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.

Potential of the Novel Slot Blot Method with a PVDF Membrane for Protein Identification and Quantification in Kampo Medicines

“…However, the proteins in the water extract of Kampo medicines (or crude drugs) are often low and may be undetectable. When researchers investigate intracellular or tissue proteins in vitro or in vivo, Western blot with SDS-PAGE [22,26] and ELISA [27,28] are commonly used to identify or quantify the individual proteins (e.g., interleukin-1, tumor necrosis factor-α, and matrix metalloproteinase) [79][80][81]. The volume of samples and reagents used is 10-30 µL per SDS-PAGE well and 50-200 µL per ELISA well.…”

“…Accordingly, we evaluated the applicability and suitability of conventional technologies for the separation, detection, identification, and quantification of proteins in Kampo medicines [22][23][24]. We found that, given the insufficient crude sample volume, column chromatography [22,25], electrophoresis [22,26], and enzyme-linked immunosorbent assay (ELISA) [27,28] may be unsuitable for detecting rare proteins within the extract in Kampo medicines. Hence, we focused on membrane chromatography, where samples can repeatedly flow against the membranes, facilitating the collection and separation of proteins [22][23][24].…”

Experimental and computational investigation of the kinetic evolution of the glutaminolysis pathway and its interplay with the glycolysis pathway

“…Clearly, the presence of glutamine potentially influences the rate of the glycolytic pathway by fuelling the TCA cycle, which in turn draws more pyruvate into the mitochondria, facilitating the processing of pyruvate generated from glycolysis. Given this interplay, along with evidence that shows an upregulation of the kinetics of both pathways in various disorders [15,16], recent fluxomic studies encourage the simultaneous monitoring of the kinetics of these two pathways [17][18][19]. Considering that cells often adapt their metabolic rates and switch between pathways such as glycolysis and glutaminolysis in response to local environmental conditions, the kinetic nature of these processes emphasises the necessity for real-time and in situ monitoring.…”