CP-25 combined with MTX/ LEF ameliorates the progression of adjuvant-induced arthritis by the inhibition on GRK2 translocation.

Graphical abstract Figure. Mechanism of CP-25 combined with MTX/LEF to ameliorate AA rats In the one hand, CP-25 alone and in combination blocked the activation of P38, ERK, and JNK. CP-25 combined with MTX/LEF exerts synergistic effects to inhibit inflammation of AA rats by inhibiting the MAPK pathway. In the other hand, CP-25 inhibited GRK2 translocation in AA rats which further restored the inhibition of GRK2 on p-ERK. CP-25 synergized with MTX/LEF through GRK2-induced inhibition of p-ERK, thereby suppressing inflammation in AA rats. Highlights • CP-25 combined with low-dose MTX/LEF showed equivalent therapeutic effects of high-dose MTX/LEF. • CP-25 decreased the expression of p-ERK through the inhibition on GRK2 membrane expression. • CP-25 has potential as a useful drug for the combined treatment of patients with RA. Abstract Objective CP-25 attenuates arthritis progression in animal models by inhibiting G protein-coupled receptor kinase 2 (GRK2) membrane expression. This study compared groups treated with high-dose methotrexate (MTX)/leflunomide (LEF) and CP-25 combined with low-dose MTX/LEF in an adjuvant-induced arthritis (AA) rat model and investigated possible mechanisms. Methods AA was induced in rats via complete Freund's adjuvant. Experimental groups were divided into a normal group; vehicle group; monotherapy groups, including CP-25 (50 mg/kg), MTX (0.25, 0.5 mg/kg), and LEF (5, 10 mg/kg); and CP-25 (50 mg/kg)-combined MTX (0.25 mg/kg)/LEF (5 mg/kg) groups. We measured cytokine levels, phosphorylation and protein expression, and interactions between proteins. The role of GRK2 on phosphorylated extracellular signal-regulated kinase (p-ERK) was determined via GRK2-siRNA using a high content imaging system. Results Therapeutic effects, including pathology and cytokine balance, were equivalent between the CP-25-combination groups and the high-dose MTX/LEF groups. P38, ERK, and c-Jun N-terminal kinase (JNK) activation in AA fibroblast-like synoviocytes (FLS) was reduced in the treatment groups; GRK2 expression was only inhibited in the CP-25 group. Interactions between GRK2 and p-ERK decreased in the vehicle group and were restored in the CP-25 group. GRK2 membrane expression and p-ERK nuclear expression increased in FLS pre-treated with tumour necrosis factor alpha and stimulated with prostaglandin E2. Nuclear expression of p-ERK increased in GRK2-siRNA FLS. Conclusion Equivalent therapeutic effects were observed between CP-25-combination groups and high-dose MTX/LEF groups. CP-25 inhibited p-ERK by reducing the membrane expression of GRK2 in FLS from AA rats. [ABSTRACT FROM AUTHOR]