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.

Global trends in research of fibroblasts associated with rheumatoid diseases in the 21st century: A bibliometric analysis

“…With the understanding of the fibroblast pathologic process, how to regulate fibroblast autophagy has become an emerging hot topic. For example, knock down of miR-218-5p could regulate the autophagy of FLSs (78) and targeting PGC-1a prevents abnormal autophagy, inhibits fibroblast activation and tissue fibrosis in SSc (79). To sum up, the understanding of fibroblasts in RDs is becoming comprehensive and is gradually being transformed into clinical practice.…”

Can pyroptosis be a new target in rheumatoid arthritis treatment?

“…By expressing different molecular patterns, FLS enhances autophagy and antiapoptosis to promote its proliferation. In addition, the development of autophagy by T cells, osteoblasts and osteoclasts has been connected to RA (163, [185][186][187]. Thus, the phenomenon of autophagy is strongly associated with the progression of RA.…”