Habbe Gule Aakh prevents glycolytic program and alleviates disease progression in a rheumatoid arthritis animal model

Arulkumaran Rithvik¹Shangomitra Bhattacharjee¹Mansi Kumari Gupta²Sudandiradoss C²Abdul Wadud³Mahaboobkhan Rasool^1*

• ¹SMV 240, Immunopathology Lab, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India
• ²Department of Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India
• ³Regional Research Institute of Unani Medicine, Srinagar, India

Fibroblast-like synoviocytes (FLS) are critical mediators of rheumatoid arthritis (RA) pathogenesis. The aberrant glycolytic program of resident synoviocytes has been shown to promote the plasticity or tumorigenic phenotype of these cells, resulting in cartilage degradation or joint damage. Toll-like receptor 4 (TLR4) signalling has been shown to improve glycolysis in dendritic cells, monocytes, and macrophages, resulting in a sustained proinflammatory milieu. In this study, we demonstrated the effectiveness of Habbe Gule Aakh (HGA), a polyherbal unani formulation, in modulating the glycolytic pathway in RA human synoviocytes or SW 982 cells through the inhibition of TLR4, as well as its role in preventing cartilage breakdown in an experimental arthritis model. Our results demonstrate that HGA led to downregulation of the rate-limiting glycolytic markers hexokinase 2 (HK2), glucose transporter 1 (GLUT1), and pyruvate kinase M2 (PKM2) via TLR4 inhibition. Furthermore, we also decoded a previously unknown mechanism of glucose uptake via tissue inhibitors of matrix metalloproteinase 1 or TIMP1. HGA diminished TIMP1 expression by blocking the TLR4-p65 signalling axis. TIMP1 knockdown or treatment with the p65 inhibitor (Bay 11-7082) inhibited glucose uptake and sustained the proliferation of SW 982 cells via diminished glycolysis. To validate our findings, we utilized a network pharmacology approach to uncover the interactome of HGA against rheumatoid arthritis targets. Our in-silico analysis revealed the interaction of the key phytoconstituents with TLR4, TIMP1, and CD63, thus reinforcing our in vitro observations. Ultimately, we leveraged in vivo models to support the anti-arthritic claims of HGA. HGA treatment effectively diminished cartilage degradation and bone damage by regulating the expression of matrix metalloproteinases and inflammatory cytokines. In conclusion, pharmacological intervention with HGA can rescue the exacerbation of disease severity via the TLR4 signaling axis.