Cilostazol enhances apoptosis of synovial cells from rheumatoid arthritis patients with inhibition of cytokine formation via Nrf2-linked heme oxygenase 1 induction.

OBJECTIVE

To assess the effects of cilostazol in inhibiting proliferation and enhancing apoptosis in synovial cells from patients with rheumatoid arthritis (RA).

METHODS

Synovial cell proliferation was measured by MTT assay. The expression of NF-kappaB, IkappaBalpha, Bcl-2, Bax, heme oxygenase 1 (HO-1), and Nrf2 was determined by Western blotting.

RESULTS

Cilostazol suppressed synovial cell proliferation by arresting the G(2)/M phases of the cell cycle, and this was reversed by KT5720, an inhibitor of protein kinase A. Cilostazol increased the number of TUNEL-positive cells, with increased cytochrome c release and apoptosis-inducing factor translocation as well as increased caspase 3 activation. Cilostazol (10 microM) and cobalt protoporphyrin IX (CoPP) increased HO-1 messenger RNA and protein expression. These effects were suppressed by zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor. Cilostazol and CoPP significantly increased IkappaBalpha in the cytosol and decreased NF-kappaB p65 expression in the nucleus. Increased expression of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 induced by lipopolysaccharide was attenuated by cilostazol and CoPP, and this was reversed by ZnPP. In mice with collagen-induced arthritis treated with cilostazol (10 and 30 mg/kg/day), paw thickness was decreased with increased apoptotic cells in the joints. In synovial cells transfected with small interfering RNA (siRNA) targeting HO-1, cilostazol did not suppress expression of TNFalpha, IL-1beta, and IL-6, in contrast to findings with negative control cells. Cilostazol- and CoPP-induced HO-1 expression was diminished in cells transfected with Nrf2 siRNA.

CONCLUSION

Cilostazol suppressed proliferation of synovial cells from RA patients by enhancing apoptosis, and also inhibited cytokine production via mediation of cAMP-dependent protein kinase activation-coupled Nrf2-linked HO-1 expression.