Paeoniflorin derivative ameliorates methotrexate resistance in treating rheumatoid arthritis through targeting GRK2-A2AAR axis

Introduction: Methotrexate (MTX), a cornerstone disease-modifying anti-rheumatic drug, exhibits an efficacy rate of approximately 45%-60% in both rheumatoid arthritis (RA) patients and relevant animal models. Elucidating the mechanisms underlying MTX non-responsiveness and developing effective strategies to ameliorate it are of significant clinical importance for advancing precision medicine in RA treatment.

Objectives: Our study aimed to elucidate the role of membrane GRK2 expression in the immunosuppressive effect of MTX mediated by the A_2AAR, and to demonstrate that the co-administration of paeoniflorin derivative effectively enhance the MTX response in the treatment of RA.

Methods: The expression of GRK2 and A_2AAR on peripheral blood lymphocytes (PBLs) from RA patients were evaluated, along with its correlation with disease activity. The association between the expression of these proteins and MTX efficacy was examined using immunofluorescence and correlation analysis. Collagen-induced arthritis (CIA) rats were treated with MTX and categorized into MTX-effective (MTX-E) and MTX-ineffective (MTX-N) groups based on their treatment response. The MTX-N rats subsequently received a combination therapy of MTX and a paeoniflorin derivative. Furthermore, by detecting GRK2 distribution on the cell membrane of PBLs in CIA rats, rats with high GRK2 expression (GRK2^High) were identified. These GRK2^High rats were then treated with either MTX alone or MTX combined with the paeoniflorin derivative, allowing for the observation of the combination therapy's efficacy.

Results: The study found a negative correlation between the membrane expression of GRK2 on PBLs and the efficacy of MTX, whereas a positive correlation was observed between the membrane expression of A_2AAR and MTX efficacy in both RA patients and CIA rats. Knocking down GRK2 decreased the IC₅₀ of MTX and promoted MTX-induced Apoptosis of Jurkat cells, while A_2AAR deficiency produced the opposite effect. Elevated GRK2 promotes ERK pathway activation, which inhibits MTX-induced Apoptosis and impairs its immunosuppressive function. In CIA rats that were unresponsive to MTX, additional treatment with a paeoniflorin derivative in combination with MTX increased the therapeutic response rate to 77.79%-88.89%. Furthermore, administering MTX alone to CIA rats with low GRK2 membrane expression on PBLs, or combining MTX with paeoniflorin derivative in those with high GRK2 membrane expression, both resulted in significantly improved therapeutic outcomes.

Conclusion: The excessive expression of membrane GRK2 impairs the membrane distribution of A_2AAR, which weakens the anti-inflammatory effects of MTX. Therefore, combining MTX with a paeoniflorin derivative may significantly enhance the therapeutic response in RA.

A(2A) adenosine receptor; Combination therapy; G protein-coupled receptor kinase 2; Methotrexate; Rheumatoid arthritis.