Inflammation is a vital defense mechanism for the organism in response to pathogen stimuli. Monocytes and macrophages are dominant at the locations of lipopolysaccharide (LPS) induced inflammation. Macrophages become active upon LPS stimulation, and several cellular mediators such as tumor necrosis factor-α (TNF-α), IL-1β, nitric oxide (NO) and cyclooxygenase (COX)-2 are released to regulate inflammation (
NO is also known as a short-lived free radical and one of key cellular mediator for inflammatory responses. There are three isoforms of NO synthases (NOS) in tissues to generate NO (
Over-reactive and uncontrolled inflammatory responses can cause the tissues to remain in a chronic inflammatory status, which leads to a variety of diseases including rheumatoid arthritis, pulmonary fibrosis and even cancer (
Celebrex, acetone, dimethylsulfoxide (DMSO), lipopolysaccride (LPS), and
The leaves of
Before analysis by HPLC, CIL extract was filtered through a 0.2 μm Millipore filter, and then total volume of 20 μl was loaded into the HPLC column. External standards were prepared as concentration of 100 μg/ml in HPLC grade-methanol and used to calculate the concentration of examined compounds. Reverse phase HPLC was performed on a Perkin-Elmer HPLC system (Perkin-Elmer, Waltham, MA, USA) equipped with Perki-Elmer Series 200 pump, Perki-Elmer 785A UV/VIS detector and Perk-Elmer Series 200 autosampler. Separations were accomplished on LiChroCART 250-4 C18 HPLC-cartridge (5 μm; Merck, Whitehouse Station, NJ, USA). The separation conditions of HPLC analysis of examined compounds are described in
The murine macrophage RAW 264.7 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. Cells were incubated with CIL extract as indicated for 1 h and then stimulated with 1 μg/ml lipopolysaccride (LPS) (
Cell viability of RAW 264.7 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described elsewhere (
Nitric oxide (NO) production was determined by measurement of the accumulation of nitrite, the stable metabolite of NO in the culture medium. Nitrite was assayed colorimetrically after reaction with the Griess reagent as described previously (
Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNA (1 μg) was heated at 70°C for 10 min and reversely transcribed using reverse transcriptase 200 U (Promega, Madison, WI, USA). The mixture was then incubated at 37°C for 60 min, heated at 95°C for 10 min and stored at −20°C until use. Real-time PCR was performed 40 cycles with primers for iNOS and β-actin as an internal control using the ABI PRISM 7300 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). Cycles consisted of 30 sec of denaturation at 95°C, 30 sec of annealing at 60°C, 1 min of extension at 72°C, followed by 10 min of elongation at 72°C. Data were collected by the Sequence Detection Software (SDS; Version 1.3.1, Applied Biosystems) and analyzed using the threshold cycle relative quantification method. Primers were designed with computer assistance according to the gene bank. The sequence of the primers are as follows; iNOS sense primer 5′-CAT CAA CCA GTA TTA TGG CTC CT-3′, and iNOS anti-sense 5′-TCC TGT TGT TTC TAT TTC CTT TGT T-3′; β-actin sense 5′-CTA AGG CCA ACC GTG AAA AG-3′, and β-actin antisense 5′-ACC AGA GGC ATA CAG GGA CA-3′. The cycle threshold (Ct) values were determined in at least three independent experiments for each sample. Results were normalized to the endogenous gene β-actin.
TaqMan miRNA assays (Applied Biosystems) were used to quantify mature miRNA of miR-146a, miR-155 and miR-424. RNU6B was used as a reference gene control. Quantitative assays were performed in the 7900HT system according to the manufacturer’s instructions (Applied Biosystems). Briefly, 10 ng of RNA samples extracted either normal parts or tumor of same oral patient were mixed and reacted with reverse transcription master mix reagents (Applied Biosystems) at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Each of PCR cycle contained the denaturation step at 95°C for 15 sec and the extension step at 60°C for 1 min for a total of 40 cycles. miRNA expression values were normalized using RNU6B following the 2−ΔΔCt method and analyzed by using ABI 7900HT SDS 2.2 software. All reactions were run in triplicate.
Transfection followed the manufacturer’s protocol (Thermo Scientific Open Biosystems, Huntsville, AL, USA). Briefly, 3×105 RAW 264.7 cells/well were incubated in 6-well plates overnight. Medium was changed into the DMEM without 10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin before transfection. Solution A (2 μg of DNA and 50 μl of DMEM) was mixed with solution B (10 μl of Arrest-In transfection reagent and 50 μl of DMEM) at room temperature for 20 min. Transfection reagents were equally added into cells. Cells were harvested after 48-h transfection.
pCOX-2-LUC plasmid was used to quantify COX-2 promoter activity. pRL-CMV, a renilla luciferase reporter plasmid under the control of the cytomegavirus promoter (Promega), was used as an internal control to normalize the reporter gene activity. Plasmids were transfected into RAW264.7 cells. After 24-h transfection, cells were treated with CIL extract for 1 h followed by stimulation with LPS and analyzed the luciferase activity after 48-h transfection. The luciferase activity was determined by a luminometer using a dual-luciferase reporter assay system (Promega) according to the instructions of the manufacturer. Briefly, cells were lysed with 1× PLB (passive lysis buffer) for 15 min. PLB lysate (20 μl) was added into a 96-well plate and mixed with 80 μl of LAR II (luciferase assay substrate in luciferase assay buffer II) to measure luciferase activity by using the SpectraMax L spectrometer (Molecular Devices, Sunnyvale, CA, USA). The values were normalized with the measurement of renilla luciferase activity.
The nuclear extract was prepared as described previously (
Proteins were separated by SDS-PAGE and transferred onto PVDF (Millipore, Billerica, MA, USA) as described previously (
Data are expressed as mean ± standard deviations (SD) from three different experiments. Statistical analysis was carried out using the Student’s t-test. It was considered statistically significant at *p<0.05, and **p<0.01.
After 24-h treatment with CIL extract at the indicated concentrations (0–25 μg/ml) in RAW264.7 cells, CIL extract was found to inhibit RAW264.7 cell proliferation in a dose-dependent manner, and the IC50 value of CIL extract for 24 h was 14 μg/ml (
We investigated the inhibitory effect of CIL extract on LPS-induced NO production in RAW264.7 cells. Cells were preincubated with CIL extract at the indicated concentrations for 1 h and then stimulated with LPS for 23 h. Supernatants were collected for determination of nitrite production. CIL extract inhibited NO production in RAW264.7 cells in a dose-dependent manner as compared to controls, and the anti-inflammatory agent
QPCR was performed to determine whether the inhibitory effects of the CIL extract on the pro-inflammatory mediators (NO) were related to the modulation of the expression of iNOS and COX-2. After the transient transfection of RAW 264.7 cells with pCOX-2-LUC and pRL-CMV, the expressions of firefly luciferase and Renilla luciferase, respectively, were used to quantify the COX-2 promoter activity. RAW264.7 cells were treated with the various concentrations of CIL extract (0, 1, 2.5, 5 and 10 μg/ml). As shown in
Since p65 and p50 are the major components of NF-κB activated by LPS in the macrophage (
When RAW264.7 cells were exposed to LPS, expressions of microRNA-146a and miR-155 were up-regulated 6.97- and 77.23-fold, respectively (
Two of the marked components of CIL extract, including amentoflavone and oleanolic acid, were identified by HPLC analysis to be indicator compounds for quality check of extraction procedure of each batch (
Macrophage activation is important to the progression of multiple diseases through the release of inflammatory mediators. Lipopolysaccharide (LPS)-induced RAW264.7 macrophages are widely used
In the present study, we prepared acetone extract from CIL extract and examined its effects on the LPS-induced inflammation in a murine macrophage cell line RAW 264.7 model. First, the cytotoxicity of CIL extract in RAW 264.7 cells were evaluated by MTT assay, and it was observed that CIL extract did not affect cell viability <2.5 μg/ml. Many lines of evidence have indicated that NO is a potent proinflammatory mediator and may have a multi-faceted role in mutagenesis and carcinogenesis (
NF-κB is a transcription factor that plays a critical role in inflammatory and immune responses. It is present in the cytoplasm, binding to the inhibitory protein IκB in unstimulated cells (
Up to date, miRNAs have been demonstrated to be dysregulated in cancer (
Furthermore, amentoflavone and oleanolic acid are two major components isolated from CIL extract (
Collectively, we have demonstrated that CIL extract inhibits LPS-induced NO production and iNOS expression which mediated through the inhibition of NF-κB activation in RAW 264.7 macrophages. CIL extract exerts its potent anti-inflammatory activity by suppressing COX-2 promoter activity and expression. Our results demonstrate the strong anti-inflammatory properties of CIL extract by inhibition of iNOS and COX-2 expression as well as miR-146a expression.
This study was supported by grants from the China Medical University and National Science Council (NSC98-2815-C-039-080-B) (CMU96-116).
nuclear factor kappaB
nitric oxide
dimetylsulfoxide
microRNA
cyclooxygenase-2
lipopolysaccride
Effects of
The inhibitory effect of CIL extract on production of nitric oxide (NO) and the expression of iNOS in LPS-induced inflammation. (A) The NO production of CIL extract in RAW 264.7 cells. LPS+ indicated cells were induced by LPS (100 ng/ml) for a 24-h treatment. LPS− indicated cells were treated in the absence of LPS. iNOS inhibitor (1400W) at 10 μM was the positive control. *p<0.05; **p<0.01 as compared with the LPS-induced cells. (B) Real-time RT-PCR analysis of iNOS mRNA expression the LPS-induced cells were determined and data were normalized to β-actin control. iNOS inhibitor (1400W) at 10 μM was the positive control.
Effect of CIL extract on LPS-induced COX-2 activation. The p COX-2-LUC and the pRL-CMV-LUC were co-transfected into RAW 264.7 cells for 24 h and then cells were treated with DMSO or the indicated concentrations (μg/ml) of CIL extract for 1 h before stimulation with LPS (1 μg/ml) for another 24 h. The firefly and Renilla luciferase activities in the cell lysates were determined. The former activity was normalized to the respective latter activity. Data represent as the mean ± SD from at least three independent experiments. *p<0.05 as compared with the LPS-induced cells.
Inhibitory effect of CIL extract on LPS-induced NF-κB activation. (A) RAW 264.7 cells were cultured with the indicated concentrations of CIL extract and 100 ng/ml of LPS. Nuclear extract fraction of cell lysates were analyzed by western blot analysis. PCNA was the internal control. (B) The pNF-κB-LUC and the pRL-CMV-LUC co-transfected cells were treated with DMSO or the indicated concentrations (μg/ml) of CIL extract for 1 h before stimulation with LPS for another 24 h. The firefly and Renilla luciferase activities in the cell lysates were determined. The former activity was normalized to the respective latter activity. Values are expressed as the mean % of three independent experiments. *p<0.05 as compared with the LPS-induced cells.
miR-146a, miR-424, and miR-155 expression levels in response to CIL extract in LPS-induced RAW264.7 cells. RAW264.7 cells were stimulated with LPS (100 ng/ml) in the presence (+) or absence (-) of CIL extract for 24 h. Expression of miR-146a (A), microR-155 (B), and miR-424 (C) was assayed by real-time PCR, and data were normalized to U6 control. The fold increase in the expression of these miRNAs versus non-stimulated cells is shown. Data represent the mean ± SD from at least three independent experiments. *p<0.05 as compared with the LPS-induced cells.
Representative HPLC chromatogram of the marked compounds in CIL extract. (A) HPLC chromatogram of amentoflavone standard (left, 100 μg/ml and CIL extract (right, 10 mg/ml) (UV-spectrum 330 nm), respectively. (B) HPLC chromatogram of oleanolic acid standard (left, 100 μg/ml) and CIL extract acetone extracts (right, 10 mg/ml) (UV-spectrum 215 nm), respectively. The examination conditions and monitoring wavelength of HPLC analysis are described in
HPLC separation conditions for identifying marked components within CIL extract.
Compounds | Mobile phase | Wavelength (nm) | RT (min) | Contents (mg/g of CIL) |
---|---|---|---|---|
Amentoflavone | MeOH:H3PO4 (0.11%, pH 2.2) = 25:75 | 330 | 10.39 | 84.72±1.46 |
Oleanolic acid | ACN:H3PO4 (0.11%, pH 2.2) = 28:72 | 215 | 10.15 | 1.05±0.01 |
ACN, acetonitrile; RT, retention time. All samples were loaded at a total volume of 10 μl into the HPLC cartridge; a flow rate of 1.0 ml/min was used to perform HPLC analysis.