Effect And Mechanism Of Methotrexate On Serum Metabolic Profile In Collagen-Induced Arthritis Rats

Objective:In this study,a collagen-induced arthritis(CIA)model was established and non-targeted metabolomics was used to investigate the effects of MTX on serum metabolism in CIA rats,so as to clarify the potential regulatory mechanism of MTX intervention in CIA rats.Methods:A total of 24 healthy female Wistar rats were randomly divided into normal,model,and MTX treatment groups(n = 8).Collagen-induced arthritis(CIA)models were constructed in all groups except the normal group,and rats in the MTX treatment group were treated with MTX(1.0 mg/kg)once a week for 4 weeks,and rats in the other two groups were treated with saline.Serum contents of TNF-α,IL-1β,IL-6,IL-4,IL-10 were determined by enzyme-linked immunosorbent assay(ELISA),histological changes in the joints of rats in each group were observed by HE and Masson staining,and non-targeted gas chromatography-mass spectrometry metabolomics technique was used to screen the serum differential metabolite expression profiles and cluster analysis and KEGG enrichment analysis were performed to screen the differential metabolic pathways.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the key enzymes in the differential metabolic pathways.Results:MTX significantly improved the joint inflammatory response and arthritis score and increased the body weight of CIA rats(P<0.05).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,IL-1β and IL-6 in the serum and increased the contents of IL-4 and IL-10 in the serum of CIA rats(P<0.05).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 and sphingolipid metabolism in CIA rats.q RT-PCR results showed that MTX could down-regulate the expression levels of Plb1,Gpcpd1,Chka,Chkb and Phospho1,which are key enzymes in the glycerophospholipid metabolic pathway,and also down-regulate the expression levels of Kdsr,Plpp1 and Degs1,which are key enzymes in the sphingomyelin metabolic pathway,in the synovial membrane of CIA rats(P<0.05).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.