Hormetic effect of fisetin against oxidative stress in healthy rat chondrocytes

Abstract

Introduction

Oxidative stress and chondrocyte senescence contribute to cartilage degradation and to joint diseases such as osteoarthritis, for which no treatment halts progression. Fisetin, a flavonoid with antioxidant and senolytic properties, may modulate the chondrocyte response to oxidative damage; however, its safety profile and its effect on the chondrocyte phenotype in healthy cells remain poorly characterized.

Objective

To evaluate the antioxidant effect of fisetin on healthy young-rat chondrocytes under oxidative stress, and its impact on viability and the chondrogenic phenotype.

Methods

Primary chondrocytes from young rats were used. The cultured phenotype was characterized by immunofluorescence for aggrecan (ACAN), type II collagen (COL2), and LC3-II. Chondrogenic gene expression (SOX9, RUNX2, COMP, ACAN, COL2A1, COL1A1, COL10A1) was quantified by SYBR Green qPCR, normalized to the reference gene RPL27, across three passages (P0, P1, P2) and after 24 h of stimulation with fisetin (1–80 µM). Viability was determined by the alamarBlue assay at 24 and 48 h, and total ROS by flow cytometry after exposure to tert-butyl hydroperoxide (tBHP, 50 µM) with or without fisetin. Viability, ROS, and gene expression were analyzed by the Kruskal-Wallis test with Dunn post hoc (Holm correction); the ROS dose-response was additionally fitted with a non-linear model.

Results

Immunofluorescence confirmed the chondrocyte phenotype (ACAN- and COL2-positive) with LC3-II, indicating basal autophagic activity. Fisetin was not cytotoxic: viability remained at 88–102 % of control at 24 and 48 h, and only the 80 µM dose reduced it significantly (Dunn p<0.001), whereas doses up to 50 µM did not differ from control. By qPCR, significant differences were shown for SOX9 (p=0.010) and COMP (p=0.006); fisetin at 1 and 10 µM increased COMP expression relative to tBHP (Dunn p=0.016), while the remaining chondrogenic genes were unchanged; across passages, only COL1 increased (p=0.011), consistent with early dedifferentiation. tBHP raised total ROS 3.5-fold over basal (Dunn p=0.001); fisetin reversed this effect in a hormetic, inversely dose-dependent manner. Low doses (1–10 µM) significantly lowered ROS relative to tBHP (Dunn p<0.05) and normalized them to basal levels (fold 0.78–1.05), whereas high doses (25–80 µM) did not differ from tBHP. The dose-response fit confirmed this biphasic pattern (R²≈0.88), with maximal effect between 1 and 10 µM.

Conclusion

Fisetin exerts a cytoprotective and antioxidant effect on healthy rat chondrocytes with a hormetic profile, in which low doses are more effective at containing oxidative damage without compromising viability or the chondrocyte phenotype. These preliminary findings support its potential as a chondroprotective agent and provide a basis for its evaluation in osteoarthritis models, with a view to preserving joint function and preventing musculoskeletal disability.