Background: Osteoarthritis (OA) is a unmet medical need with no disease-modifying drugs (DMOAD) available. Recent findings from our group pointed towards Peroxisome Proliferator-Activated Receptor α (PPARα) as a potential DMOAD target mechanism for knee OA, through its activation with Fenofibrate (FN), a PPARα agonist commonly used for dyslipidemias treatment in humans. In addition, levels of PPARα were reduced in patients with OA and the treatment with FN improved OA clinical conditions of subjects with knee OA [1]. This drug excelled over other candidates due to its senolytic and pro-autophagic properties which conferred protection by reducing pain and structural changes in a post-traumatic OA model in mice after an intra-articular injection FN by extended-release during 3 months. Besides these positive outcomes, the molecular mechanism in which PPARα is involved on joint health and disease remains unclear. Interestingly, PPARα plays an important role in the regulation of circadian rhythm (CR) and metabolism, although it has not been explored in the context of joint homeostasis. Previous evidences support CR being one of the most dysregulated pathway in human OA cartilage [2].

Objectives: Here, we investigate the potential relevance of PPARα on circadian rhythm to guide the molecular insights of the future DMOADs in development for OA treatment.

Methods: The genetic deletion of PPARα was performed in human chondrocytes T/C28a2 by knocking-down PPARα by using a small-interference RNA delivery with Lipofectamine RNAiMAX in presence or absence of interleukin 1β (IL1β, 5 ng/ml). We induced a circadian synchronization 48h after transfection by a short term exposure to Dexamethasone (1h, 100 nM), a potent synthetic glucocorticoid, allowing the expression levels to align their phases, for monitoring the periodic rhythm of cells. After 24h period post-synchronization, cells were harvested at 4-hours intervals from 0 to 24 hours or at 12-hours intervals from 0 to 72 hours to evaluate effects at shorter and longer period, respectively. To investigate the consequences of PPARα absence to the circadian rhythmicity, core circadian clock components, macroautophagy and senescence markers were analyzed by using Taqman probes technology and Western Blot (WB) analysis, respectively. In addition, to identify genomic alterations that were dependent uniquely on PPARα loss of function, we performed a transcriptomic analysis by mRNAseq at 4 different timepoints (0, 8, 16 and 24 hours). An enrichment analysis was conducted as well as an assessment of CR to the genes by computational algorithms related to CR, such as JTK-Cycle, RAIN and Cosinor Fit Analysis.

Results: Knockdown of PPARα was efficient up to 96h post synchronization with Dexamethasone. Diminished PPARα notably disrupts the circadian rhythm of the chondrocytes by altering the rhythmicity of core clock components BMAL1 and CLOCK either at baseline or upon stress-induced by IL1β in chondrocytes with PPARα knock-down, suggesting a role of PPARα on the regulation of CR. Moreover, a significant reduction in these two CR proteins was detected by WB (q-value<0.05), suggesting that BMAL1 and CLOCK might be interesting checkpoints in OA. The functional consequences of CR dysregulation by PPARα loss of function in human chondrocytes at baseline were associated with decreased LC3II expression and increased phosphorylation of rpS6 (q-value<0,0005), which indicates a disabled macroautophagy, as well as, with cellular senescence by increasing p21 expression, suggesting that overall, PPARα is essential for maintaining chondrocyte homeostasis. Moreover, under stress-induced pro-inflammatory environment, same tendencies were observed, but the most remarkable feature was the complete abolition of periodic expression patterns which was a clear signal of the strong influence of OA-like environment has on CR. Interestingly, the mRNAseq analysis of PPARα Knock-down detected 193 differentially expressed genes (DEG) (96 genes upregulated and 97 genes downregulated) based on adjusted p-value (<0,05) and a fold change >1. From this list, we selected a top candidate list of 11 based on enrichment analysis which are closely linking PPARα molecular function to specific key pathways such as Wnt signaling, CR, HedgeHog, Metabolism, and Homeostasis. Further validation is ongoing to demonstrate the relevance of these molecular pathways in relation to the disease.

Conclusion: These findings provide a novel approach to study molecular function of PPARα in the context of CR, which are intended to 1) explain the implication of PPARα on the disease, and 2) understand the molecular basis of the beneficial effect of PPARα activation.

REFERENCES: [1] Nogueira-Recalde U, et al. Fibrates as drugs with senolytic and autophagic activity for osteoarthritis therapy. EBioMedicine. 2019;45:588-605.

[2] Fisch KM, Gamini R, et al. Identification of transcription factors responsible for dysregulated networks in human osteoarthritis cartilage by global gene expression analysis. Osteoarthritis Cartilage. 2018;26:1531-1538.

Acknowledgements: PI20/00643, Instituto de Salud Carlos III, Spain.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.