Formononetin attenuates IL-1β-induced apoptosis and NF-κB activation in INS-1 cells

Abstract

Several studies suggest that the inflammation plays a role in the pathogenesis of some glucose disorders in adults. Exposure of pancreatic β-cells to cytokines, such as interleukin-1β (IL-1β), is thought to contribute to β-cell apoptosis. One important event triggered by IL-1β is induction of nitric oxide synthase (iNOS), an enzyme that catalyzes intracellular generation of the cytotoxic free radical NO. Recent work have suggested that formononetin, as an O-methylated isoflavone found in a number of plants and herbs like Astragalus membranaceus, inhibited some pro-inflammatory cytokine production in macrophages. However, the roles of formononetin in pancreatic beta cells have not been fully established. The aim of the present study was to assess possible in vitro effects of formononetin on cell apoptosis induced by IL-1β in the rat insulinoma cell line, INS-1. Our results demonstrate that formononetin significantly prevents IL-1β-increased INS-1 cell death and blocks cytokine-induced apoptotic signaling (the reduction of Bax/Bcl-2 ratio and caspase-3 activity). Formononetin also inhibited the activation of nuclear factor-kappaB (NF-κB), which is a significant transcription factor for iNOS, so as to decease nitric oxide (NO) formation in a dose dependent manner in vitro. Our observations indicated that formononetin could protect against pancreatic β-cell apoptosis caused by IL-1β and therefore could be used in the future as a new drug improving diabetes mellitus.

Figure 1

Formononetin inhibited IL-1β-induced apoptosis in INS-1cells. (A) Cells were treated with the indicated concentrations of formononetin and IL-1β for 24 h, analyzed by MTT assay. (B) Cells werestained with Annexin V-FITC and PI, analyzed by flow cytometry. Data are expressed as % of Annexin V-FITC-positiveand PI-negative cells (early stage of apoptosis). (C) Tunel staining of apoptotic cells, IL-1β (50ng/mL) and formononetin (1 μmol/L). Apoptotic cells were marked with green fluorescence, the nuclei of cells are stained by blue fluorescence. Values are the means ± SD (n = 3) of three individual experiments. * p < 0.05, ** p < 0.01 vs. ctr(DMSO); ^# p < 0.05, ^## p < 0.01 vs. group treated with IL-1β excluding formononetin.

Figure 2

The effect of formononetin on IL-1β-induced activation of NF-κB in INS-1cells. Cells were treated with the indicated concentrations of IL-1β and formononetin for 24h. (A) Formononetin dose-dependently reduced NF-κB activation in luciferase reporter assay. Values are the means ± SD (n = 3) of three individual experiments. * p < 0.05, ** p < 0.01 vs. ctr (DMSO); ^# p < 0.05, ^## p < 0.01 vs. group treated with IL-1β excluding formononetin. (B) Protein level of the p65 subunit of NF-κB, as determined by Western blot. (C) Representative immunofluorescence localization for p65 subunit. IL-1β increased NF-κB translocation from the cytoplasm to nucleus, IL-1β (50 ng/mL) and formononetin (1 μmol/L). All IL-1β-induced increases were reversed by formononetin.

Figure 3

The effect of formononetin on IL-1β-induced NO formation and apoptotic pathway in INS-1 cells. Cells were treated with the indicated concentrations of IL-1β and formononetin for 24h. (A) The level of NO in medium of INS-1 cells was determined using Griess method. Values are the means ± SD (n = 3) of three individual experiments. * p < 0.05, ** p < 0.01 vs. ctr (DMSO); ^# p < 0.05, ^## p < 0.01 vs. group treated with IL-1β excluding formononetin. (B) The expression of apoptotic genes were analyzed by western blot in INS-1 cells.