| Background:Rheumatoid arthritis(RA)is a chronic inflammatory joint disease that affects all races and ethnicities and affects patients worldwide,most often in temperate,subtropical and boreal regions.It can lead to cartilage and bone damage as well as disability.Early diagnosis is the key to optimal treatment success,especially in patients with high disease activity,the presence of autoantibodies and early joint damage,and other risk factors for poor prognosis.Rheumatoid arthritis(RA)is characterized by systemic inflammation,joint swelling and deformity,and synovitis.The conventional and biological therapies currently used for rheumatoid arthritis do not meet the needs of patients and are only partially effective.Although some drugs such as methotrexate,tumor necrosis factor(TNF-α)inhibitors and other disease-modifying anti-rheumatic drugs(DMARDs)are effective in improving patients’ symptoms,their adverse effects and limited efficacy make alternative treatments possible.Objectives:The combined transcriptomics and metabolomics analysis were used to screen for significant differential genes and differential metabolites,map relevant signaling pathways,verify gene function through in vivo and in vitro experiments,illustrate the therapeutic mechanism of mannose in RA,and provide further evidence for the development of D-mannose therapy for RA.Methods:1.LPS induced the activation of RAW 264.7 cells.After treatment with Dmannose,the cells were collected for RNA extraction and then transcriptome sequencing.The sequencing results were analyzed bioinformatically to screen out the differential genes.2.Centrifugation to collect peripheral blood serum from CIA rats and D-mannose treated CIA rats,GC-MS was used to decete changes in RA metabolites.PLS-DA was applied to analyze and screen differential metabolites.3.Combined metabolomics and transcriptomics analysis were performed to construct gene pathways using Protein-Protein Interaction Networks(PPI).4.The functions of differential genes and metabolites were validated and explored the interactions and signaling mechanisms of differential genes.5.siRNA transfection was used to interferes with the expression of switch genes.The cell phenotype and the changes of upstream and downstream signaling pathways were verified by WB,QP,flow and NADPH assays.6.The antirheumatic effect of D-mannose was explored using collagen-induced rheumatoid arthritis(CIA)rat animal model,foot swelling and arthritic pathological changes,histopathological assessment,and diagnostic imaging(MRI,CT).Results:1.After treatment of polarized RAW 264.7 cells with D-mannose,RNA was extracted for eukaryotic with reference transcriptome sequencing analysis to screen for differential gene expression.2.After D-mannose treatment of polarized RAW 264.7 and THP-1 cells,q PCR results showed that the markers of M1 polarization and the expression of proinflammatory factors such as NOS2,CD11 c,IL-6,TNF-α and CD86 significantly decreaed,indicating that D-mannose could effectively inhibit inflammatory response in macrophages.3.siRNA transfection interfered with the expression of Tigar gene,and the WB results showed down-regulation of Tigar expression.WB results also showed that key genes in glycolysis were significantly decreaed.4.Result of flow detection of cell cycle showed an increase in cell S phase after mannose treatment compared to LPS induced macrophage.5.Result of NADPH kit assay showed that NADPH level was significantly increased after D-mannose treatment.6.The animal model of collagen-induced rheumatoid arthritis(CIA)in rats was successfully constructed,and there were obvious histopathological changes of arthritis including edema,bone/chondral destruction,synovial hyperplasia in the joint parts of the model group.After treatment with D-mannose,the arthritis score,histopathological assessment,and diagnostic imaging(MRI,CT)showed that treatment with D-mannose almost completely prevented the histopathological changes in the joints of CIA rats.The levels of cytokines in the serum of rats were detected by rat inflammatory factor antibody microarrays,and the levels of TNF-α,IL-1α,IL-1β and IL-10 were elevated in the serum of rats in the model group,and treatment of CIA rats with D-mannose significantly suppressed the levels of these inflammatory cytokines.Conclusion:1.The therapeutic effect of D-mannose on rheumatoid arthritis in CIA rats was clarified,and D-mannose could effectively alleviate the joint inflammation in CIA rats.2.D-mannose regulates intracellular glucose metabolism through Tigar,inhibits glycolysis and enhances the PPP pathway,thus inhibiting the inflammatory response induced by RA. |
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