The mechanism of methotrexate in the treatment of rheumatoid arthritis using untargeted metabolomics / 中华风湿病学杂志

ABSTRACT

Objective:To investigate the mechanism of methotrexate in the treatment of rheumatoid arthritis (RA) by constructing a rat model of collagen-induced arthritis (CIA) and using non-targeted metabolomics.Methods:Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of TNF-α, IL-1β, IL-6, IL-4 and IL-10 in serum. HE staining and Masson staining were used to observe the histological changes of joints in each group. Non-targeted gas chromatography-mass spectrometry metabolomics technique was used to screen the expression profiles of differential metabolites in serum and cluster analysis and KEGG enrichment analysis were performed to screen the differential metabolic pathways, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the key enzymes in the differential metabolic pathways. All experimental data conforming to the normal distribution were compared between groups using one-way ANOVA, and P<0.05 was considered statistically significant. Results:MTX significantly improved the joint inflammatory response and arthritis score and increased the body weight of CIA rats. The results of HE and Masson staining showed that MTX could ameliorate the erosion of articular cartilage by synovial tissue in CIA rats. ELISA results showed that MTX significantly decreased the contents of TNF-α [(191.2±17.4)pg/ml, F=40.31, P<0.001], IL-1β[(28.4±1.2)pg/ml, F=10.11, P=0.012] and IL-6[(118.7±1.4)pg/ml, F=829.40, P<0.001] in the serum and increased the contents of IL-4 [(49.3±3.3)pg/ml, F=33.44, P<0.001] and IL-10 [(30.2±0.7)pg/ml, F=33.44, P<0.001] in the serum of CIA rats. Non-targeted metabolomics technique showed MTX had an effect on metabolites such as phosphocholine, palmitic acid, oleic acid, and choline in the serum of CIA rats. KEGG pathway enrichment analysis showed that MTX had an effect on glycerophospholipid metabolism( P<0.01)and sphingolipid metabolism( P<0.05)in CIA rats. qRT-PCR results showed that MTX could down-regulate the expression of the key enzymes such as Plb1 [(1.00±0.49), F=8.23, P=0.019], Gpcpd1[(1.10±0.09), F=8.19, P=0.019], Chka [(1.33±0.19), F=33.00, P<0.001], Chkb [(2.07±1.21), F=8.20, P=0.019]and Phospho1 [(1.07±0.14), F=13.58, P=0.006]in the glycerophospholipid metabolic pathway in the synovial membrane of CIA rats, and can also down-regulate the expression of the key enzymes Kdsr [(1.24±0.32), F=13.85, P=0.006], Plpp1 [(1.61±0.32), F=11.95, P=0.003) and Degs1 [(1.21±0.15, F=46.55, P<0.001]in the sphingolipid metabolic pathway. Conclusion:The biological mechanism of MTX in the treatment of rheumatoid arthritis may be related to the down-regulation of glycerophospholipid metabolism and sphingolipid metabolism pathway metabolic levels in the body.