Suppressive Effects of Flavonoids on Macrophage-Associated Adipocyte Inflammation in a Differentiated Murine Preadipocyte 3T3-L1 Cells Co-Cultured with a Murine Macrophage RAW264.7 Cells

The suppressive effects of flavonoids on macrophage-associated adipocyte inflammation in a differentiated murine preadipocyte cell line (3T3-L1) co-cultured with a murine macrophage cell line (RAW264.7) were evaluated. Extracellular lipid accumulation was investigated via Oil Red O staining. The expression levels of adipogenesis- and inflammation-associated proteins, including CCAAT/enhancer-binding protein (C/EBP)-α, inducible nitric oxide synthase (iNOS), C/EBPβ, peroxisome proliferator-activated receptor γ (PPARγ), and cyclooxygenase-2 (COX-2), were determined via Western blotting. Proinflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1) and interleukin-6 (IL-6), were assessed using enzyme-linked immunosorbent assay kits. We found that silybin, formononetin, and diosmetin inhibited lipid accumulation and production of proinflammatory cytokines in the co-cultures of 3T3-L1 and RAW264.7 cells. Moreover, they inhibited the protein expression of PPARγ, C/EBPα, COX-2, C/EBPβ, and iNOS in the co-cultures of 3T3-L1 and RAW264.7 cells. These data support that silybin, formononetin, and diosmetin inhibit macrophage-associated adipocyte inflammation and lipid accumulation.


Introduction
Excessive fat accumulation is closely related to an increased hazard of various metabolic diseases, that is, type II diabetes, inflammation, heart disease, and hypertension [1]. Chronic inflammation in adipose tissue, characterized by the disturbed infiltration of macrophages and secretion of proinflammatory cytokines, is related to obesity [2]. The step of differentiation of preadipocytes into adipocytes starts with the exponential growth phase of adipoblasts [3]. Cells go through a limited number of cell divisions to enter cell cycle arrest and then re-enter the cell cycle. In the final stage, they differentiate into mature adipocytes [4]. Adipogenesis and adipocyte hypertrophy induces inflammatory signaling [5]. The macrophage-associated adipocyte inflammation is a risk factor for obesityrelated metabolic disorders, which are caused by the accumulation of macrophages in adipose tissue [6].
A polyphenolic flavonoid, silybin, was obtained from the milk thistle, Silybum marianum (L.). The pharmacological activities of silybin have been extensively studied. Its hepatoprotective effects are associated with antioxidant and anti-inflammatory effects [36]. The isoflavone formononetin, found in Astragalus membranaceus, exerts anti-obesity,
In this experiment, consistent with the inhibitory effects of silybin, formononetin, and diosmetin on lipid accumulation, silybin (100 µM), formononetin (50 and 100 µM), and diosmetin (50 and 100 µM) inhibited the levels of proinflammatory cytokines (MCP-1 and IL-6) and inducible enzymes (COX-2 and iNOS), which produce NO that causes inflammation in co-cultures of RAW264.7 and 3T3-L1 cells. It is assumed that 3T3-L1 cells participate in inflammatory crosstalk upon cell-cell contact with RAW264.7 macrophages. These inhibitory effects were also observed by increased levels of proinflammatory cytokines and inducible enzymes, as reported in previous studies [35,38,39]. These results indicate that silybin, formononetin, and diosmetin inhibit macrophage-associated adipocyte inflammation. In addition, silybin, formononetin, and diosmetin inhibited the protein expression levels of adipogenesis-associated proteins (C/EBPβ, C/EBPα, and PPARγ) in co-cultures of RAW264.7 and 3T3-L1 cells. Among PPARs, PPARγ is predominately expressed in adipose tissue and has been known as a critical modulator of adipogenesis. In the intracellular molecular pathways involved in adipogenesis, C/EBPβ activates the transcription of master regulators, such as PPARγ and C/EBPα, which together enhance lipid accumulation and adipogenesis [40]. Inhibition of C/EBPα by silybin was reported by Ka et al. [41], but its effects on PPARγ and C/EBPβ were not identified yet. The inhibitory effect of diosmetin on inflammation and lipolysis in the co-culture of adipocytes and macrophages was recently reported by Lee et al. [35], but the involvement of PPARγ was not identified. However, formononetin exerted PPARγ-driven reporter-gene activity and induced differentiation of 3T3-L1 preadipocytes [42]. This difference is thought to be due to the difference in the experimental conditions and concentration of the formononetin, which is different from our results.
All flavonoids have two six-membered rings and one three-carbon unit linked through the linear three-carbon chain [43,44]. The glycosides are usually attached at position 3 or 7, and the most common carbohydrates are arabinose, L-rhamnose, galactose, glucorhamnose, or D-glucose [45]. However, glycosides had little effect in comparison to the effects of the flavonoids used in this study. Therefore, the effect of aglycone without sugar was good, and the relationship with efficacy can be confirmed through structural comparison of aglycones. The other factors related to the various chemical properties of flavonoids include their hydroxylation patterns, methoxy groups, and conjugation between aromatic rings [45]. Diosmetin is an O-methylated flavone is also known as 5,7,3trihydroxy-4 -methoxyflavone. Apigenin is a trihydroxyflavone that is flavone substituted by hydroxy groups at positions 4 , 5 and 7. As a result of comparing diosmetine, which showed the best effect, and apigenin, which had a similar structure but had no effect, it was confirmed that 4 -methoxy was an important structure to exert the effect. Formononetin (7hydroxy-4 -methoxyisoflavone) is an O-methylated isoflavone. Formononetin was difficult to compare its effect because it did not have a similar structure among the tested compounds, Plants 2022, 11, 3552 6 of 9 but similarly to diosmetin, 4 -methoxy group seem to be important. However, the 7-hydoxyl group contained in both flavonoids cannot be ignored. It is necessary to further investigate which receptors these flavonoids act on.
In conclusion, our results revealed that silybin, formononetin, and diosmetin inhibited the inflammatory responses stimulated by the interaction of RAW264.7 and 3T3-L1 cells via downregulating the inflammatory biomarkers. These results can be used to develop potential strategies to alleviate and prevent obesity-related inflammatory disorders. However, further evidence for this result should be evaluated in animal models of obesity.

Cell Viability Assay
The cytotoxicity of 3T3-L1 preadipocytes and RAW264.7 cells was evaluated using an Ez-Cytox cell viability assay kit (Daeil Lab Service Co., Seoul, Republic of Korea). The two cell lines were seeded and incubated with flavonoids (100 µM) for 24 h. After treating the two cell lines with 10% (v/v) Ez-Cytox reagent at 37 • C for 1 h, the viability of the two cell lines was assessed via measuring the optical density (450 nm) using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

Oil Red O Staining
Differentiated 3T3-L1 adipocytes co-cultured with RAW264.7 cells were fixed with paraformaldehyde solution (4%) and stained with Oil Red O solution (0.3%) at room temperature for 1 h. After extraction with isopropanol (100%), the absorbance (490 nm) of the stained oil droplets (red color) was determined using a microplate reader (PowerWave XS).

Measurement of Proinflammatory Cytokine Levels
The levels of MCP-1 and IL-6 in the cell culture supernatant were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's protocol.