3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Griess reagent and Neutral Red were purchased from MilliporeSigma. Inhibitors, such as C29 [Toll-like receptor (TLR)2 inhibitor], TAK-242 (TLR4 inhibitor), PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) were purchased from MilliporeSigma. The primers for inducible nitric oxide synthase (iNOS), IL-1β, IL-6 and GAPDH were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The primary antibodies individually raised against phosphorylated (p-)ERK1/2 (cat. no. 4377), ERK1/2 (cat. no. 9102), p-p38 (cat. no. 4511), p38 (cat. no. 9212), p-JNK (cat. no. 9251), JNK (cat. no. 9252), CCAAT enhancer binding protein α (CEBPα; cat. no. 8178), proliferator-activated receptor γ (PPARγ; cat. no. 2430), adipose triglyceride lipase (ATGL; cat. no. 2138), p-hormone-sensitive lipase (HSL; cat. no. 4137), HSL (cat. no. 4107), perilipin-1 (cat. no. 9349), p-adenosine monophosphate activated protein kinase (AMPK; cat. no. 2535), AMPK (AMPK, cat. no. 5831), uncoupling protein 1 (UCP-1; cat. no. 14670) and β-actin (cat. no. 5125) were purchased from Cell Signaling Technology, Inc. Other primary antibodies, such as anti-peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α; cat. no. sc-518025) and anti-PR domain containing 16 (PRDM16; cat. no. ab106410), were purchased from Santa Cruz Biotechnology, Inc. and Abcam. Secondary antibodies, such as anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody (cat. no. 7074) and anti-mouse IgG HRP-linked antibody (cat. no. 7076), were purchased from Cell Signaling Technology, Inc.

A. himalaicum leaves (AHL) used in this study were provided as dry powder by the National Institute of Forest Science, Korea. A. himalaicum is not listed in the IUCN Red List of Threatened Species, so it is neither an endangered nor a protected species. Water extraction from A. himalaicum leaves was performed by adding 20 times the weight of distilled water to 10 g of A. himalaicum leaves and extracting them in a water bath at 40˚C for 24 h. After 24 h, the extract was lyophilized, dissolved in distilled water and added to the cells.

The murine macrophage cell line RAW264.7 and mouse pre-adipocyte 3T3-L1 cells used in this study were purchased from the American Type Culture Collection. RAW264.7 cells were cultured in a CO₂ incubator (37˚C, 5% CO₂) using a DMEM/F-12 medium (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 unit/100 µg/ml). Subculture of RAW264.7 cells was performed once every 2 days. 3T3-L1 cells were cultured in a CO₂ incubator (37˚C; 5% CO₂) using DMEM/F-12 medium containing 10% bovine calf serum and penicillin/streptomycin (100 unit/100 µg/ml). The 3T3-L1 cells were subcultured once every 3 days.

Toxicity to cells was measured using an MTT assay. RAW264.7 cells were treated with AHL and cultured for 24 h at 37˚C in a 96-well plate. MTT solution (1 mg/ml) was then added and incubated for an additional 4 h. After 4 h, the DMSO solution was added and incubated for 10 min. Absorbance was measured at 570 nm using a UV/visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

The chemical inhibitors, such as C29, TAK-242, PD98059, SB203580 and SP600125, were used to pre-treat RAW264.7 cells for 2 h at 37˚C, 2 h prior to AHL treatment.

RAW264.7 cells were cultured in a 96-well plate using DMEM/F-12 medium supplemented with 10% FBS and treated with AHL for 24 h. To measure the amount of NO, the cell culture medium and Griess solution were mixed 1:1 and allowed to react for 15 min. The absorbance was then measured at 540 nm using a UV/Visible spectrophotometer (Humancorp; Xma-3000PC). Values were calculated using NaNO₂ as a standard.

Phagocytosis of RAW264.7 cells was performed using Neutral Red assay (11). RAW264.7 cells were cultured in a 96-well plate using DMEM/F-12 medium supplemented with 10% FBS and treated with AHL for 24 h. To measure the phagocytosis in RAW264.7 cell line, cells were stained with 0.01% Neutral Red solution for 2 h and then Neutral Red was eluted with lysis buffer containing 50% ethanol and 1% acetic acid at a ratio of 1:1. The absorbance of the eluted Neutral Red was measured at 540 nm using a UV/visible spectrophotometer (Humancorp; Xma-3000PC).

Two days (D0) after culturing 3T3-L1 cells in a 6-well plate to 100% confluency, the 3T3-L1 cells were differentiated for 2 days in a DMEM/F-12 medium containing DMI (0.05 mM IBMX + 1 µM dexamethasone + 10 µg/ml insulin) (D2). Then, the 3T3-L1 cells were cultured for 2 days in a DMEM/F-12 medium containing 10 µg/ml insulin (D4). 3T3-L1 cells were further cultured for 4 days, with the DMEM/F-12 medium replaced once every 2 days (D6-D8). AHL were administered from D0 to D8 and experimental analysis was performed on D8.

Lipid accumulation in 3T3-L1 cells was confirmed using ORO staining. The 3T3-L1 cells were washed with PBS, fixed with 10% formalin and stained with the ORO staining reagent for 20 min at room temperature. After staining, the staining solution was removed, the cells were washed with distilled water and the degree of lipid accumulation was observed under a light microscope (Olympus Corporation). After microscopic observation, the stained ORO was eluted with 100% isopropanol and the absorbance was measured at 500 nm using a UV/visible spectrophotometer (Humancorp; Xma-3000PC). Lipid accumulation in ORO staining was calculated by measuring the absorbance of released ORO dye in cells.

To obtain RNA from RAW264.7 cells, total RNA was extracted from RAW264.7 cells using a RNeasy Mini kit (Qiagen GmbH). The extracted RNA was synthesized into cDNA using 1 µg of total RNA through quantitative analysis and Verso cDNA kit (Thermo Fisher Scientific, Inc.). RT-PCR of iNOS, IL-1β, IL-6, TNF-α and GAPDH genes was performed using the synthesized cDNA and PCR Master Mix kit (Promega Corporation). The primer sequences used in this study are listed in Table I. PCR reaction (PCR Thermal Cycler Dice; Takara Bio, Inc.) was performed for 30 cycles under the following conditions: Denaturation at 94˚C for 30 sec, annealing at 55˚C for 1 min and extension at 72˚C for 1 min. After the PCR, the products were electrophoresed on a 1% agarose gel at 100 V for 15 min. The results presented are not mRNA, but rather DNA. Therefore, the brightness of the expressed DNA bands was quantified using UN-SCAN-IT (Silk Scientific, Inc.).

After washing the cells with PBS, RIPA buffer was added to extract proteins, the recovered cells were centrifuged at 25,200 x g for 30 min at 4˚C and the protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). The quantified proteins (25 µg/well) were separated using SDS-PAGE (10% polyacrylamide gel) and transferred onto a nitrocellulose blotting membrane (Thermo Fisher Scientific, Inc.). The transferred membranes were blocked with 5% skimmed milk for 1 h at room temperature. For the primary antibody reaction, the membrane was incubated at 4˚C overnight with a 5% BSA solution containing the primary antibodies (1:1,000). Then, a 5% BSA solution containing secondary antibodies (1:1,000) was added to the membrane and allowed to react at room temperature for 1 h. To activate the horseradish peroxidase molecules bound to the secondary antibody, western blotting substrate was added to the membrane (Cytiva) and protein expression was confirmed using a LI-COR C-DiGit Blot Scanner (LI-COR Biosciences). Protein bands were quantified using UN-SCAN-IT gel software version 5.1 (Silk Scientific, Inc.). Actin was used as a loading control for normalization in western blot analysis.

All experiments were repeated at least three times. Statistical analyses were verified using GraphPad Prism version 5.0 (Dotmatics) and data are presented as mean ± standard deviation. Each data point was analyzed using one-way analysis of variance and the data was analyzed using the Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of AHL on macrophage activation, the production of immunostimulatory factors and the activation of phagocytosis were analyzed in AHL-treated RAW264.7 cells. The production of NO was increased in AHL-treated RAW264.7 cells. DNA levels of immunostimulatory factors such as iNOS, IL-1β, IL-6 and TNF-α were increased in AHL-treated RAW264.7 cells (Fig. 1A). In addition, AHL increased the phagocytosis of RAW264.7 in a concentration-dependent manner (Fig. 1B). However, AHL had no effect on the viability of RAW264.7, indicating that AHL is not cytotoxic to RAW264.7 cells (Fig. 1C). Taken together, these results suggested that AHL activates macrophages.

To investigate the involvement of TLR2 and TLR4 in AHL-mediated macrophage activation, the production of immunostimulatory factors and phagocytosis was measured in AHL-treated RAW264.7 cells in the presence of C29 (TLR2 inhibitor) and TAK-242 (TLR4 inhibitor). The NO level and DNA level for iNOS, IL-1β and IL-6 were increased in both AHL treatment alone and AHL treatment in the presence of C29 with little difference (Fig. 2A). However, inhibition of TLR4 by TAK-242 markedly reduced the AHL-mediated increase in The NO level and DNA level for iNOS, IL-1β and IL-6 (Fig. 2A). Furthermore, inhibition of TLR2 by C29 did not reduce AHL-mediated phagocytosis of RAW264.7 cells, whereas inhibition of TLR4 by TAK-242 significantly blocked AHL-mediated phagocytosis (Fig. 2B). Taken together, these results indicated that AHL-mediated activation of macrophages may be dependent on TLR4.

To analyze the effect of AHL on MAPK activation, the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2), p38 and c-JNK were investigated in AHL-treated RAW264.7 cells. AHL increased the phosphorylation of ERK1/2, p38 and JNK, indicating that AHL can activate ERK1/2, p38 and JNK signaling (Fig. 3A). To analyze the effect of ERK1/2, p38 and JNK signaling on AHL-mediated activation of RAW264.7 cells, the present study investigated the level of immunostimulatory factors in AHL-treated RAW264.7 cells in the presence of specific ERK1/2, p38 and JNK signaling inhibitors. Inhibition of p38 by SB203580 and inhibition of JNK by SP600125 significantly reduced AHL-mediated increase in the NO level and DNA level for iNOS, IL-1β and IL-6 (Fig. 3B). However, inhibition of ERK1/2 by PD98059 did not reduce AHL-mediated increase in the NO level and DNA level for iNOS, IL-1β and IL-6. These results indicate that the activation of p38 and JNK signaling may be important for AHL activation in macrophages. Finally, the effect of TLR4 on AHL-mediated activation of p38 and JNK signaling was investigated. Inhibition of TLR4 by TAK-242 reduced the phosphorylation of p38 and JNK by AHL (Fig. 3C), indicating that the activation of p38 and JNK signaling by AHL may depend on TLR4. Taken together, these results suggested that AHL activates macrophages by activating p38 and JNK signaling in a TLR4-dependent manner.

To analyze whether AHL has anti-obesity activity, 3T3-L1 cells were treated with AHL during the differentiation of 3T3-L1 cells with DMI and insulin and the degree of lipid accumulation was examined by ORO staining. Intracellular lipid accumulation was reduced in AHL-treated cells compared to that in untreated cells (Fig. 4). These results suggested that AHL may have anti-obesity effects.

The present study investigated whether the inhibitory effect of AHL on lipid accumulation could be attributed to the regulation of proteins related to adipogenesis, lipolysis and browning of white adipocytes. AHL reduced the protein levels of CEBPα and PPARγ, which induce adipogenesis (Fig. 5). By contrast, AHL increased ATGL protein and the phosphorylation level of HSL and decreased perilipin-1 protein, which is essential for lipolysis. Additionally, AHL increased the phosphorylation level of AMPK and increased UCP-1, PGC-1α and PRDM16 proteins, which induce browning of white adipocytes. Taken together, these results suggested that AHL inhibits lipid accumulation in mature adipocytes by inhibiting adipogenesis and inducing lipolysis and browning of white adipocytes.