Methotrexate and theaflavin-3,3’ di gallate synergistically restore the balance between apoptosis and autophagy in synovial fibroblast of RA: An ex vivo approach with cultured human RA FLS

Abstract Background: Rheumatoid arthritis (RA) is characterized by inflammation mediated angiogenesis in synovial tissue, leading to apoptotic retardation and enhanced cell survival in synovial fibroblasts. Methotrexate (MTX) can reduce selective pro-inflammatory cytokines but unable to restore disrupted homeostasis between autophagy and apoptosis in fd-FLS.Objective: To evaluate the effect of black tea compound TF3 along with MTX upon fluid derived (fd)-FLS to induce apoptosis and inhibit autophagy through ER stress-mediated pathways.Methods: FLS sourced from synovial fluid (SF) of patients with RA (n=11) and osteoarthritis (OA) (n=10) were cultured following treatment with MTX/TF3 or in combination and underlying mechanisms were investigated. Extracellular inflammatory markers like CRP and cytokines (TNF-α, IL-6), angiogenic markers (VEGF, ANG-1) were quantified by ELISA. Cell viability of cultured fd-FLS was determined by MTT assay. fd-FLS treated with MTX/TF3 or combination of MTX(125nM) and TF3(10µM), followed by apoptosis measurement by flow cytometry. ER stress associated markers were quantified by RT-PCR (IRE1A and spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF1-α). Apoptotic (Bcl-2, Bax, and Caspases) and autophagic proteins (Beclin1, LC3b and p62) were quantified by immunoblot study. Results: MTX and TF3 both in single doses (IC25) could down-regulate the levels of pro-inflammatory and angiogenic markers. Combination treatment modulated ER stress response and blocked the auto-phagmosomal proteins in fd-FLS and induced apoptosis.Conclusion: Disruption in homeostasis between apoptosis and autophagy might be an underlying phenomenon in the progression of pathophysiology in fd-FLS. The combined administration of MTX and TF3 successfully balanced the homeostasis by inducing apoptosis.