Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

EFFECT OF STAT3 SIGNALLING PATHWAY MODULATOR ON SENESCENCE OF NUCLEUS PULPOSUS CELLS   (#170)

Jolita Pachaleva 1 , Ilona Uzieliene 1 , Gailute Kirdaite 2 , Raminta Vaiciuleviciute 1 , Gunaras Terbetas 3 4 , Denis Evseenko 5 6 , Eiva Bernotiene 1 7
  1. Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania
  2. Department of Personalised medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania
  3. Department of Neurosurgery, Republican Vilnius University Hospital, Vilnius, Lithuania
  4. Clinic of Neurology and Neurosurgery, Faculty of Medicine, Vilnius University, Vilnius, Lithuania
  5. Department of Orthopaedic Surgery, Keck School of Medicine of USC, University of Southern California (USC), Los Angeles, USA
  6. Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of USC, University of Southern California (USC), Los Angeles, USA
  7. VilniusTech, Faculty of Fundamental Sciences, Vilnius, Lithuania

Introduction: Intervertebral disc degeneration (IDD) is one of the main causes of low back pain and the development of effective regenerative technologies for IDD repair remains a challenge [1]. Intervertebral disc (IVD) cells produce large amounts of cytokines such as interleukin IL-6, which is a classic homeostatic cytokine closely related to nucleus pulposus (NP) degeneration [2]. IL-6 family members modulate the signal transducer and activator of transcription 3 (STAT3) signalling pathway by binding to its receptor (IL-6R) and subunit (gp130). A novel small-molecule regulator of cartilage growth and differentiation (RCGD423) has been shown to suppress cartilage tissue degeneration via STAT3 modulation [3]. As cartilage tissue and IVD NP share a similar structure, we hypothesised that the application of STAT3 modulator in IVD cells will lead to the development of an effective IDD progression prevention method.

The aim of this study was to evaluate the effects of RCGD423 on the senescence and expression of cytokines in human NP cells. 

Methods: This study was performed in compliance with all the bioethical requirements. NP cells were isolated from human IVD samples (grade I degeneration, Pfirrmann). IVD cells were treated with 10 µM RCGD 423 w/wo 10 ng/ml IL-6+sIL-6R. Untreated cells were used as controls. Measurements were performed 72 h after treatment with RCGD 423, IL-6+sIL-6R. To determine the levels of iCa2+, cells were stained with the calcium-specific fluorescent dye Cal-520 and measured using a flow cytometer. Cell senescence was measured using a β-Galactosidase assay. The Seahorse XF-HS mini was used to evaluate the effect of RCGD423 w/wo IL-6 on cell metabolic activity. ProcartaPlex panel was used to analyse the levels of cytokines, chemokines, and growth factors. 

Results: To induce senescence in IVD cells, stimulation with IL-6 was used, and senescence in NP cells was determined by evaluation of β-Galactosidase activity by flow cytometry. The addition of RCGD423 reduced NP cell senescence, as evidenced by significantly lower SA-β levels, as compared to IL-6 stimulated cells and control. Metabolic activity measurement in NP cells using glycolysis stress test showed increased glycolysis in NP cells after stimulation with IL-6, while RCGD423 counteracted these effects and resulted in downregulation of the elevated glycolysis levels. In addition, RCGD423 reduced iCa2+ levels upregulated by stimulation with IL-6. RCGD423 was also found to modulate the levels of chemokines, cytokines and growth factors, including CXCL1, IL-6, LIF (leukemia inhibitory factor), vascular endothelial growth factor (VEGF) and others.

Discussion: Previous study on the effect of STAT3 modulator on chondrocytes from osteoarthritis patients showed promising results: RCGD 423 reduced hypertrophy and degeneration of articular cartilage chondrocytes and improved chondrocyte proliferation in vitro and promoted regeneration of cartilage in vivo [3]. Results of our study are in line with this positive effect for NP cells, as the RCGD423 significantly reduced IL-6 mediated cell senescence, inflammation and down-regulated iCa2+ level. The similarities of IVD with articular cartilage and the obtained results suggest that RCGD423 may appear as a promising therapeutic tool for the treatment of IDD.

Acknowledgements: funded by Lithuanian Research Council Nr.P-MIP-23-464 (Agreement Nr.S-MIP-23-138).

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