Supplementary file 1_Habbe Gule Aakh prevents glycolytic program and alleviates disease progression in a rheumatoid arthritis animal model.docx

Background<p>The acquired tumorigenic phenotype of the resident fibroblast like synoviocytes (FLS) is cornerstone to exacerbating rheumatoid arthritis (RA) disease progression. Toll like receptor 4 (TLR4) signalling can sustain the proliferative and invasive phenotype of these synoviocytes resulting in cartilage degradation and bone damage. A marked increase in glycolytic activity also contributes to the malignant character of these cells. Herein, we aim to study the prospects of TLR4 activation leading to improved glycolytic flux. Further, we also strategize the therapeutic modality of Habbe Gule Aakh (HGA), a polyherbal unani formulation to rescue disease progression via blockade of TLR4 activation.</p>Methods<p>We activated TLR4 signaling in SW982 cells, cultured in high glucose medium. Initially, the expression profile of glycolytic rate limiting enzymes- hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2) was assessed. Next, we evaluated the ability of HGA to regulate the expression of these enzymes via ablation of TLR4 activation. Further, we investigated the pathway of glucose uptake via tissue inhibitor of matrix metalloproteinase 1 or TIMP1 and counterintuitively investigated HGA to arrest the uptake of glucose via p65-TIMP1 signaling axis. To sufficiently validate our findings, we utilised network pharmacology approach, to uncover the interactome of HGA against rheumatoid arthritis targets. Ultimately, we leveraged in-vivo models to support the anti-arthritic claims of HGA.</p>Results<p>HGA regulated the proliferation and invasive phenotype of SW982 cells cultured in high glucose medium via blockade of TLR4 activation. Further, in-silico and in-vivo approaches suggest a mechanistic insight to the anti-arthritic activity of HGA upon blockade of TLR4-mediated glycolytic flux in resident synoviocytes.</p>Conclusion<p>Pharmacological intervention with Habbe Gule Aakh can rescue exacerbation of rheumatoid arthritis disease severity via TLR4 signaling axis. The findings of this study strengthen the rationale for the use of HGA in clinical settings involving RA patients.</p>