Hydroxychloroquine inhibits the PI3K/AKT pathway in synovial fibroblasts of rheumatoid arthritis and alleviates collagen-induced arthritis in mice.

Abstract

Objectives: To investigate the effects of hydroxychloroquine (HCQ) in rheumatoid arthritis (RA), particularly on fibroblast-like synoviocytes (FLS).

Methods: We analysed the R (-) and S (+) enantiomers of HCQ. Cell viability and proliferation inhibition were quantified using CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays, respectively. Flow cytometry was employed to analyse cell cycle arrest and apoptosis induction. Transwell assays were conducted to evaluate cellular motility. Molecular docking simulations with key targets predicted binding interactions. The therapeutic efficacy was validated in a collagen-induced arthritis (CIA) mouse model.

Results: The study finds that these enantiomers in a racemic combination, known as Rac-HCQ, are potent in arresting the growth of RA-FLS by inhibiting cell migration and invasion through downregulation of vimentin expression, inducing apoptosis via increased bax expression, and promoting cell cycle arrest by suppressing CDK1, cyclins A2, and B1. In vivo experiments showed that Rac-HCQ significantly reduced symptom severity in collagen-induced arthritis (CIA) mice. RNA sequencing suggests that Rac-HCQ alters the disease pathway in RA-FLS by blocking the PI3K/AKT pathway.

Conclusions: Our molecular docking studies indicate KIT, SRC, and PIK3A may potentially serve as the targets of Rac-HCQ in RA. These findings reveal the potential of Rac-HCQ to modulate the functions of RA-FLS and mitigate CIA manifestations, principally through its interaction with the pathway PI3K/AKT.