Aprotinin, leupeptin, LPS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), penicillin, streptomycin and other chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and supplements for cell culture were purchased from Gibco-BRL (Gaithersburg, MD, USA). Antibodies targeting phosphorylated Erk1/2 (p-Erk1/2), total Erk1/2 (t-Erk1/2), phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated p38 (p-p38), total p38 (t-p38), IκBα and NF-κB (p65 subunit) were purchased from Cell Signaling Technologies (Beverly, MA, USA). The antibody targeting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Sigma-Aldrich. HRP-conjugated secondary antibodies targeting mouse and rabbit IgGs were purchased from Abcam (Cambridge, UK). The murine macrophage RAW264.7 cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA).

Perilla frutescens plants were purchased from a certified herbal pharmacy (Chung-Yi Chinese Herbal Medicine Pharmacy, Taichung, Taiwan). After dehydration, 100 g of the leaves were homogenized, and the powder was passed through a mesh (0.05 mm). The filtered powder was resuspended into 1 liter of 100% methanol and stirred at room temperature for 24 h. Following filtration on a Whatman no. 1 filter paper, the solution was lyophilized. Stock solution (20 mg/ml) of the extract was prepared in dimethylsulfoxide, and stored at −20°C until further use.

The RAW264.7 cells were incubated in DMEM supplemented with 0.1% sodium bicarbonate, 2 mM glutamine, penicillin G (100 U/ml), streptomycin (100 μg/ml) and 10% FBS, and were maintained at 37°C in a humidified incubator containing 5% CO₂. Following pre-incubation with different concentrations of PLE for 4 h, 1 μg/ml LPS was added and then incubated for 2 h (NO assay), 3 h (RT-PCR and qRT-PCR analysis), or 24 h (cell viability assay and cell morphology analyses).

Cell viability was determined by an assay based on the mitochondrial-dependent reduction of MTT to formazan. Briefly, 10 μl of MTT solution (5 mg/ml in DMEM) were added to the cell supernatant and incubated for 4 h at 37°C. After removal of the medium, 2-propanol was added to lyse the cells and to solubilize the formazan. The optical density of formazan was measured at 570 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Hercules, CA, USA). The optical density of formazan generated by untreated cells was used to determine the 100% viability.

Total RNA was isolated from individual samples, according to the manufacturer’s instructions, using the RNeasy kit (Qiagen, Valencia, CA, USA). The purified RNA was used as a template to generate cDNA using the RevertAid™ First Strand cDNA Synthesis kit (Fermentas Life Sciences, St. Leon-Rot, Germany). The primer sequences used for RT-PCR and qRT-PCR are listed in Table I. RT-PCR experiments were performed in triplicates for each sample. Amplifications were performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA). For mRNA quantification, the FastStart Universal SYBR-Green Master mix (Roche Applied Science, Mannheim, Germany) was used. The cycle threshold (Ct) values were calculated using the ΔΔCT method, and relative expression values were expressed by normalizing to the expression of GAPDH. qRT-PCR experiments were performed in duplicates for each sample. The size of the PCR products was confirmed by agarose gel electrophoresis.

The NO level in cell culture supernatants was determined using the Griess test. Briefly, cells were treated with 1, 5, or 15 μg/ml PLE for 1 h, followed by incubation with 1 μg/ml LPS for 24 h. Nitrite in the culture supernatants was mixed with the same volume of Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid]. Absorbance was measured at 540 nm, and the nitrite concentration was determined using sodium nitrite (NaNO₂) as a standard (17).

To analyze the production of pro-inflammatory mediators, cells were seeded in 6-well plates at an initial density of 5×10⁵ cells/ml, starved in serum-free medium for 16 h, pre-incubated with different concentrations of PLE (5, 10 and 20 μg/ml) for 1 h, then treated with 1 μg/ml LPS for 24 h. The supernatants were collected, and the concentrations of IL-6, IL-8 and TNF-α, as well as of prostaglandin E₂ (PGE₂) were determined using DuoSet ELISA kits and the Prostaglandin E₂ Parameter Assay kit (R&D Systems, Abingdon, UK) respectively, following the manufacturer’s instructions.

Cells were washed with physiological saline and incubated with lysis buffer [10 mM HEPES, pH 7.6, containing 15 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 0.05% v/v IGEPAL^® CA-630, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin] for 10 min. Following centrifugation at 2,500 × g for 10 min at 4°C, the supernatant was transferred into a new Eppendorf tube, further centrifuged at 20,000 × g for 15 min at 4°C, and the new supernatant was collected as the cytosolic fraction. The pellets containing the nuclei were washed with physiological saline, incubated with a nucleus lysis buffer (25 mM HEPES, pH 7.6, 0.1% v/v IGEPAL^® CA-630, 1 M KCl, 0.1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 2 mM sodium fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin), and then centrifuged at 10,000 × g for 15 min at 4°C. The resulting supernatants were collected as the nuclear fraction.

Cells (5×10⁵ cells/ml) were harvested, washed twice with ice-cold phosphate-buffered saline (PBS), and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% v/v IGEPAL^® CA-630, 1 mM PMSF, 1 mM sodium fluoride, and 10 μg/ml aprotinin and leupeptin). The cell lysates were centrifuged at 14,000 × g for 10 min at 4°C to remove the debris. The supernatants were collected and crude protein concentrations were determined using the BCA™ protein assay kit (Pierce, Rockford, IL, USA). Crude proteins (30 μg/lane) were electrophoresed and transferred onto a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking with 5% w/v skimmed milk in PBS, the membrane was incubated for 2 h with a 1/1,000 dilution of the specific primary antibodies. Bound antibodies were detected using a 1/2,000 dilution of peroxidase-conjugated secondary antibodies (Abcam) and ECL chemiluminescence reagent (Millipore) as the substrate. Three independent experiments were performed, and results were quantified by densitometry.

Data were expressed as means ± standard deviation (SD) of the mean from three independent experiments. Statistical comparisons were performed by a one-way analysis of variance (ANOVA), followed by a Duncan multiple comparison test. Differences were considered statistically significant at P<0.05.

The cytotoxic effect of PLE on viability of RAW264.7 cells was investigated by the MTT assay. As shown in Fig. 1, the viability of RAW264.7 cells was determined following incubation with different concentrations (1, 5, 15, 30, 50 and 100 μg/ml) of PLE for 24 h. The results revealed that treatment with concentrations of 50 and 100 μg/ml PLE significantly decreased the viability of RAW264.7 cells (P<0.05) compared to control (untreated) cells, while lower concentrations (1, 5, 15 and 30 μg/ml) of PLE had no significant effect on viability of RAW264.7 cells.

LPS is known to induce morphological transformation of macrophage RAW264.7 cells (18). Therefore, whether PLE affects the LPS-induced morphological changes of RAW264.7 cells was investigated. As shown in Fig. 2, LPS alone significantly induced the morphological transformation of RAW264.7 cells to dendritic cells, and this transformation was attenuated by PLE pretreatment in a dose-dependent manner. Further quantitative analysis revealed that LPS significantly increases the number of cells having a dendritic morphology to 27.0±0.5% (P<0.05 as compared to the control). When cells were pretreated with 10 or 15 μg/ml PLE and then treated with LPS, the number of cells with dendritic morphology was decreased to 13.6±1.6 and 9.4±0.6%, respectively (P<0.05 as compared to LPS treatment alone).

To investigate whether PLE affects the expression of pro-inflammatory cytokines in LPS-stimulated macrophages, the mRNA levels of IL-6, IL-8 and TNF-α were determined in LPS-treated RAW264.7 cells. RT-PCR analysis revealed that LPS alone enhances the expression of IL-6, IL-8 and TNF-α, and this increase is reverted by pretreatment with PLE in a dose-dependent manner (Fig. 3A). Further quantitative analysis using qRT-PCR showed that treatment with LPS alone significantly increases the mRNA levels of IL-6, IL-8 and TNF-α, 3.9±0.4-, 13.8±1.5- and 5.4±0.3-fold respectively (Fig. 3B), as compared to the control (untreated cells). In RAW264.7 cells pretreated with 20 μg/ml PLE and then treated with 1 μg/ml LPS, the mRNA levels of IL-6, IL-8 and TNF-α were reduced 1.4±0.2-, 5.1±0.3- and 3.4±0.2-fold as compared to the control, respectively (P<0.005 as compared to LPS alone).

In addition to mRNA expression, LPS alone increased the protein production of IL-6, IL-8, and TNF-α in RAW264.7 cells (Fig. 3C) up to 135.7±18.9, 145.9±21.3 and 488.2±33.2 pg/ml/10⁴ cells, respectively (P<0.05 as compared to the control). PLE pretreatment dose-dependently reduced the protein production of the tested cytokines (Fig. 3C) up to 59.3±5.6 (IL-6), 38.2±3.9 (IL-8) and 228.7±13.3 pg/ml/10⁴ cells (TNF-α), respectively (P<0.05 as compared to LPS alone).

COX-2 and iNOS are both critical enzymes associated with macrophage-mediated development and progression of inflammation (19–21); therefore, effects of PLE on the mRNA level of COX-2 and iNOS were analyzed and quantified in LPS-stimulated RAW264.7 cells. RT-PCR analysis showed that LPS treatment alone enhances the expression of COX-2 and iNOS, and this increase was reverted by pretreatment with PLE in a dose-dependent manner (Fig. 4A). Further quantitative analysis using qRT-PCR revealed that LPS alone significantly increases the mRNA levels of COX-2 and iNOS, 41.6±2.6- and 24.9±4.3-fold, respectively, as compared to the control (P<0.05). In RAW264.7 cells pretreated with 20 μg/ml PLE and then treated with 1 μg/ml LPS, the mRNA levels of COX-2 and iNOS were dose-dependently reduced (Fig. 4B) 11.6±1.5- and 6.6±1.2-fold as compared to the control, respectively (P<0.05 as compared to LPS alone).

Since mRNA expression of COX-2 and iNOS in RAW264.7 cells treated with LPS was inhibited by PLE pretreatment, the production of PGE₂ and NO was further assessed. As shown in Fig. 4C and D, treatment with LPS alone significantly increased the concentrations of PGE₂ and NO up to 310.9±22.5 pg/ml and 25.3±1.6 μM, respectively. Upon pretreatment with 10 or 20 μg/ml PLE, the concentration of PGE₂ was reduced down to 168.1±9.2 pg/ml and 118.7±3.1 pg/ml, respectively, and the concentration of NO was decreased to 14.7±0.7 and 5.5±1.3 μM, respectively (P<0.05 as compared to LPS alone).

Activation of MAPKs is known to be involved in LPS-induced production of pro-inflammatory cytokines (22–24). To investigate whether PLE regulates activation of MAPKs in response to LPS, the levels of phosphorylated ERK1/2, JNK and p38 were determined by immunoblotting in LPS-stimulated RAW264.7 cells. As shown in Fig. 5A, LPS alone considerably increased the phosphorylation of ERK1/2, JNK and p38 in RAW264.7 cells, and pretreatment with 5, 10 or 20 μg/ml PLE inhibited the LPS-induced phosphorylation of ERK1/2, JNK and p38 in a dose-dependent manner.

The transcription factor NF-κB plays a pivotal role in regulation of pro-inflammatory factors, and its nuclear translocation is associated with the expression of these factors (25). Thus, the effects of PLE on the cytosolic level of IκBα (that inhibits the nuclear translocation of NF-κB) and the nuclear level of NF-κB were subsequently investigated in RAW264.7 cells exposed to LPS. As shown in Fig. 5B, exposure of RAW264.7 cells to LPS led to an important decrease in the cytosolic IκBα level, accompanied by the expected increase in the nuclear NF-κB level. However, PLE pretreatment restored the LPS-decreased cytosolic IκBα level and reduced the nuclear NF-κB level that had been increased by LPS treatment, in a dose-dependent manner. These findings revealed that PLE inhibits not only the degradation of IκBα, but also the nuclear translocation of NF-κB.