Abstract
Background
Imbalance between apoptosis and autophagy in fibroblast-like synoviocytes (FLS) is one of the pathogenic mechanisms responsible for their abnormal proliferation in rheumatoid arthritis (RA). Methotrexate (MTX) demonstrated limited efficacy in amending this imbalance in fluid-derived (fd)-FLS. The active compound of black tea Theaflavin 3,3′-digallate (TF3) may be effective in restoring apoptosis–autophagy imbalance in (fd)-FLS. The combined effect of MTX + TF3 upon the same is yet to be elucidated.
Objective
To evaluate the effect of MTX + TF3 on fd-FLS to induce apoptosis and inhibit autophagy through Endoplasmic Reticulum (ER) stress-mediated pathways.
Methods
FLS from synovial fluid of 11 RA and 10 osteoarthritis patients were cultured after treatment with MTX/TF3 or a combination of MTX (125 nM) and TF3(10 µM) and the following parameters were evaluated. C-reactive protein, cytokines (TNF-α, IL-6), angiogenic markers were quantified by ELISA. fd-FLS viability was determined by MTT assay and apoptosis by flow cytometry. ER stress markers were estimated by RT-PCR (IRE1A, spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF-1α). Immunoblot studies were done to evaluate apoptotic (Bcl-2, Bax, Caspases) and autophagic (Beclin1, LC3b, p62) proteins.
Results
MTX (IC25) and TF3 (IC50) both in single doses could down-regulate the levels of pro-inflammatory and angiogenic markers. Combinatorial treatment modulated autophagosomal proteins in fd-FLS and induced apoptosis by regulating ER stress response.
Conclusion
Disruption in homeostasis between apoptosis and autophagy in fd-FLS might be an underlying phenomenon in the progression of pathophysiology in RA. Co-administration of MTX + TF3 successfully restored the homeostasis by inducing apoptosis.
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Abbreviations
- ANG-1:
-
Angiopoietin 1
- ACR/EULAR:
-
American College of Rheumatology/European League Against Rheumatism
- ANOVA:
-
Analysis of variance
- Caspase:
-
Cysteine-aspartic proteases
- CRP:
-
C-reactive protein
- CHOP:
-
C/EBP homologous protein
- cDNA:
-
Complementary DNA
- DMARDs:
-
Disease-modifying anti-rheumatic drugs
- DEPC:
-
Diethyl pyrocarbonate
- ELISA:
-
Enzyme-linked immunosorbent assay
- ER-Stress:
-
Endoplasmic reticulum stress
- ERAD:
-
Endoplasmic-reticulum-associated protein degradation
- FBS:
-
Fetal Bovine serum
- FITC:
-
Fluorescein isothiocyanate
- fd-FLS :
-
Fluid-derived fibroblast-like synoviocytes
- GAPDH:
-
Glyceraldehyde–3 phosphate dehydrogenase
- GRP-78:
-
78-KDa glucose-regulated protein
- HIF-1 α:
-
Hypoxia-inducible factor 1-alpha
- HSP70:
-
Heat shock protein 70
- IL-6:
-
Interleukin 6
- IRE1A:
-
Serine/threonine-protein kinase/endoribonuclease A
- LC3b:
-
Microtubule-associated proteins 1A/1B light chain 3B
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- MTX:
-
Methotrexate
- NSAIDs:
-
Non-steroidal anti-inflammatory drugs
- OA:
-
Osteoarthritis
- PBS:
-
Phosphate-buffered saline
- p-62:
-
Sequestosome 1(SQSTM1)
- PCRP:
-
Polymerase chain reaction
- RA:
-
Rheumatoid Arthritis
- RT-PCR:
-
Real-time polymerase chain reaction
- RPM:
-
Revolutions per minute
- RT:
-
Room temperature
- RNA:
-
Ribonucleic acid
- RASF:
-
Rheumatoid arthritis synovial fibroblast
- SF:
-
Synovial fluid
- SD:
-
Standard deviation
- TF3:
-
Theaflavin-3,3′-digallate
- TNF-α:
-
Tumor necrosis factor-alpha
- UPR:
-
Unfolded protein responses
- VEGF:
-
Vascular endothelial growth factor
- 3-MA:
-
3-Methyladenine
- XBP-1:
-
X box-binding protein 1
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Acknowledgements
The work was supported by National Tea Research Foundation, ICMR Senior Research Fellowship programme (ID:3/1/2/10/Ortho/2019-NCD-I) Govt. of India, DST-Inspire fellowship programme, Department of Science and Technology, Govt. of India, ICMR Research Associate Programme, Govt. of India.
Funding
This work was carried out with the support of the funding agency National Tea Research Foundation (NTRF 174/2015).
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SM, AS, SM, PSM, AG, PC and MC initiated the concept and designed the experiments. SM, AB, DB, SC, SC, AS performed the experiments. SN assisted in confocal experiments. SM, AS, SM, AB, PSM and AG wrote the manuscript. AC extracted the synovial fluid from patients and assisted in manuscript preparation.
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This study was approved by the Institutional Ethics Committee (No: Inst/IEC/24.02.2014) dated 24th February, 2014.
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This study was conducted using the biological samples collected from selected patients visited our OPD. For every patient, prior written consent was taken before enrolling him/her in the study.
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Misra, S., Bagchi, A., Sarkar, A. et al. Methotrexate and theaflavin-3, 3′-digallate synergistically restore the balance between apoptosis and autophagy in synovial fibroblast of RA: an ex vivo approach with cultured human RA FLS. Inflammopharmacol 29, 1427–1442 (2021). https://doi.org/10.1007/s10787-021-00857-0
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DOI: https://doi.org/10.1007/s10787-021-00857-0