Zerumbone Suppresses High Glucose‐ and Lipopolysaccharide‐Induced Pro‐inflammatory Effects through Regulation of Histone Deacetylase in Differentiated Human THP‐1 Monocytes

Abstract

Hyperglycemia contributes to diabetes and leads to several diabetic complications. Moreover, hyperglycemia plays a primary role in inducing atherosclerosis in patients with diabetes. Inflammation is thought to play a crucial role in atherosclerosis. Since previous studies suggested that persistent low‐grade infection by bacteria is associated with increased atherosclerosis among diabetic patients, we hypothesized that macrophages may respond to lipopolysaccharide (LPS) challenge in a more exaggerated manner under hyperglycemic conditions than under normal glucose conditions. Zerumbone is a major compound isolated from Zingiber zerumbet, which has been reported to exert anti‐inflammatory, anti‐ulcer, and antihyperglycemic effects. However, the specific mechanisms through which zerumbone exerts its anti‐inflammatory effects are not fully understood. The aim of this study was to determine whether zerumbone treatment suppressed pro‐inflammatory cytokine secretion through the nuclear factor (NF)‐κB signaling pathway by altering the histone acetylation/histone deacetylation balance under high‐glucose conditions in differentiated THP‐1 cells in response to LPS. THP‐1 cells were differentiated into macrophages by treatment with phorbol 12‐myristate 13‐acetate (PMA, 1 μM) for 48 h. Differentiated THP‐1 cells were cultured under normoglycemic (NG, 5.5 mM glucose) or hyperglycemic (HG, 25 mM glucose) conditions in the absence or presence of zerumbone (5–50 μM) for 48 h and then treated with 100 ng/mL LPS 6 h prior to harvesting. In this study, under in vitro conditions mimicking diabetes complications, the release of inflammatory cytokines and expression of genes encoding interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α were increased compared with those under NG conditions. Western blotting, reverse transcription polymerase chain reaction (RT‐PCR), and enzyme‐linked immunosorbent assays (ELISA) showed that zerumbone treatment suppressed the expression and activity of inflammatory markers in high glucose‐ and LPS‐treated differentiated THP‐1 cells. Moreover, western blotting and RT‐PCR also showed that zerumbone inhibited NF‐κB expression in high glucose‐ and LPS‐treated differentiated THP‐1 cells. We further observed that increased NF‐κB and acetyl‐NF‐κB levels in high glucose‐ and LPS‐treated differentiated THP‐1 cells were downregulated by zerumbone treatment through immunofluorescence. Histone deacetylases (HDACs) have been found to play important roles in the regulation of several inflammatory and diabetic target genes by removing an acetyl group from lysine residues of target proteins. Zerumbone restored histone acetylase (HAT) and HDAC activities, similar to NG conditions, in high glucose‐ and LPS‐treated differentiated THP‐1 cells. We examined HDAC3, SIRT3 and SIRT6 expresson in differentiated THP‐1 cells using western blotting. Although high glucose and LPS conditions induced SIRT3, SIRT6, and HDAC3, our results showed that zerumbone suppressed SIRT3, SIRT6, and HDAC3. Hence, we assumed that SIRT3, SIRT6, and HDAC3 could be potent HDAC targets in the context of inflammation, infection, diabetes, and diabetic complications. Analysis of post‐transcriptional histone modifications showed that high glucose and LPS increased H3K9ac levels, whereas zerumbone suppressed H3K9ac levels. Based on these findings, zerumbone may be a potential target for the treatment and prevention of diabetes and its complications.