Paeonol, an Ingredient of Kamishoyosan, Reduces Intracellular Lipid Accumulation by Inhibiting Glucocorticoid Receptor Activity in 3T3-L1 Cells

Abstract

Excessive triglyceride accumulation in lipid-metabolizing tissues is associated with an increased risk of a variety of metabolic diseases. Kamishoyosan (KSS) is a Kampo composed of 10 constituent herbs, and contains moutan cortex (MC) and paeonol (PN) as the major ingredient of MC. Here, we demonstrate the molecular mechanism underlying the effect of KSS on the differentiation of mouse preadipocytes (3T3-L1 cells). KSS inhibited the accumulation of triglycerides in a dose-dependent manner in 3T3-L1 cells that were induced to differentiate into adipocytes. We also found that MC and PN were responsible for the anti-adipogenetic effect of KSS and significantly suppressed the expression of CCAAT/enhancer-binding proteins-δ (C/EBP-δ) mRNA 3 days after the induction of differentiation. Thus, PN may contribute to the anti-adipogenetic property of MC in 3T3-L1 cells. In addition, PN inhibited dexamethasone (Dex)-induced glucocorticoid receptor (GR) promoter activity. Taken together, these results suggest that PN suppresses C/EBP-δ expression by inhibiting Dex-induced GR promoter activity at the early stage of differentiation and, consequently, delays differentiation into mature adipocytes. Our results suggest that the habitual intake of Kampo-containing PN contributes to the prevention of the onset of metabolic diseases by decreasing the excessive accumulation of triglycerides in lipid-metabolizing tissues.

Keywords: C/EBP-δ; Kamishoyosan; Kampo medicine; Moutan cortex; adipocyte; glucocorticoid receptor; paeonol.

Figure 1

Kamishoyosan (KSS) reduces lipid accumulation. (A) Induction time course of 3T3-L1 cell differentiation. 3T3-L1 cells were seeded on a 24-well plate at 2 × 10⁴ cells per well and cultured for 3 days. After confluent (0 day), 1 μM dexamethasone (Dex; D), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; M), and 10 μg/mL insulin (I) were added to induce differentiation for 3 days followed by additional 2 days culture with 5 μg/mL insulin (day 3–5). Thereafter, the medium was changed to normal Dulbecco’s modified Eagle’s medium (DMEM) every 2 days (day 5–8). Kampo medicines were administered at a concentration of 1 mg/mL after the onset of differentiation (day 0–8). (B) Images of Oil-Red-O staining of 3T3-L1 cells on day 8 are shown. (Ba) None (no DMI differentiation induction). (Bb–f) control and Kampo medicine administration groups ((Bb); H₂O (Bc); KSS, (Bd); Hochuekkito (HET), (Be); Shoseiryuto (SST), (Bf); Goreisan (GRS) with DMI differentiation induction). The scale bar indicates 200 μm. (C) KSS suppresses lipid accumulation. (Ca) Oil-Red-O dye was extracted with isopropanol to measure intracellular lipid accumulation, and the absorbance was measured at 490 nm as compared with H₂O (with DMI differentiation induction). (Cb) Cell viability after 8 days of culture was evaluated with the CCK-8 assay. Absorbance measured at 450 nm is shown as relative absorbance to H₂O (with DMI differentiation induction). Data are shown as the mean ± standard deviation (n = 3). * p < 0.05 vs. H₂O (with DMI differentiation induction).

Figure 2

KSS reduces lipid accumulation in a dose-dependent manner. (A) KSS inhibited lipid accumulation in 3T3-L1 cells induced by the DMI method in a dose-dependent manner. Oil-Red-O staining images of cells cultured for 8 days is shown. (Aa) KSS 0 mg/mL, (Ab) 0.1 mg/mL, (Ac) 1 mg/mL, (Ad) 2 mg/mL, (Ae) 5 mg/mL, and (Af) 10 mg/mL. The scale bar indicates 200 μm. (B) KSS inhibited the lipid accumulation in 3T3-L1 cells cultured for 8 days in a dose-dependent manner. As in Figure 1, (Ba) the amount in lipid accumulation and (Bb) the cell viability was measured. (C) KSS suppressed lipid accumulation after 21 days of administration. (Ca,b) Oil-Red-O staining images of cultured cells are shown. (Ca) KSS (0 mg/mL) and (Cb) KSS (2 mg/mL). The scale bar indicates 200 μm. As in (B), (Cc) the amount of lipid accumulation was measured and is expressed as relative absorbance. (Cd) Cell viability. Data are shown as the mean ± standard deviation (n = 3). * p < 0.05 and ** p < 0.01 vs. H₂O.

Figure 3

Bupleuri Radix (BR), Paeoniae Radix (PR), Moutan Cortex (MC), Glycyrrhizae Radix (GLR), and Menthae Herb (MH) in KSS reduce lipid accumulation. During differentiation induction by the DMI method, KSS or the 10 constituent herbs (2 mg/mL) were administered and cultured for 8 days. As in Figure 1, the amount of lipid accumulation was measured and is shown as relative absorbance. Data are shown as the mean ± standard deviation (n = 3). * p < 0.05 and ** p < 0.01 vs. H₂O.

Figure 4

Paeoniflorin (PNF), Paeonol (PN), Saikosaponin B1 (SSb1), and Saikosaponin B2 (SSb2) reduce lipid accumulation in a dose-dependent manner. (A) (Aa) DMSO (DMI method only), (Ab) KSS 2 mg/mL, (Ac) PNF 1000 μM, (Ad) PN 1000 μM, (Ae) SSb1 300 μM, or (Af) SSb2 100 μM was added in culture media and cultured for 8 days. Images of Oil-Red-O staining are shown. The scale bar indicates 200 μm. (B) PNF, PN, SSb1, and SSb2 were administered during differentiation induction by the DMI method and cells were cultured for 8 days. As in Figure 1, the amount of lipid accumulation was measured and is shown as relative absorbance. (Ba) PNF, (Bb) PN, (Bc) SSb1, and (Bd) SSb2. Data are shown as mean ± standard deviation (n = 3). * p < 0.05 and ** p < 0.01 vs. DMSO.

Figure 5

KSS inhibits early differentiation into adipocytes through the inhibition of Dex action. (A) 3T3-L1 cell differentiation stage and KSS administration time are shown. (i) No KSS administration, (ii) 0–8 days, (iii) 0–3 days (early-stage), (iv) 3–5 days (middle-stage), and (v) 5–8 days (late-stage). (B) KSS was administered at each differentiation stage. As in Figure 1, the amount of lipid accumulated at 8 days is measured and shown as relative absorbance. (Ba) KSS (2 mg/mL), (Bb) KSS (5 mg/mL). (C) 3T3-L1 cells were cultured for 3 days in the presence of two of the three types of differentiation inducers (1 μM Dex, 0.5 mM IBMX, and 10 μg/mL insulin). KSS (2 mg/mL) was administered for 8 days from the start of differentiation. The amount of lipid accumulated was measured as in Figure 1, and the relative absorbance is shown for the DMI method and KSS (-). (D) Oil-Red-O stained images of the cells cultured for 8 days. Upper: KSS (-), Lower: KSS (+), (Da) DMI (-), (Db) DMI (+), (Dc) MI (+), (Dd) DI (+), and (De) DM (+). The scale bar indicates 200 μm. Data are shown as mean ± standard deviation (n = 3). * p < 0.05 and ** p < 0.01 vs. (i) KSS (-) administration (B) or DMI method and KSS (-) (C). † p < 0.05 vs. KSS (-) (C).

Figure 6

KSS, MC, and PN suppress C/EBP-δ gene expression. (A) KSS suppresses differentiation marker gene expression in a time-dependent manner. The gene expression level was quantified by the qPCR method. (Aa) C/EBP-δ, (Ab) C/EBP-β, (Ac) C/EBP-α, (Ad) PPAR-γ. (B,C) During differentiation induction, MC (1 mg/mL), PNF (1000 μM), and PN (1000 μM) were administered, and the expression level of the C/EBP-δ and C/EBP-β genes was quantified as in (A). (a) MC, (b) PNF, (c) PN. (D) Alteration of the expression level of (Da) Adiponectin and (Db) FoxO1 during differentiation induction with or without KSS. Data are shown as mean ± standard deviation (n = 3). * p < 0.05 and ** p < 0.01 vs. H₂O or DMSO.

Figure 7

KSS, MC, PNF, and PN inhibit GR promoter activity. In the presence of 1 μM Dex, 2 mg/mL KSS, 1 mg/mL MC, 1000 μM PNF, 1000 μM PN, or 10 μM RU486 was administered, and a luciferase reporter assay was performed. Dex was used as a positive control for the GR promoter activity, and the relative activity was analyzed. Data are shown as the mean ± standard deviation (n = 3). * p < 0.05 vs. H₂O (Dex added). † p < 0.05 and †† p < 0.01 vs. DMSO (Dex added).

Figure 8

KSS, MC, and PN suppress lipid accumulation by suppressing Dex-induced GR activation and controlling early differentiation in 3T3-L1 cells. 3T3-L1 cells are differentiated into mature adipocytes by the addition of Dex, IBMX, and insulin (DMI method). As a result of differentiation, inducers bind to the receptor and the signals are transferred to the nucleus. KSS, MC, and PN inhibit the promoter activity of GR induced by Dex. This suppression of the promoter activity reduces the expression of the transcription factor C/EBP-δ gene and consequently inhibits the initial differentiation of 3T3-L1 cells. By suppressing early differentiation, the expression levels of C/EBP-α and PPAR-γ genes are reduced, and lipid accumulation is suppressed.