Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Abstract Background Synovial fibroblasts in rheumatoid arthritis (RAFLS) exhibit a pathological aberration of glycolysis and glutaminolysis. Henceforth, we aimed to investigate if dual inhibition of these pathways by phytobiological compound c28MS has the potential of synergistic therapy for arthritis by targeting both glucose and glutamine metabolism. Methods The presence of HK2 and GLS across various cell types and associated gene expression in human synovial cells and a murine model of arthritis was evaluated by scRNA-seq. The metabolic profiling of RAFLS cells was done using H1-nuclear magnetic resonance spectroscopy under glycolytic and glutaminolytic inhibitory conditions by incubating with 3-bromopyruvate, CB839, or dual inhibitor c28MS. FLS functional analysis was conducted under similar conditions. ELISA was employed for the quantification of IL-6, CCL2, and MMP3. K/BxN sera was administered to mice to induce arthritis for in vivo arthritis experiments. Results scRNA-seq analysis revealed that many fibroblasts expressed Hk2 along with Gls with several genes including Ptgs2, Hif1a, Timp1, Cxcl5, and Plod2 only associated with double-positive fibroblasts, suggesting that dual inhibition can be an attractive target for fibroblasts. Metabolomic and functional analysis revealed that c28MS decreased the aggressive behavior of RAFLS by targeting both upregulated glycolysis and glutaminolysis. c28MS administered in vivo significantly decreased the severity of arthritis in the K/BxN model. Conclusion Our findings imply that dual inhibition of glycolysis and glutaminolysis could be an effective approach for the treatment of RA. It also suggests that targeting more than one metabolic pathway can be a novel treatment approach in non-cancer diseases

Additional file 3 of Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Additional file 3: Figure S3. Effect of c28MS on viability of RAFLS (n=3) A glucose medium (25mM of glucose and 6mM of glutamine) and B low glucose medium (2mM of glucose and 6mM of glutamine)

Additional file 2 of Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Additional file 2: Figure S2. Color scale encoded heat maps illustrating variations in the concentration of various metabolites between control and treatment groups in RAFLS A glucose medium (25mM of glucose and 6mM of glutamine) and B low glucose medium (2mM of glucose and 6mM of glutamine) respectively

Additional file 1 of Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Additional file 1: Figure S1. A scRNA-seq UMAP of all cells sequenced as described in Zhang et al., 2019 [25]. B,C Segregation of synovial fibroblasts from Zhang et al., 2019 [25] in to GLS positive, HK2 positive, double negative and double positive cells

Additional file 5 of Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Additional file 5. Details of genes found in double negative, Hk2 positive, Gls positive and double positive fibroblasts in murine STIA

Additional file 6 of Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Additional file 6. List of up-and down regulated genes in double positive fibroblasts for the different disease stages

Additional file 4 of Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis

Additional file 4: Figure S4. Expression of Ccl2, Mmp3 and Il6 in double positive and negative fibroblasts at different stages of the disease