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.
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Table-1: Clinical data of RA (n=11) and OA (n=10) patients from synovial fluid. Data expressed as mean ± SD. * represents p value <0.05 which is considered as significant. **represents median value (inter-quartile range). Anti-CCP = Anti-citrullinated protein antibody; CRP = C-reactive protein; DAS 28-CRP Disease activity Score 28.
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Posted 12 Mar, 2021
Received 10 Mar, 2021
On 07 Mar, 2021
Invitations sent on 07 Mar, 2021
On 26 Feb, 2021
Posted 12 Mar, 2021
Received 10 Mar, 2021
On 07 Mar, 2021
Invitations sent on 07 Mar, 2021
On 26 Feb, 2021
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
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