Brassinin, a phytoalexin in cruciferous vegetables, suppresses obesity‐induced inflammatory responses through the Nrf2‐HO‐1 signaling pathway in an adipocyte‐macrophage co‐culture system

Abstract

The aim of this study was to investigate the effect of brassinin (BR), a phytoalexin found in plants belonging to the Brassicaceae family, on the obesity‐induced inflammatory response and its molecular mechanism in co‐culture of 3T3‐L1 adipocytes and RAW264.7 macrophages. BR effectively suppressed lipid accumulation by down‐regulating the expression of adipogenic factors, which in turn, were regulated by early adipogenic factors such as CCAAT‐enhancer‐binding protein‐β and Kruppel‐like factor 2. Production of inflammatory cytokines and reactive oxygen species, induced by adipocyte‐conditioned medium, was significantly decreased in BR‐treated cells. This effect of BR was more prominent in contact co‐culture of adipocytes and macrophages with a 90% and 34% reduction in IL‐6 and MCP‐1 levels, respectively. BR also restored adiponectin expression, which was significantly reduced by culturing adipocytes in macrophage‐conditioned medium. In the transwell system, BR increased the protein levels of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target molecule, hemoxygenase‐1 (HO‐1), by 55%–93% and 45%–48%, respectively, and also increased Nrf2 translocation into the nucleus. However, knockdown of Nrf2 or HO‐1 in RAW264.7 cells restored this BR‐mediated inhibition of IL‐6 and MCP‐1 production. These results indicated that BR inhibited obesity‐induced inflammation via the Nrf2‐HO‐1 pathway.

Mechanisms Underlying Biological Effects of Cruciferous Glucosinolate-Derived Isothiocyanates/Indoles: A Focus on Metabolic Syndrome

“…Glucoraphanin supplementation had no effect in HFD fed Nrf2 knockout mice, suggesting that the anti-obesity effects of glucoraphanin (SFN) are due to the activation of the Nrf2 pathway. Similarly, using an adipocyte (3T3-L1)-macrophage (RAW264.7) co-culture system, Kang et al described that brassinin (an indole) suppressed inflammation through Nrf2-HO-1 signaling pathway activation ( 260 ).…”

Using Immune Cell/Adipocyte Co-Culture Models to Identify Inflammatory Paracrine Signaling Mechanisms: A Process Attenuated by Long-Chain N-3 Polyunsaturated Fatty Acids

“…The adipocyte -immune cell (either macrophages, CD8 + or CD4 + T cells) co-culture model we developed [27][28][29]31,32,[37][38][39], utilizes the murine 3T3-L1 preadipocyte cell line, which requires differentiation into lipid-laden mature adipocytes prior to co-culture with immune cells and provides a standardized component of the model between co-culture studies that is combined with varying immune cell populations [1]. To recapitulate a critical feature of the obese phenotype and provide an inflammatory stimulus, co-cultures are stimulated with a physiologically relevant dose of lipopolysaccharide (LPS, 10 ng/mL) derived from Escherichia coli serotype 055:B5, which reproduces the level of endotoxin units reported in obese humans and rodents (5-6 endotoxin units/mL) [40][41][42], that is not utilized in other adipocyte-immune cell co-culture models [43][44][45][46][47]. Moreover, we have conducted studies involving the pre-stimulation of adipocytes with LPS for 24 hr to recapitulate already inflamed AT prior to co-culture with either macrophages [27,28] or CD8 + T cells [31], as this would be the AT microenvironment that newly infiltrating immune cells would encounter in vivo [2,9].…”

“…Moreover, we have conducted studies involving the pre-stimulation of adipocytes with LPS for 24 hr to recapitulate already inflamed AT prior to co-culture with either macrophages [27,28] or CD8 + T cells [31], as this would be the AT microenvironment that newly infiltrating immune cells would encounter in vivo [2,9]. Our initial co-culture work utilized the RAW264.7 macrophage cell line [38], which is commonly employed by other groups in adipocyte co-cultures treated with dietary bioactives [43][44][45][46][47]. More recently, we have utilized mouse primary splenic CD11b + macrophages in co-culture with 3T3-L1 mature adipocytes [28], which increases the translational potential of this model compared to studies using primary splenocytes (comprised of multiple undefined cell types) [48], or immortalized cell lines (e.g.…”

“…More recently, we have utilized mouse primary splenic CD11b + macrophages in co-culture with 3T3-L1 mature adipocytes [28], which increases the translational potential of this model compared to studies using primary splenocytes (comprised of multiple undefined cell types) [48], or immortalized cell lines (e.g. RAW264.7 macrophages) [43][44][45][46][47]. In this connection, our co-culture work with CD8 + and CD4 + T cells has exclusively utilized primary cells purified from the spleen of both lean and obese mice [29,31,32,37,39].…”

Murine in vitro cellular models to better understand adipogenesis and its potential applications

“…This 3T3-L1 co-culture system was also used to screen plant-derived components for biological activity. For example, Brassinin (BR) was recognized for inhibition of obesityinduced inflammation via Nrf2-HO-1 signaling pathway (Kang, Kim, Hwang et al, 2019). The 3T3-L1 cell line was also used to further evaluate the effects of saponin fraction from red ginseng on treatment of obesity through a co-culture system (Kim, Kang, Suh et al, 2018).…”