| Objective: Autoimmune thyroid diseases(AITDs),consisting of Hashimoto’s thyroiditis(HT)and Graves’ disease(GD),are T cell-mediated organ-specific autoimmune diseases that affect approximately 10% of the Chinese population.Thyroid autoimmunity,determined by the presence of thyroid antigen-specific antibodies,is even more prevalent.Although genetic and environmental factors may lead to AITDs,the pathogenesis remains unclear,and few biomarkers for predicting AITD progression are available.Metabolomics is an emerging field in the systems biology realm in which most of the studied subjects are small molecules with a relative molecular mass of less than 1 kDa.Metabolomics research aims to measure altered metabolic pathways in diseases and seeks new biomarkers involved in the diseases.Among metabolomes,polyamines are an indispensable polycation in all living cells and play a role in the regulation of thyroid growth and nucleic acid metabolism.The most common and functional polyamines are putrescine(PUT),spermidine(SPD)and spermine(SPM).Polyamines,particularly SPM and SPD,play important physiological roles in living cells,including the regulation of ion channels,gene transcription and translation and protection from oxidative damage.Some polyamine metabolites have been associated with autoimmune disorders,such as multiple sclerosis(MS),rheumatoid arthritis(RA),systemic lupus erythematosus(SLE)and type 1 diabetes mellitus(T1DM).However,little is known about the relationship between polyamine metabolites and AITD.Autoimmune diseases share some common features(e.g.,sex bias,adult onset,and genetic and epigenetic modifications),suggesting similar underlying mechanisms.Hence,we speculate that polyamines may also play a crucial role in the development of AITDs.In the present study,we aimed to evaluate polyamine metabolite levels in serum and thyroid from AITD patients and healthy controls(HCs)and to predict whether thyroid hormone,thyroid autoantibodies,chemokines or disease progression is associated with polyamine metabolites.Furthermore,the animal model of spontaneous autoimmune thyroiditis NOD.H-2h4 mice were treated with polyamine(spermine and spermidine)intervention,to observe the effects of polyamines on spontaneous autoimmune thyroiditis and the changes of thyroid antibody levels,and to provide experimental evidence for polyamine clinical intervention.Methods: Part I: We recruited 98 patients from the First Affiliated Hospital of China Medical University,including 33 untreated HT patients,36 untreated GD patients,29 euthyroid patients who were positive for thyroid autoantibodies(pTAb)and 38 sex-and age-matched HCs.In addition,thyroid tissues from 20 HT patients and 20 healthy controls were collected.Serum TgAb,TPOAb,TRAb,free T3(FT3),FT4 and thyroid-stimulating hormone(TSH)were measured using an electrochemiluminescence immunoassay(Roche Diagnostics Ltd.,Switzerland).Fourteen serum polyamine metabolites,including polyamine precursors(SAM,L-ARG,L-ORN,LYS),polyamines(PUT,SPD,SPM,CAD,AGM,DAP,NSPD,NPUT,NSPM)and polyamine catabolite(GABA),were measured by UFLC-MS/MS.Three polyamines(PUT,SPM and SPD)of thyroid tissues were measured by UFLC-MS/MS.Serum chemokines(MCP-1,IP-10,IL-8,RANTES and MIG)were measured using a cytometric bead array(CBA)human chemokine kit(BD Bioscience).Total RNA were extracted from thyroid tissues and PBMC.Real-time PCR was used to detect mRNA expression levels of SSAT,SMS,SDS,ODC and AMD,as well as DNMT1 and G9 A.Enzyme-linked immunosorbent assay(ELISA)was used to detect global DNA and H3K9 methylation in PBMC and thyroid tissues.Part Ⅱ: Four weeks old NOD.H-2h4 mice were randomly divided into control group(normal drinking water)and high iodine drinking water group(1000 times high iodine drinking water).The high iodine group was given 1000 times of high iodine water for 8 weeks and divided into 3 groups: SAT+SPD group,SAT+SPM group,and SAT group,10 mice in each group.Experimental animals in each group were anesthetized and sacrificed after 4 weeks of polyamine supplementation and specimens were collected.The lymphocyte infiltration was observed by HE staining in the thyroid tissue of mice.Serum levels of thyroglobulin(TgAb)were determined by ELISA.Determination of polyamines in spleen by ultrahigh performance liquid chromatography-tandem mass spectrometry.Inflammatory cytokines were detected by ELISA.Real-time PCR was performed to detect the mRNA expression levels of SSAT,SMS,SDS,ODC and AMD in mice spleen.Results: Part I: 1.Fourteen serum polyamine metabolites were measured.For most polyamine metabolites,similar changes were observed in GD and HT patients relative to HCs.GD and HT patients had significantly higher L-ORN,L-ARG and LYS,and AGM and lower PUT,NPUT,SPM,and DAP than the HCs(all P<0.05).Some polyamine metabolite levels were different in GD and HT patients from those in HCs: GD patients had significantly lower CAD and higher SPD,NSPD and GABA than HCs(all P<0.05),whereas NSPM was significantly decreased in HT patients compared with HCs(P<0.05).Only SPM and NSPM were significantly lower in pTAb patients than in controls.Other polyamine metabolites did not differ between pTAb patients and controls.2.IP-10 was significantly higher in all AITD patients,including GD,HT and pTAb patients,than in HCs(all P<0.05).The GD patients exhibited higher levels of MCP-1(P<0.05),whereas no differences in MCP-1 were observed in HT patients or pTAb patients compared to the HCs.3.We also calculated the ratio of SPM to SPD(SPM/SPD),which was significantly reduced in all AITD patients(GD,HT,pTAb)compared to HCs(all P<0.05).SPM/SPD was strongly negative correlated with TPOAb(r=-0.569,P<0.01).4.Multivariable ordinal logistic regression analysis showed that SPM was negatively correlated with thyroid-specific antibodies grade(TPOAb and TgAb)in both the unadjusted and adjusted models(OR=0.989,95%CI=0.981-0.998,P=0.013 and OR=0.988,95%CI=0.979-0.997,P=0.009).5.Multivariate logistic regression analysis showed that NSPD may be a risk factor for the progression of pTAb to overt hypothyroidism(OR= 1.269;95%CI: 1.023-1.574;P=0.031).6.SSAT mRNA levels in PBMC of HT and GD groups were significantly higher than those of controls.SMS mRNA levels in the HT and GD groups were significantly lower than those in controls.7.The content of spermine in thyroid tissues of the HT group was significantly lower than that of the control group,and there was a significant negative correlation between spermine and TPOAb(r=-0.332,P<0.05).The level of SSAT mRNA in thyroid tissues of the HT group was significantly higher than that of the control group(P=0.019<0.05).8.There were no statistically significant differences in DNA and H3K9 methylation levels and methylation metabolic enzymes in either PBMC or thyroid tissues.Part Ⅱ:1.The relative weight of thyroid in the SAT group was significantly higher than that in the control group(P<0.05),and the relative weight of thyroid in the SAT+SPD group was significantly lower than that in the simultaneous SAT group(P<0.05).There was no statistical difference in the relative weight of thyroid between the SAT+SPM group and the SAT group.2.The TgAb titer of SAT group was significantly higher than that of control group(P<0.05).The TgAb titer of the SAT+SPD group was significantly lower than that of the SAT group(P<0.05),and there was no statistical difference between the TgAb titer of the SAT+SPM group and that of the SAT group.3.Histological score of the SAT+SPD group was significantly lower than that of the SAT group(P<0.05),and there was no significant difference between the SAT+SPM group and the SAT group(P>0.05).The follicular cavity of the SAT+SPD group decreased compared with that of the SAT group,but did not return to the pre-iodine level,the morphology of follicular epithelial cells and nuclei was recovered,the degree of lymphocyte infiltration was significantly reduced.Lymphocyte infiltration was also reduced in the SAT+SPM group.4.TNF-a,IL-12 and IP-10 in the SAT+SPD group were significantly lower than those in the SAT group(P<0.05).IL-12 and IP-10 in the SAT+SPM group were significantly lower than those in the SAT group(P<0.05).5.SSAT mRNA expression level in SAT group was significantly higher than that in control group(P<0.01),SAT+SPD group(P<0.01)and SAT+SPM group(P<0.05).Conclusion: 1.Most metabolites of GD and HT showed similar pattern compared with the controls,suggesting the possibility of a common pathophysiological basis or metabolic pathway between these two diseases.2.NSPD might be a potential risk factor for predicting pTAb progression to overt hypothyroidism.Thyroid autoimmunity was associated with low SPM levels.3.The results of our study showed that SPM/SPD was significantly lower in all AITD patients than in healthy controls,indicating a decrease in protective polyamines.Moreover,SPM/SPD was negatively correlated with TPOAb(r=-0.569,P<0.01)and inflammatory chemokines IP-10(r=-0.336,P<0.05).Decreased SPM/SPD might be related to the consumption of inflammation.4.Abnormal SSAT expression may be the cause of abnormal polyamine metabolism in serum and thyroid tissues of AITD patients.5.Although peripheral blood and thyroid tissues of AITD patients showed evidence of polyamine circulation disorder,it was not sufficient to cause abnormal DNA/ histone methylation.6.SPD intervention can significantly reduce iodine-induced goiter in NOD mice.The inflammatory levels and serum TgAb level of NOD mice with autoimmune thyroiditis were significantly decreased after SPD supplementation.7.TNF-a,IL-12,and IP-10 in spleen of spm-supplemented mice were significantly decreased.These results suggest that SPD and SPM have anti-inflammatory effects.8.The mRNA level of SSAT,a key polyamine enzyme,was significantly higher in the SAT group than in the control group,suggesting that abnormal SAT polyamine metabolism may be related to abnormal SSAT expression. |
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