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

Yang XY, Zheng KD, Lin K, Zheng G, Zou H, Wang JM, Lin YY, Chuka CM, Ge RS, Zhai W, Wang JG - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China.

ABSTRACT

Objectives: To explore the pathogenesis of rheumatoid arthritis (RA), the different metabolites were screened in synovial fluid by metabolomics.

Methods: 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.

Results: 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 t-test (p<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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Verification of different metabolites and enzymes in synovial fluid and synovial tissues from RA patients and normal subjects.(A) Real-time PCR amplification was performed, the results were normalized for the amount of β-actin 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.
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pone.0132695.g002: Verification of different metabolites and enzymes in synovial fluid and synovial tissues from RA patients and normal subjects.(A) Real-time PCR amplification was performed, the results were normalized for the amount of β-actin 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.

Mentions: 46 enzymes of energy metabolism were identified in FLS cells from synovial membrane in our previous competitive proteomics study [17]. Among them 12 enzymes were different between RA patients and normal subjects. PFKP and P4HA1 were increased and the others were decreased in RA patients, including DLST, PGD, CS, ACO1, GFPT2, G6PD, MDH1, ACSL4, HADHA and ACADVL (Table 2). 7 of the 12 differentially expressed proteins were verified by real-time PCR and western blot in synovial tissues of RA patients and normal subjects. The real-time PCR result showed that the expression of PFKP and LDHA were elevated, and CS, DLST, PGD, ACSL4, HADHA and ACADVL were reduced in the synovial tissues of RA patients as compared to normal subjects (Fig 2A). The RA/normal ratio was 2.32, 2.23, 0.29, 0.22, 0.37, 0.38, 0.34 and 0.46, respectively. These results were strongly consistent with the results obtained by western blot, in which the RA/normal ratio was 2.21, 2.16, 0.47, 0.35, 0.30, 0.57, 0.44 and 0.27 (Fig 2B).


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

Yang XY, Zheng KD, Lin K, Zheng G, Zou H, Wang JM, Lin YY, Chuka CM, Ge RS, Zhai W, Wang JG - PLoS ONE (2015)

Verification of different metabolites and enzymes in synovial fluid and synovial tissues from RA patients and normal subjects.(A) Real-time PCR amplification was performed, the results were normalized for the amount of β-actin 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.
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Related In: Results  -  Collection

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pone.0132695.g002: Verification of different metabolites and enzymes in synovial fluid and synovial tissues from RA patients and normal subjects.(A) Real-time PCR amplification was performed, the results were normalized for the amount of β-actin 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.
Mentions: 46 enzymes of energy metabolism were identified in FLS cells from synovial membrane in our previous competitive proteomics study [17]. Among them 12 enzymes were different between RA patients and normal subjects. PFKP and P4HA1 were increased and the others were decreased in RA patients, including DLST, PGD, CS, ACO1, GFPT2, G6PD, MDH1, ACSL4, HADHA and ACADVL (Table 2). 7 of the 12 differentially expressed proteins were verified by real-time PCR and western blot in synovial tissues of RA patients and normal subjects. The real-time PCR result showed that the expression of PFKP and LDHA were elevated, and CS, DLST, PGD, ACSL4, HADHA and ACADVL were reduced in the synovial tissues of RA patients as compared to normal subjects (Fig 2A). The RA/normal ratio was 2.32, 2.23, 0.29, 0.22, 0.37, 0.38, 0.34 and 0.46, respectively. These results were strongly consistent with the results obtained by western blot, in which the RA/normal ratio was 2.21, 2.16, 0.47, 0.35, 0.30, 0.57, 0.44 and 0.27 (Fig 2B).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China.

ABSTRACT

Objectives: To explore the pathogenesis of rheumatoid arthritis (RA), the different metabolites were screened in synovial fluid by metabolomics.

Methods: 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.

Results: 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 t-test (p<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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus