Paeonol protects against TNF-α-induced proliferation and cytokine release of rheumatoid arthritis fibroblast-like synoviocytes by upregulating FOXO3 through inhibition of miR-155 expression.

Abstract

Fibroblast-like synoviocytes (FLS) play an essential role in the pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis. Paeonol (Pae) is a phenolic compound found in many traditional Chinese medicine remedies. However, the effects of Pae on TNF-α-stimulated FLS and the underlying molecular mechanism are unknown. In this study, we aimed to investigate the anti-proliferative and anti-inflammatory effect of Pae against activated FLS. Rheumatoid arthritis FLS (RA-FLS) were pre-treated with different doses (25, 50, and 100µM) of Pae or miR-155 inhibitor for 30min or transfected with miR-155 mimic, and then treated with 50ng/mL of tumor necrosis factor alpha (TNF-α) for 1h. Cells that were untreated served as control. At 24h after drug pretreatment, the proliferation of FLS was detected using the MTT assay. The concentrations of interleukin IL-6 and IL-1β in cell culture supernatant were examined by enzyme-linked immunosorbent assay (ELISA), and mRNA levels of Foxo3 and miR-155 expression in FLS were quantified by reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions of forkhead box O3 (FOXO3), cyclin D1, and c-Myc were detected by Western Blot. TNF-α induced the proliferation of FLS, whereas Pae inhibited this proliferation in a dose-dependent manner. Pae attenuated TNF-α-induced production of IL-6 and IL-1β, and inhibited the expression of miR-155 in a dose-dependent manner. In addition, miR-155 inhibitor decreased TNF-α-induced proliferation of FLS, and attenuated TNF-α-induced production of IL-6 and IL-1β. In addition, pretreatment with different doses of Pae or miR-155 inhibitor markedly attenuated TNF-α-induced decrease in protein expression of FOXO3 in FLS. Mechanistic studies revealed FOXO3 as miR-155-5p direct target and inhibition of FOXO3 led to the abolishment of Pae protective effects. Paeonol protected against TNF-α-induced proliferation and cytokine release of FLS by decreasing the expression of miR-155 and upregulating its target FOXO3.