Therapeutic role of aripiprazole in cartilage defects explored through a drug repurposing approach

Abstract

Articular cartilage has a limited regenerative capacity, resulting in poor spontaneous healing of damaged tissue. Despite various scientific efforts to enhance cartilage repair, no single method has yielded satisfactory results. With rising drug development costs, drug repositioning has emerged as a viable alternative. This study aimed to identify a drug capable of improving cartilage defects by analyzing chondrogenesis-related microarray data from the Gene Expression Omnibus (GEO) public database. We utilized datasets GSE69110, GSE107649, GSE111822, and GSE116173 to identify genes associated with cartilage differentiation, employing StringTie for differential gene expression analysis and extracting drug data from the Drug-Gene Interaction database. Additionally, we aimed to verify the cartilage regeneration potential of the identified drug through experiments using cellular and animal models. We evaluated the effects of aripiprazole on adipose-derived mesenchymal stem cells (ADMSCs) and chondrocytes using qRT-PCR and a 3D pellet culture system. In vivo, we assessed cartilage restoration by combining aripiprazole with a scaffold and implanting it into artificially induced cartilage defects in Sprague-Dawley rats. Subsequent mRNA sequencing provided insights into the mechanistic pathways involved. Our results showed that aripiprazole significantly increased mRNA expression of COL2A1 and SOX9, markers of chondrogenesis, and promoted chondrogenic condensation in vitro. Furthermore, aripiprazole effectively enhanced cartilage regeneration in the rat model. KEGG pathway and Gene Ontology Biological Processes (GOBP) analyses of the mRNA sequencing data revealed that aripiprazole upregulated genes related to ribosomes and cytoplasmic translation, thereby facilitating chondrogenesis. In conclusion, our findings suggest that aripiprazole is a promising candidate for improving damaged cartilage, offering a novel approach to cartilage regeneration.

Fig. 1

Differences in the expression of genes involved in chondrogenesis. Hierarchical clustering analysis of differentially expressed genes from GSE107649 (A), GSE116173 (B), GSE69110 (C), and GSE111822 (D).

Fig. 2

Functional screening of drugs promoting chondrogenic differentiation (A) First Screening of Cartilage Differentiation-Promoting Drugs: Adipose-derived mesenchymal stem cells (ADMSCs) were treated with 10 µM of each drug for 72 h. The expression levels of COL2A1 (top) and SOX9 (bottom) mRNA were assessed using qRT-PCR. Dotted lines indicate drugs that significantly increased SOX9 and COL2A1 expression compared to DMSO control. (B) Second Screening: The Venn diagram shows the overlap of drugs that increased COL2A1 and SOX9 expression. Bar graphs present relative mRNA expression levels with statistical significance indicated. The cartilage-differentiation effect of seven drugs selected from the first screening was re-evaluated by measuring changes in mRNA levels of COL2A1, SOX9, and aggrecan (ACAN).: *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3). (C) ADMSCs were cultured in pellet form and treated with DMSO, aripiprazole, or irinotecan for three weeks. Aripiprazole treatment showed increased cartilage differentiation compared to DMSO and irinotecan. (D) Hematoxylin and Eosin (H&E) staining and Alcian blue staining of pellet cultures. Aripiprazole treatment resulted in larger pellet size and more intense staining, indicating enhanced cartilage matrix production. Scale bars represent 100 μm.

Fig. 3

Restorative effect of aripiprazole in a rat model of articular cartilage defect. (A) Representative images of distal femur cartilage in the defect, DMSO, and aripiprazole groups taken at 8 weeks post-establishment of a cartilage defect model. The histology score and gross appearance were quantified and are presented as bar graphs. The histology score (left graph) and gross appearance (right graph) show significant improvement in the aripiprazole group compared to the defect and DMSO groups. Statistical significance is indicated as **p < 0.01, ***p < 0.001. (B) Histological analysis of cartilage defects in euthanized rats using H&E, Alcian blue, and safranin-O staining. The staining results demonstrate better cartilage regeneration and matrix formation in the aripiprazole-treated group compared to the defect and DMSO groups. Scale bars represent 100 μm.

Fig. 4

Gene expression changes in cartilage differentiation following aripiprazole treatment (A) KEGG pathway enrichment analysis of genes modulated in chondrocytes upon aripiprazole treatment. The pathways with significant enrichment are shown, with the horizontal axis representing fold enrichment and the size and color of the circles indicating the enrichment score (− log10(p-value)). (B) GO biological process (GOBP) enrichment analysis on upregulated and downregulated genes in chondrocytes treated with aripiprazole. The horizontal axis represents fold enrichment, and the size and color of the circles indicate the enrichment score (− log10(p-value)). Prominent biological processes include cytoplasmic translation, translation, tRNA processing, and ribosomal small subunit biogenesis.