The leaves of
Inflammation is a response of organisms to pathogens and chemical or mechanical injury. Inflammatory response and tissue damage are induced by inflammatory mediators generated through upregulation of a number of inducible genes, including inducible nitric oxide (
Macrophages play a crucial role in eliciting NF-κB-related cascades at the acute phase of the inflammatory response. LPS stimulation of mouse macrophages leads to increased phosphorylation and activation of mitogen-activated protein kinases (MAPKs), such as extracellular-signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) (
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).
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
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 (
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
LPS is known to induce morphological transformation of macrophage RAW264.7 cells (
To investigate whether PLE affects the expression of pro-inflammatory cytokines in LPS-stimulated macrophages, the mRNA levels of
In addition to mRNA expression, LPS alone increased the protein production of IL-6, IL-8, and TNF-α in RAW264.7 cells (
COX-2 and iNOS are both critical enzymes associated with macrophage-mediated development and progression of inflammation (
Since mRNA expression of
Activation of MAPKs is known to be involved in LPS-induced production of pro-inflammatory cytokines (
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 (
Macrophages were reported to be directly involved in the inflammatory response (
NO is an important mediator and regulator involved in inflammatory responses. In activated inflammatory cells, NO is produced at high levels by iNOS. COX-2 is regarded as a central mediator of inflammation, and regulation of COX-2 was suggested to be useful in the development of a therapeutic target (
LPS is known to activate several signaling kinases, including ERK1/2, MEK (
In conclusion, the results from the present study indicate that PLE displays important anti-inflammatory activity in LPS-stimulated macrophage RAW264.7 cells, through inhibition of the expression of pro-inflammatory cytokines, inhibition of MAPK activation, and of NF-κB nuclear translocation in response to LPS.
This study was partly supported by grants from the National Science Council, Taiwan (nos. NSC99-2320-B-040-003-MY3 and NSC99-2632-B-040-001-MY3).
Effect of PLE on viability of RAW264.7 cells. Cells were incubated with different PLE concentrations (1, 5, 10, 20, 50 and 100 μg/ml) for 24 h, and the cell viability was determined by the MTT assay. *P<0.05 as compared to the control (C; untreated cells). PLE,
Effects of PLE on dendritic transformation of LPS-stimulated RAW264.7 cells. Cells were pre-incubated with the indicated concentrations of PLE in the DMEM culture medium for 4 h, and then stimulated with 1 μg/ml LPS for 24 h. Cell morphology was monitored under a light microscope at ×400 magnitude. Activated RAW264.7 cells are indicated by arrows. The activation index percentage was expressed as the number of cells with activated morphology relative to the total number of cells, quantified in 5 random fields (number of total cells >100). #P<0.05 as compared to the control (C; untreated cells); *P<0.05 as compared to LPS alone. PLE,
PLE reduces the mRNA level and the protein production of pro-inflammatory cytokines IL-6, IL-8 and TNF-α in LPS-stimulated RAW264.7 cells. Cells were pre-incubated with the indicated concentrations of PLE in the DMEM culture medium for 4 h, and then stimulated with 1 μg/ml LPS for 6 or 24 h. Following treatment, the cells were lysed for mRNA extraction, and the gene expression level was analyzed by (A) RT-PCR and quantified by (B) qRT-PCR, while the culture medium was collected to quantify the production of corresponding proteins by (C) ELISA. Bars denote standard deviation (SD) of the mean from three independent experiments. #P <0.05 as compared to the control (untreated cells); *P<0.05 as compared to LPS alone. PLE,
PLE reduces the mRNA level of
PLE reduces the phosphorylation of MAPKs and the nuclear translocation of NF-κB in LPS-stimulated RAW264.7 cells. Cells were pre-incubated with the indicated concentrations of PLE in the DMEM culture medium for 4 h, and were then stimulated with 1 μg/ml LPS for 1 h. Following treatment, the cells were lysed and fractionated for protein extraction. (A) Phosphorylation and protein level of MAPKs and (B) protein level of cytosolic IκBα and nuclear NF-κB visualized by immunoblot assays using specific antibodies and a chemiluminescent substrate. The levels of GAPDH and histone H1 were used as controls. PLE,
Primer sequences used for reverse transcription polymerase chain reaction (RT-PCR) and quantitative (q)RT-PCR.
Gene name | RT-PCR | qRT-PCR |
---|---|---|
F: 5′-ATGAACTCCTTCTCCACAAGCGC-3′ | F: 5′-GTAGTGAGGAACAAGCCAGAGC-3′ | |
R: 5′-GAAGAGCCCTCAGGCTGGACTG-3′ | R: 5′-GGCATTTGTGGTTGGGTCA-3′ | |
F: 5′-AGATATTGCACGGGAGAA-3′ | F: 5′-CTCTTGGCAGCCTTCCTGATTT-3′ | |
R: 5′-GAAATAAAGGAGAAACCA-3′ | R: 5′-CGCAGTGTGGTCCACTCTCAAT-3′ | |
F: 5′-CTGAGGGCTCTGTTGAGGTC-3′ | F: 5′-GGCAGCCTGTGAGACCTTTG-3′ | |
R: 5′-CCTTGTTCAGCTACGCCTTC-3′ | R: 5′-GCATTGGAAGTGAAGCGTTTC-3′ | |
F: 5′-GGAGAGACTATCAAGATAGTGATC-3′ | F: 5′-CAGAACCGCATTGCCTCTG-3′ | |
R: 5′-ATGGTCAGTAGACCTTTACAGCTC-3′ | R: 5′-TTGTAACTTCTGGTCCTCATGTCGA-3′ | |
F: 5′-GCGACGTGGAACTGGCAGAAG-3′ | F: 5′-GACCCTCACACTCAGATCATCTTCT-3′ | |
R: 5′-TCCATGCCGTTGGCCAGGAGG-3′ | R: 5′-CCTCCACTTGGTGGTTTGCT-3′ | |
F: 5′-ACCACAGTCCATGCCATCAC-3′ | F: 5′-ATGCCTCCTGCACCACCA-3′ | |
R: 5′-TCCACCACCCTGTTGCTGTA-3′ | R: 5′-CCATCACGCCACAGTTTCC-3′ |