Melittin enhances apoptosis through suppression of IL-6/sIL-6R complex-induced NF-kappa B and STAT3 activation and Bcl-2 expression for human fibroblast-like synoviocytes in rheumatoid arthritis

Abstract

Objective: Resistance to apoptosis of fibroblast-like synoviocytes (FLS) is considered as a major characteristic in RA. This study was designed to identify whether melittin has a pro-apoptotic effect in IL-6/sIL6R-stimulated human FLS by investigating the expression of mitochondrial apoptosis-related genes, nuclear factor-kappa B (NF-kappa B), and signal transducer and activators of transcription (STAT) activation. Methods: Cell viability was determined using a MTT assay after melittin treatment. Expressions of STAT3 and mitochondrial apoptosis-related genes induced by the IL-6/sIL-6R complex were determined by real time-polymerase chain reaction and western blotting. The expression of NF-kappa B p65 following IL-6 stimulation was determined by western blot analysis. The effects of melittin on the expression of apoptosis-related genes and the transcription factors NF-kappa B p65 and STAT3 were assessed in FLS. Apoptosis of FLS was determined by TUNEL-labeling to detect DNA strand breaks and DNA fragmentation assays. Caspase-3 activity was determined by a colorimetric assay. Results: IL-6/sIL-6R induced the activation of the transcription factors, STAT3, NF-kappa B p65 (nucleus), and Bcl-2. Melittin increased the expression of pro-apoptosis-related molecules, namely caspase-3, caspase-9, Apaf-1, and cytosolic cytochrome c, in a dose-dependent manner after treatment with IL-6/sIL-6R. Melittin inhibited STAT3 activation, translocation of NF-kappa B p65 into the nucleus, and expression of anti-apoptotic genes such as Bcl-2 and mitochondrial cytochrome c. Conclusions: The pro-apoptotic effects of melittin likely result from inhibition of the activation of the transcription factors, STAT3 and NF-kappa B p65, and regulation of mitochondrial apoptosis-related genes. Melittin is thus a promising therapeutic option for RA as it induces apoptosis in apoptosis-resistant synoviocytes.