Energy Metabolism Disorder as a Contributing Factor of Rheumatoid Arthritis: A Comparative Proteomic and Metabolomic Study

<div><p>Objectives</p><p>To explore the pathogenesis of rheumatoid arthritis (RA), the different metabolites were screened in synovial fluid by metabolomics.</p><p>Methods</p><p>Synovial fluid from 25 RA patients and 10 normal subjects were analyzed by GC/TOF MS analysis so as to give a broad overview of synovial fluid metabolites. The metabolic profiles of RA patients and normal subjects were compared using multivariate statistical analysis. Different proteins were verified by qPCR and western blot. Different metabolites were verified by colorimetric assay kit in 25 inactive RA patients, 25 active RA patients and 20 normal subjects. The influence of hypoxia-inducible factor (HIF)-1α pathway on catabolism was detected by HIF-1α knockdown.</p><p>Results</p><p>A subset of 58 metabolites was identified, in which the concentrations of 7 metabolites related to energy metabolism were significantly different as shown by importance in the projection (VIP) (VIP≥1) and Student’s <i>t</i>-test (<i>p</i><0.05). In the 7 metabolites, the concentration of glucose was decreased, and the concentration of lactic acid was increased in the synovial fluid of RA patients than normal subjects verified by colorimetric assay Kit. Receiver operator characteristic (ROC) analysis shows that the concentration of glucose and lactic acid in synovial fluid could be used as dependable biomarkers for the diagnosis of active RA, provided an AUC of 0.906 and 0.922. Sensitivity and specificity, which were determined by cut-off points, reached 84% and 96% in sensitivity and 95% and 85% in specificity, respectively. The verification of different proteins identified in our previous proteomic study shows that the enzymes of anaerobic catabolism were up-regulated (PFKP and LDHA), and the enzymes of aerobic oxidation and fatty acid oxidation were down-regulated (CS, DLST, PGD, ACSL4, ACADVL and HADHA) in RA patients. The expression of HIF-1α and the enzymes of aerobic oxidation and fatty acid oxidation were decreased and the enzymes of anaerobic catabolism were increased in FLS cells after HIF-1α knockdown.</p><p>Conclusion</p><p>It was found that enhanced anaerobic catabolism and reduced aerobic oxidation regulated by HIF pathway are newly recognized factors contributing to the progression of RA, and low glucose and high lactic acid concentration in synovial fluid may be the potential biomarker of RA.</p></div

Metabolic patterns in RA patients and normal subjects.

<p>(A) The heat map shows the standard score for each metabolite of each RA patient and each normal subject. The standard score shows how the concentration of each metabolite is related to the mean value of the control group. Red color indicates that the metabolite is increased compared to the mean of the control group; green color indicates that the metabolite is decreased. Metabolites are sorted according to the initial letter of the metabolite. The standard score is truncated to -7.31/7.31 for clarity. (B) Orthogonal PLS-DA (OPLS-DA) score plot of the first two principal components of an analysis of metabolites from RA patients (R) and Normal subjects (N). The number after letter means the number of the sample. The horizontal axis and the vertical axis mean values of the first and the second principal components. The ellipse denotes the 95% significance limit of the model, as defined by Hotelling's t-test.</p

Verification of different metabolites and enzymes in synovial fluid and synovial tissues from RA patients and normal subjects.

<p>(A) Real-time PCR amplification was performed, the results were normalized for the amount of <i>β-actin</i> as internal control. Each value represents the average from 50 RA patients or 20 normal subjects. (B) Western blot of different enzymes. Protein samples were analyzed on 10% SDS–PAGE, followed by immunoblotting, left panel is the band image of enzymes from 3 RA patients and 3 normal subjects. The mean intensity of corresponding protein expression in RA patients (20 cases) and normal subjects (20 cases) was shown in the right panel. (C) The concentration of glucose and lactic acid were verified in synovial fluid from inactive RA patients (25 cases), active RA patients (25 cases) and normal subjects (20 cases) by colorimetric assay kit and ROC curve analysis. ROC curve analysis showed that the concentration of glucose and lactic acid in synovial fluid could be used as dependable biomarkers for the diagnosis of active RA, provided an AUC of 0.906 and 0.922. Sensitivity and specificity, which were determined by cut-off points, reached 84% and 96% in sensitivity and 95% and 85% in specificity, respectively.</p

Different enzymes in RA patients and normal subjects related to energy metabolism.

<p>^aACC: Accession number in Uniprot</p><p>^bEC: Enzyme commission number.</p><p>Different enzymes in RA patients and normal subjects related to energy metabolism.</p

Different Metabolites in RA patients and normal subjects (VIP≥1, <i>p</i><0.05).

<p>^aPathway was consulted from KEGG</p><p>^b R.T.: retention time</p><p>^cThe number of column Normal and RA mean the relative concentration to the internal reference and expressed with mean±SE</p><p>Different Metabolites in RA patients and normal subjects (VIP≥1, <i>p</i><0.05).</p

The effect of HIF-1α knockdown on the expression of PFKP, LDHA, CS, DLST, PGD, ACSL4, HADHA and ACADVL in FLS cells.

<p>(A) Real-time PCR of enzymes in FLS cells transfected with scrambled siRNA or HIF-1α siRNA for 24hr, β-actin was used as a loading control. (B) Western blot of enzymes in FLS cells transfected with scrambled siRNA or HIF-1α siRNA for 48hr, β-actin was used as a loading control. All experiments were performed at least in triplicates, the data are presented as mean ± SD, *p<0.05, compared with control, the asterisks represent significant differences.</p