Background: Recently it has been established that osteoarthritis (OA) involves the whole joint with not only loss of cartilage, but also changes in the subchondral bone, synovium, tendons, ligaments and muscles. Current pharmacological treatment of OA is inadequate and guidelines for OA advocate non-pharmacologic treatments followed by pharmacological interventions which focus on symptom management but without effective treatment. Accumulation of senescent cells contributes to OA where chondrocyte senescence is thought to play a role in the initiation and progression of OA. Senescence is proposed as a potential therapeutic target for OA. Senotherapeutics are a class of compounds (senomorphics and senolytics) that are designed to eliminate or delay the adverse effects of cellular senescence. Our previous data showed that APPA, a combination of Apocynin (AP) and Paeonol (PA), has the capacity to modulate several parameters related with OA process in human chondrocytes including reduced expression of cartilage degradation enzymes

Objectives: Study the effect of APPA on cellular senescence using human articular chondrocytes.

Methods: Chondrocytes cell line TC/28a2 and chondrocytes isolated from human OA cartilage were used. APPA, AP and PA effect on cell viability was analyzed using MTT and DRAQ7TM. A combination between Etoposide (5µM or 20 µM respectively) and 10 ng/ml oncostatin –OSM- (Eto+OSM) was used to induce cellular senescence. The percentage of senescent cells was evaluated by measuring Fluorescein di-_-D-galactopyranoside (FDG) by flow cytometry. CDKN1A gene expression was analyzed by RT-PCR. The number of viable cells were evaluated using Hoechst stain and the number of nuclei counted. The susceptibility of cells to apoptosis was analyzed by flow cytometer using Annexin-V and Propidium Iodide (PI). Unpaired Mann Whitney test was used to evaluate differences between groups. Data are presented as the mean ± standard error mean (SEM). Statistical analyses were performed with GraphPad Prism version 8.

Results: Data obtained from the MTT experiments showed that higher concentration than 20 µg/mL for AP and 30 µg/mL for PA decreased the cell viability. IC50 values were calculated for AP (16.56 µg/mL) and for PA (55.63 µg/ml). The percentage of non viable cell was estimated using DRAQ7TM never was higher than 5.05%. In TC/28a2 cells, Eto+OSM increased the number of senescent cells determined by FDG positive flow cytometry (3.42±0.91 vs 8.56±1.62; p=0.03). Interestingly, APPA 10 µg/ml significantly reduced the number of senescent cells induced by Eto+OSM (1.144±0.07 vs 0.63±0.10; p= 0.017) and the presence of PA 7.7 µg/ml also reduce the same parameter (1.144±0.07 vs 0.86±0.07; p= 0.054) When the senotherapeutic index was evaluated analyzing the number of cells, data showed that Eto+OSM reduced the total number of cells but the combination of Eto+OSM with APPA 10 µg/ml increased the number of positive nuclei (0.99±3.17e-5 vs 1.54±0.23, p=0.062). The preliminary analysis of apoptotic cells data did not show any modulation of apoptosis in any of the conditions evaluated. These data suggest that APPA, at least in this chondrocyte cell line (TC/28a2) could have senomorphic effects.

These results were partially replicated in human articular OA chondrocytes, where APPA decreased the ß-galactosidase activity induced by Eto+OSM (0.99±2.7e-5 vs 0.89±0.04, p=0.028), however AP and PA did not modulate this parameter. CDKN1A gene expression was increased by Eto+OSM, an effect that was decreased by APPA 10 µg/ml (1.89±0.48 vs 0.90±0.07, p=0.026) and by PA 10 µg/ml (1.89±0.48 vs 0.86±1.72, p=0.015). The low proliferation capacity of OA chondrocytes from elderly patients conditioned the develop of the senotherapeutic index experiments in these cells.

Conclusion: APPA reduced the number of senescent cells and increased the number of cells without causing apoptosis, which indicates that APPA may be senomorphic. The data indicated that both components of APPA need to be present to fully obtain these effects.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Francisco J. Blanco AKLRD, Mercedes Fernandez-Moreno AKLRD, Tamara Hermida Gómez: None declared, Nicholas Larkins AKLRD, AKLRD, AKLRD, Alan Reynolds AKLRD, AKLRD, AKLRD