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

Misra, Sanchaita; Bagchi, Aniruddha; Sarkar, Avik; Niyogi, Sougata; Bhattacharjee, Dipanjan; Chatterjee, Sulagna; Mondal, Sumantro; Chattopadhyay, Arghya; Saha, Ayindrila; Chatterjee, Sudipta; Sinhamahapatra, Pradyot; Chakrabarti, Partha; Chatterjee, Mitali; Ghosh, Alakendu

doi:10.1007/s10787-021-00857-0

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

Original Article
Published: 05 August 2021

Volume 29, pages 1427–1442, (2021)
Cite this article

Inflammopharmacology Aims and scope Submit manuscript

Sanchaita Misra¹,
Aniruddha Bagchi²,
Avik Sarkar¹,
Sougata Niyogi³,
Dipanjan Bhattacharjee¹,
Sulagna Chatterjee¹,
Sumantro Mondal¹,
Arghya Chattopadhyay¹,
Ayindrila Saha¹,
Sudipta Chatterjee¹,
Pradyot Sinhamahapatra¹,
Partha Chakrabarti³,
Mitali Chatterjee² &
…
Alakendu Ghosh ORCID: orcid.org/0000-0003-2956-9703¹

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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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IL-17-mediated mitochondrial dysfunction impairs apoptosis in rheumatoid arthritis synovial fibroblasts through activation of autophagy

Article Open access 19 January 2017

Dimethyl Fumarate Inhibits Fibroblast Like Synoviocytes-mediated Inflammation and Joint Destruction in Rheumatoid Arthritis

Article 17 October 2022

Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs

Article Open access 29 January 2019

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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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Authors and Affiliations

Department of Clinical Immunology and Rheumatology, Institute of Postgraduate Medical Education and Research, SSKM Hospital, 244 B, Acharya J C Bose Road, Kolkata, 700020, India
Sanchaita Misra, Avik Sarkar, Dipanjan Bhattacharjee, Sulagna Chatterjee, Sumantro Mondal, Arghya Chattopadhyay, Ayindrila Saha, Sudipta Chatterjee, Pradyot Sinhamahapatra & Alakendu Ghosh
Department of Pharmacology, Institute of Postgraduate Medical Education and Research, SSKM Hospital, Kolkata, India
Aniruddha Bagchi & Mitali Chatterjee
Division of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, Kolkata, India
Sougata Niyogi & Partha Chakrabarti

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Sanchaita Misra
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Contributions

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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Correspondence to Alakendu Ghosh.

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This study was approved by the Institutional Ethics Committee (No: Inst/IEC/24.02.2014) dated 24^th 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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Received: 27 February 2021
Accepted: 26 July 2021
Published: 05 August 2021
Issue Date: October 2021
DOI: https://doi.org/10.1007/s10787-021-00857-0

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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

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IL-17-mediated mitochondrial dysfunction impairs apoptosis in rheumatoid arthritis synovial fibroblasts through activation of autophagy

Dimethyl Fumarate Inhibits Fibroblast Like Synoviocytes-mediated Inflammation and Joint Destruction in Rheumatoid Arthritis

Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs

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