HJ was purchased from Gangwon Herbs (Wonju, Korea) in July, 2014. The voucher specimen was identified by Professor W.-K. Oh, and a voucher specimen (SNU-2014-0004) was deposited at the College of Pharmacy, Seoul National University, Seoul, Korea. Then, the HJ extract was prepared and supplied by the Korea Bioactive Natural Material Bank (Seoul, Korea). Briefly, the dried aerial parts of HJ were soaked in 100% methanol in an extraction container for 2 days at room temperature. The methanol-soluble extract was then filtered through cheesecloth, concentrated exhaustively, and dried under reduced pressure to afford a methanol extract. This HJ extract was suspended in 0.5% carboxymethyl cellulose (CMC) at a concentration of 50 mg/ml as a stock solution, and the working solution of HJ was adjusted to the intended concentrations for use in the in vitro and in vivo experiments in the present study.

The RAW 264.7 cells, a murine macrophage cell line, were purchased from the American Type Cell Culture (ATCC; Manassas, VA, USA), cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics (Gibco, Carlsbad, CA, USA), and maintained at 37°C in a humidified 5% CO₂ incubator. The cells were seeded in 12-well plates (1×10⁵ cells/well), incubated overnight, and thereafter, co-treated with different concentrations of HJ (200 and 400 µg/ml) and lipopolysaccharide (LPS; 1 µg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The doses of HJ were determined by preliminary tests using various concentrations (50–500 µg/ml) without cell toxicity to examine the anti-inflammatory effect.

The RAW 264.7 cells were treated with HJ extract for 1 h, and then stimulated with LPS or vehicle for 24 h. The total RNA from the cells was extracted and the samples were reverse transcribed using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). The resulting cDNA was amplified using the Exicycler 96 quantitative Real-Time PCR system and SYBR Premix Ex Taq (both from Bioneer, Daejeon, Korea) according to the manufacturer's instructions. Oligonucleotide primers [for TNF-α, IL-1β, IL-6, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)] were designed by Primer Express 3.0 software (Applied BioSystems, Carlsbad, CA, USA); the primer sequences used in the experiments are listed in Table I. The cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 1 min. To detect and remove possible primer-dimer artifacts, a dissociation curve was generated for the following cycling conditions: 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. The results were normalized to 18s mRNA levels (reference gene).

The secretion levels of TNF-α, IL-1-β and IL-6 were determined using culture supernatants from the LPS-stimulated RAW 264.7 cells by capture ELISA using Nunc-Immuno™ Microwell 96-well MaxiSorp™ microplates (Nunc A/S, Roskilde, Denmark). Pro-inflammatory cytokine levels were measured according to the manufacturer's instructions using the Mouse ELISA kit, BD OptEIA™ (Pharmingen, San Diego, CA, USA). Briefly, the 96-well plates were coated overnight at 4°C with a capture antibody (anti-mouse TNF-α, IL-1β and IL-6) in coating buffer (1:250). After the plates were washed with washing buffer [0.05% Tween-20 in 1X phosphate-buffered saline (PBS)], the plates were blocked with assay diluent (10% FBS in 1X PBS) for 1 h at room temperature. After the plates were washed, standards and samples were added to the wells and incubated for 2 h at room temperature. After the plates were washed, working detector (biotinylated anti-mouse cytokine and streptavidin-horseradish peroxidase conjugate in assay diluent) were added and incubated for 1 h at room temperature. The wells were again washed and the substrate solution (tetramethylbenzidine and hydrogen peroxide) was added and incubated for 30 min at room temperature in the dark. After 30 min, stop solution (1 N H₂SO₄) was added. The absorbance was measured using a microplate reader (Thermo Multiskan Spectrum, Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. A standard curve was obtained using serial dilutions starting from 0 upto 1,000 pg/ml.

The RAW 264.7 cells were plated in a 12-well plate as mentioned above, and the concentration of nitrite was measured using Griess reagent. The culture supernatant (100 µl) was mixed with Griess reagent (100 µl) [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthyl ethylenediamine dihydrochloride], and incubated at room temperature for 15 min. The light absorbance of the mixture was read at 540 nm using a microplate reader. The concentration of nitrite was calculated from a serial dilution standard curve of sodium nitrite (NaNO₂). In addition, the PGE₂ concentration was measured by a PGE₂ assay kit according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN, USA).

Eight-week-old male apoE^−/− mice were used in the present study. All animals were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the Korea Research Institute of Bioscience and Biotechnology (KRIBB; Daejeon, Korea) and housed in a controlled temperature (22 ± 1°C) facility and maintained on a reverse 12 h light/dark cycle. The mice were randomly divided into three groups: those fed i) an atherogenic diet (no. 102571; Dyets Inc., Bethlehem, PA, USA) plus vehicle (0.5% CMC) as the vehicle/control group (n=9); ii) an atherogenic diet plus 100 mg/kg of HJ as the HJ100 group (n=5); and iii) an atherogenic diet plus 500 mg/kg of HJ as the HJ500 group (n=8). The doses of HJ were determined by preliminary tests using concentrations of 100, 300, and 500 mg/kg for selection of anti-atherogenic effect. HJ was administered daily by oral gavage for 12 weeks and changes in body weight were measured each week. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of KRIBB and were performed in accordance with the institutional guidelines at KRIBB.

At the end of the experimental period, blood samples were collected from the orbital venous sinuses of the mice to analyze the concentrations of plasma lipid as well as various biomarkers. Plasma was prepared by centrifuging the blood at 10,000 × g for 5 min at 4°C and subsequently storing it at −70°C until analysis. Plasma total cholesterol and triglyceride levels were analyzed using a kit (Asan Pharm, Seoul, Korea), and other biomarkers including aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (Crea), glucose (Glu), and non-esterified fatty acid (NEFA) were measured using an automatic blood chemical analyzer (Hitachi, Tokyo, Japan).

The mice were euthanized with CO₂ gas, and then the whole aortas were isolated from the vehicle group (n=4), the HJ100 group (n=3), and the HJ500 group (n=4), and the adventitial tissue was removed. After fixation in 10% neutral formalin, the whole aortas were longitudinally dissected, and pinned flat on a rubber plate. Lipid plaques in the whole aorta were stained with oil-red O (Sigma-Aldrich), and en face images were captured using a digital camera (Canon, Tokyo, Japan). The whole aorta surface and stained plaque areas were analyzed by digital image analysis software (Image Inside; GS Media, Daejeon, Korea).

The aortic sinuses were isolated from the vehicle group (n=8), the HJ100 group (n=4), and the HJ500 group (n=7), and fixed in 10% neutral formalin. After fixation, the aortic sinuses were embedded in a Tissue-Tek optimal cutting temperature (OCT) compound and sectioned at a thickness of 8 µm using a cryotome (both from Sakura Finetek, Tokyo, Japan). Cryostat sections of the aortic sinus were stained with oil-red O. After oil-red O staining, photographs were captured under a light microscope (BX51; Olympus Corp, Tokyo, Japan) and the atheroma areas of the aortic sinus were quantified using digital image analysis software (Image Inside).

The infiltration of monocytes and macrophages was detected using anti-MOMA-2, a mouse monocyte/macrophage-specific antibody (Abcam, Cambridge, UK). The aortic sinuses were embedded in a Tissue-Tek OCT compound, and then sections of the aortic sinus (8 µm) were incubated with anti-MOMA-2 (1:200), followed by Alexa Fluor 488 nm goat anti-rat IgG (1:250) (Invitrogen, Carlsbad, CA, USA) for visualization. Images of the aortic sinus were captured under a dual fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The positively stained areas in the aortic sinus were quantified using digital image analysis software (Image Inside).

Total RNA from the whole aorta in each group was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, quantified by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, San Diego, CA, USA), and stored at −70°C until analysis. Relative quantification of the gene expression was performed using an Exicycler 96 Real-Time PCR System (Bioneer) with SYBR-Green dye (Takara). Oligonucleotide primers (VCAM-1, ICAM-1, iNOS, COX-2, MCP-1, CD68, IL-1β, IL-6 and IL-18) were designed by Primer Express 3.0 software (Applied Biosystems), and the primer sequences used in the experiments are listed in Table I. The cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 1 min. To detect and remove possible primer-dimer artifacts, a dissociation curve was generated by adding a cycle of 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. The results were normalized to 18s RNA levels (reference gene).

All data are presented as the means ± standard error of the mean (SEM). For two-group comparisons, a Student's t-test was used. A P-value of <0.05 was considered to indicate a statistically significant difference.

To examine the anti-inflammatory effect of HJ, the mRNA expression of inflammation-related genes (TNF-α, IL-1β, IL-6, COX-2 and iNOS) was measured in HJ-treated RAW 264.7 cells stimulated with LPS. LPS stimulation significantly induced the increased expression of pro-inflammatory genes compared with the non-treated cells. The mRNA expression of TNF-α and IL-1β was significantly decreased in the HJ-treated cells (Fig. 1A and B). In addition, IL-6 mRNA expression was markedly reduced in the HJ-treated groups, particularly in the HJ400 group (by 68%) compared with IL-6 mRNA expression in the cells treated with LPS only (Fig. 1C). The expression of other inflammatory mediators, such as iNOS and COX-2, markedly decreased after HJ treatment in a dose-dependent manner. Particularly, the mRNA expression of these two genes decreased by >80% in the HJ400 group compared with that in the cells treated with LPS only (Fig. 1D and E).

In addition to assessing the mRNA expression of pro-inflammatory cytokines and inflammatory mediators, we measured the secretion levels of TNF-α, IL-1β, IL-6, nitrite and PGE₂ in the LPS-stimulated RAW 264.7 cells following HJ treatment. As expected, the TNF-α and IL-6 secretion levels were significantly decreased by HJ treatment (Fig. 2A and C). Particularly the HJ400 group showed a >50% decrease compared with the cells treated with LPS only. Notably, IL-1β secretion was completely inhibited by HJ treatment regardless of the concentration (Fig. 2B). In general, the increased expression of iNOS leads to the production of high quantities of nitrite, nitrate and superoxide (O₂⁻) (22). The release of nitrite in HJ-treated cells was markedly reduced by >50% compared with the level of nitrite release in the cells treated with LPS only (Fig. 2D). The effects of PGE₂, an inflammatory mediator, are mediated through the induction of COX-2 expression resulting in inflammation (23). The treatment of LPS-stimulated RAW 264.7 cells with 400 µg/ml of HJ significantly reduced PGE₂ levels by 12% compared with the PGE₂ levels in the LPS-only treated cells (Fig. 2E). Taken together, these findings suggest that HJ inhibits the secretion of pro-inflammatory cytokines and inflammatory mediators in the LPS-stimulated RAW 264.7 cells.

To examine the effect of HJ on the formation of atherosclerotic lesions in vivo, HJ was administered to apoE^−/− mice for 12 weeks, and the whole aorta was obtained, stained with oil-red O, and analyzed. No significant change in the number of atherosclerotic lesions in the aortic en face images of the HJ100 group was observed, whereas the number of aortic lesions was markedly reduced in the HJ500 group (72.09±14.16%), compared with the number of lesions in the vehicle group (Fig. 3). Furthermore, the number of atherosclerotic lesions in the aortic sinus was compared following oil-red O staining. The atherosclerotic area was significantly and dose-dependently decreased in the HJ treatment groups (HJ100 group, 89.71±4.75%; HJ500 group, 83.87±3.97%), compared with that in the vehicle group (Fig. 4). These results indicate that high-dose treatment with HJ clearly reduces the formation of atherosclerotic lesions.

To examine the infiltration of monocytes and macrophages in the arterial intima, we performed immunofluorescence staining for MOMA-2 in the aortic sinus (Fig. 5A). HJ decreased the levels of MOMA-2-positive areas in a dose-dependent manner. Moreover, the areas of the aortic sinus stained by MOMA-2 were quantified using image analysis software, and a significantly decreased monocyte/macrophage infiltration was observed in the HJ-treated mice, compared with that in the control apoE^−/− group (Fig. 5B). This finding suggests that HJ treatment strongly inhibits the infiltration of monocytes and macrophages in the aortic sinus.

To further examine the aortic expression of atherogenic genes in HJ-treated apoE^−/− mice, we measured the mRNA levels of adhesion molecules (VCAM-1 and ICAM-1), inflammatory mediators (iNOS and COX-2), the monocyte/macrophage marker, CD68, and MCP-1, as well as the pro-inflammatory cytokines (IL-1β, IL-6 and IL-18) in the HJ500 and the control groups. Among the analyzed parameters, the mRNA expression of ICAM-1, MCP-1, CD68 and IL-18 was significantly decreased in the HJ500 group compared with that in the control group (Fig. 6). These results indicate that HJ may reduce the expression of the pro-inflammatory cytokines and atherogenic genes, thereby regulating the migration and the infiltration of mainly monocytes/macrophages, in the aorta of HJ-treated apoE^−/− mice.

Plasma lipid and other physiological biomarkers were also determined in the HJ- or vehicle-treated apoE^−/− mice. Apart from a decrease in plasma triglyceride levels in both the HJ100 and the HJ500 groups compared with the control group, no significant changes were found in cholesterol, NEFA, AST, ALT, BUN, Crea and Glu levels (Table II). Moreover, the changes in body weight were not significantly different among groups during the whole period of the experiments (data not shown). Based on these results, it is suggested that long-term treatment with HJ, at least for 12 weeks in apoE^−/− mice, does not induce significant toxic effects.