different types of leprosy and normal controls to obtain significantly different miRNA,and the expanded samples were used for validation to determine significantly differentially expressed miRNA.The comprehensive variables of logistic binary regression model were evaluated alone or used,and ROC curve was drawn to evaluate their diagnostic value.2.A human mononuclear/macrophage and Mycobacterium leprae co-culture system was established at the cell level in vitro to verify the expressions of significantly differentially expressed miRNA in supernatant exosomes and cells.3.Verify the regulation of differentially expressed miRNA on predicted target genes by cell experiments in vitro.4.Non-targeted metabolomics was used to analyze serum exosomes of leprosy patients and normal controls,to find metabolic molecules with different expressions,and to draw ROC curve to evaluate its diagnostic efficacy.5.Serum exosomes of leprosy patients and normal controls were detected and analyzed by protein spectrum method,metabolic molecules with different expressions were searched,bio informatics analysis was performed,and ROC curve was drawn to evaluate the diagnostic efficacy of the disease.Results:1.MiRNA microarray(10 T-lep patients,10L-lep patients,10 type2 reaction patients and 10 normal individuals)detection and expanded samples validation showed that miR4485-3p(p<0.01)and miR1227-5p(p<0.05)were higher in leprosy patients than normal controls,and both of them were higher in L-lep than T-lep.The ROC curve showed that miR1227-5p was 0.648(p=0.048),while miR4485-3p was 0.747(p=0.001),and the ROC curve was 0.788(p<0.001)evaluated by the logistic binary regression model with the predicted probability values of the two miRNAs.Target gene bioinformatics prediction showed that miR4485-3p had significant regulatory effects on RAP2B,HPSE,SLC11A and TLR4 genes in L-lep,and CD40LG in T-lep.MiR1227-5p showed more significantly down-regulating the RAP2B,HPSE,CD5L,SLC11A1 and TLR4 in L-lep,and CYP27B1,S100B more prominently in T-lep.GO and KEGG analysis revealed that the pathways of miR4485-3p were enriched into the immune response pathway and T-cell receptor signaling pathway involved in macrophage activation,while the positive pathways of miR227-5p were for skeletal muscle tissue development,the cytochrome P450 enzyme metabolism pathway for exogenous organisms,and the arachidonic acid metabolism pathway.2.In the model of leprosy infection,aiming at macrophages(human monocytes/macrophages and THP1 cells)in vitro,it was found that the expression of miR4485-3p in both cell supernatant exosomes and cells showed significantly up-regulation,meanwhile the expression was time-dependent and bacterial concentration dependent.No significant changes were found in the internal reference miR16-5p.3.After transfecting miR4485-3p mimic into Mycobacterium leprae treated monocyte/macrophage and cultivating autologous T cells as well as adding IL10 found that it suppressed CD4+T cells function by down-regulated CD40L,furthermore it also inhibits the activity of macrophages by down-regulated CD40 expression,together with CD86,decreasing expressions of TNFa,IL1B,IL6 in supernatant,increasing the macrophage apoptosis,and maintain the vitality of bacteria.While inhibitor transfection shows opposite effect.4.A total of 2109 compounds were identified in serum exosome non-targeted metabolomics studies of leprosy patients(6 T-lep patients,7 L-lep patients,6 type 1 reaction patients,3 type 2 reaction patients and 6 normal controls).According to rules of FC>2 and P<0.01,up-regulated metabolites in each group were identified as CerP(d18:1/24:1(15Z)),PC(24:1(15Z)/14:1(9Z)),PS(20:4(5Z,8Z,11Z,14Z)/16:0),TG(24:1(15Z)/15:0/22:4(7Z,10Z,13Z,16Z)),PS(M onoMe(13,5)/M onoMe(13,5)),and down-regulated compound was 2-linoleoyl-sn-glycero-3-phosphocholine.The ROC curve was drawn according to the content(strength)of the samples,showing that the areas under the curves of the first three highly expressed metabolites were 0.917(p=0.002),0.992(p=0.000),and 0.939(p=0.001),while the area under the curve of the only down-regulated compound was 0.242(p=0.57).The lipids of PC,PS,PI,TG and DG were all highly expressed in the disease group,and the expressions of PE,PS,TG and DG were significantly increased in T1LR,while PC,PE and CE were significantly different in T-lep and L-lep.5.According to the screening criteria of FC>2(or<1/2)and p<0.05,a total of 1767 protein molecules were detected in serum exosomal proteomics of patients with leprosy(6 T-lep patients,7 L-lep patients,5 type 1 reaction patients,4 type2 reaction patients and 7 normal controls),showing that there were 86 up-regulated proteins and 65 down-regulated proteins in T-lep compared to normal controls.In L-lep,83 proteins were up-regulated and 59 proteins were down-regulated.In T1LR,68 proteins were up-regulated and 118 proteins were down-regulated.While in T2LR,63 proteins were up-regulated and 102 proteins were down-regulated.D9YZU5(HBB)increased in all disease groups.ROC curve was drawn with protein content of different samples showing that the areas including reaction groups was 0.740(p=0.059)and 0.78(p=0.043)without the reaction groups,while CD5L molecule significantly decreased in disease groups,especially in T-lep and reaction group(T1LR).Conclusions:1.Serum exosome miR4485-3p has the potential to become a biomarker for the diagnosis of leprosy2.After infected with Mycobacterium leprae M acrophages can regulate CD40L receptors on the surface of CD4+T cells by targeting the delivery of miR4485-3p via exosomes,thereby inhibiting their function in response.3.Non-targeted metabolic compounds CerP(d18:1/24:1(15Z)),PC(24:1(15Z)/14:1(9Z)),PS(20:4(5Z,8Z,11Z,14Z)/16:0)may become biomarkers for the diagnosis of leprosy.4.T-lep and L-lep increase protein significantly more than the increase in protein,and cut in T1LR and T2LR protein more,HBB can be elevated in patients with leprosy,speculated that it has become a diagnostic markers may,CD5L expressed in leprosy patients decreased,the T-lep and reaction group significantly,could become less bacteria diagnostic markers,leprosy reaction may also be predictive molecules. |
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