| Objective:Hashimoto Thyroiditis(HT)is an organ-specific Autoimmune disease caused by genetic,environmental and other factors.In recent years,the incidence of this disease has been increasing year by year,which has aroused people’s great concern.The main pathological changes of thyroid tissue in patients with HT were immune cell infiltration dominated by CD4+T cells.Due to the increase in the number of CD4+T cells such as Th1 cells and Th17 cells,a large number of inflammatory cytokines such as TNF-α and IL-6 were produced,which further led to the apoptosis and necrosis of thyroid cells,and finally the progress of thyroid failure.According to the new theory of Immunometabolism proposed in recent years,dormant T cells are mainly energized by fatty acid oxidation and oxidative phosphorylation.Once activated,Metabolic Reprogramming occurs as the strength of glycolysis increases and the strength of the KCA cycle decreases.Activated T cells produce inflammatory mediators,energy carriers,and metabolic intermediates by altering dominant metabolic pathways.Studies have confirmed that glycolysis is involved in many immune processes,especially in the activated effector T cell subtypes such as Th17 cells,Th1 cells and Th2 cells,glycolysis metabolism is more significant.Acetated pyruvate dehydrogenase complex(PDC),as A key rate-limiting enzyme in sugar metabolism,catalyzes irreversible oxidative decarboxylation of pyruvate into acetyl-Co A and promotes glucose tricarboxylic acid cycle.It plays A crucial role in energy metabolism of mitochondrial respiratory chain.Short chain fatty acids(SCFAs),as the main metabolites of intestinal flora,mainly include acetic acid,propionic acid,butyric acid and valeric acid,etc.,are the main energy sources of intestinal cells,which regulate the absorption of various nutrients and hormone production in the intestinal tract,and are widely involved in energy metabolism.Some studies have pointed out that patients with HT have abnormal intestinal flora.It has also been shown that butyric acid in short-chain fatty acids can activate the acetyl pyruvate dehydrogenase complex and impair the anaerobic glycolysis of tumor cells.After treatment with butyrate in animal models of rheumatoid disease,the progression of the disease can be reduced to some extent,or even reversed.Therefore,this study aims to investigate whether patients with HT have abnormal short-chain fatty acid metabolism,and whether butyric acid in short-chain fatty acid can inhibit glycolysis of T cells by activating HDAC/SIRT3/PDHA/PDC pathway,correct the imbalance between T cell subtypes,and participate in the pathogenesis of HT.Methods:1.Clinical study of HT patients: Feces and serum of HT patients and age-and sex-matched healthy controls were collected.Serum free T3,free T4,thyroid stimulating hormone,thyroglobulin antibodies and thyroid peroxidase antibodies were determined by electrochemical immunoluminescence method.At the same time,fecal targeted metabonomics,serum non-targeted metabonomics and targeted metabonomics were tested to analyze the differences in metabolites and 7 kinds of short-chain fatty acids.2.CD4+T cell study: CD4+T cells were selected from PBMC of the above-mentioned HT patients and healthy controls for primary culture.Cell inflammatory cytokines were detected by flow cytometry,and the proportion of Th17,Th1 and Tregs cell subsets was determined.Glycolysis and aerobic metabolism of CD4+T cells were detected by cell energy metabolism detector(Seahorse).WB(Western-blot)was used to analyze the expression of PDHA and other key enzymes in glycolysis.HT patients were treated with butyric acid and SIRT3 inhibitors respectively,and the effects of treatment factors on cellular inflammatory factors,Th17 and Th1,the proportion of Tregs cell subsets,CD4+T cell metabolism and pathway-related protein expression were observed.Results:(LC-MS/MS)1.Clinical study on HT patients:(1)Feces of 28 HT patients and 28 healthy controls matched for age and sex were collected for targeted metabonomics detection.There were no significant differences in TSH,FT4 and FT3 levels between the two groups(P > 0.05).TPOAb and Tg Ab in HT patients were significantly higher than those in normal control group(P<0.05).Targeted metabonomics analysis indicated that the fecal content of butyric acid in HT group was significantly lower than that in control group,but there was no statistical significance(P> 0.05).The content of caproic acid in serum of HT group was significantly lower than that of control group(P<0.05).(2)Serum samples from 30 HT patients and 30 healthy controls matched for age and sex were collected for non-targeted metabonomics detection(LC-MS/MS).TSH levels were within the normal range between the two groups,and there were no significant differences in FT4 and FT3(P > 0.05).TPOAb and Tg Ab in HT patients were significantly higher than those in the normal control group(P<0.05).The LC-MS/MS results showed that,among the top 20 metabolites,the content of 4-phenylbutyric acid in HT group was significantly lower than that in control group(P<0.05).(3)Targeted quantitative detection and analysis(GCMS)of 7 kinds of short-chain fatty acids in serum of patients in two groups were further conducted.The results indicated that the contents of butyric acid and valeric acid in serum of HT patients were significantly lower than that of normal control group(P<0.05);There was no significant difference in the contents of acetic acid,propionic acid,isobutyric acid and caproic acid(P > 0.05).2.Cell experiment:(1)Compared with normal control group,the content of IL-17 A in CD4+T cell medium in peripheral blood of HT group was significantly increased(P<0.05),IFN-R was significantly increased,but there was no statistical difference;IL-10 was decreased,but there was no statistical difference;The proportion of CD4+ IFNR +T cell subsets in peripheral blood was increased,and the proportion of CD25+Treg cells was decreased(P<0.05).The glycolytic metabolism of CD4+T cells was significantly increased(P<0.05);The expression of SIRT3 in CD4+T cells increased(P<0.05),while the expression of PDHA and PDP1 decreased(P<0.05).(2)After treatment with different concentrations of butyrate,the content of IL-17 A in CD4+T cell medium in peripheral blood of HT patients was significantly decreased(P<0.05);The proportion of CD4+ IFNR +T cell subsets in peripheral blood of HT patients decreased gradually(P<0.05),while the proportion of CD25+Treg cells increased gradually(P<0.05).The glycolytic metabolism of CD4+T cells was inhibited(P<0.05).The expression of SIRT3 protein was decreased(P<0.05),while the expression of PDHA and PDP1 protein were increased(P<0.05).All were dose-dependent.(3)After selecting an important target IN the pathway and treating cells with SIRT3 inhibitor SIRT-in-3,the increased expression of SIRT3 IN CD4+T cells IN peripheral blood of HT patients was inhibited(P<0.05),the expression of PDHA was increased(P<0.05),the expression of Foxp3 was increased(P<0.05),and the glycolysis level of CD4+T cells was inhibited(P<0.05).Conclusion:1.HT patients had abnormal metabolism of short-chain fatty acids caused by intestinal flora imbalance,and the content of butyric acid and the level of caproic acid in feces showed a significant decrease trend;The contents of butyric acid and valeric acid in serum were significantly lower than those in normal subjects.The metabolite of butyric acid,4-phenylbutyric acid,was significantly lower in HT patients.;2.The proportion of T cell subsets was unbalanced in HT patients,and the metabolism of T cell immune cells was abnormal,and the metabolism of glycolysis was significantly increased.3.Short-chain fatty acids dominated by butyric acid can change the T cell metabolism pathway,inhibit glycolysis,correct the imbalance of T cell subsets and delay the onset of HT by activating the HDAC/SIRT3/PDHA/PDC pathway. |
PDF Full Text Request