Antiinflammatory Activity of Gynura bicolor (紅鳳菜 Hóng Fèng Cài) Ether Extract Through Inhibits Nuclear Factor Kappa B Activation

Abstract

This study investigated effects of the Gynura bicolor (Roxb. and Willd.) DC. ether extract (GBEE) on nitric oxide (NO) and prostaglandin (PG)E₂ production on the lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 cells. A composition analysis of GBEE showed that the major compounds were b-carotene, chlorophyll, and quercetin, respectively. Furthermore, NO and PGE₂ levels of 120 μg/ml GBEE-treated cells were 70% and 9.8%, respectively, than those of cells treated with LPS alone. Immunoblots assays showed that the GBEE dose-dependently suppressed LPS-induced inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 protein levels. The GBEE significantly decreased cytosolic phosphorylated (p)-IκBa and nuclear p65 protein expressions. Electrophoresis mobility shift assays indicated that the GBEE effectively inhibited nuclear factor kappa B (NF-κB) activation induced by LPS. These results support a role of the GBEE in suppressing activation of NF-κB to inhibit NO and PGE₂ production in the LPS-induced inflammatory response by RAW 264.7 cells.

INTRODUCTION

Gynura bicolor (紅鳳菜 Hóng Fèng Cài; the leaves of Gynura bicolor [Roxb. and Willd.] DC.) is a common vegetable in Taiwan and Far Eastern. The top and bottom sides of G. bicolor leaves, respectively, appear dark-green and purple. The major constituents of G. bicolor related to it pigment sources and physiological effects are possibly the rich flavonoids.[1] G. bicolor has been shown as antioxidant, antiinflammatory, and antihyperglycemic effect.[2,3] To date, limited studies have examined the biological activities of G. bicolor, and the working mechanisms are not yet elucidated.

Inflammation is a physiological defense response of the body to various types of injurious stimuli. Chronic inflammation is characterized by a proliferation of fibroblasts and the formation of blood vessels (angiogenesis), as well as an influx of chronic inflammatory cells, namely granulocytes (neutrophils, eosinophils, and basophils), lymphocytes, plasma cells, and macrophages.[4] In the inflammatory response process, nitric oxide (NO) and prostaglandin (PG)E₂, acts as a molecular messenger for various physiologic functions and pathologic processes.[5] Expressions of iNOS and COX-2, two major regulate enzymes of NO and PGE₂ production, respectively,[5,6] are primarily controlled at the transcriptional stage, and nuclear factor kappa B (NF-κB) is well recognized to play a key role.[7]

In the present study, we compared the efficacy of G. bicolor in modulating NO and PGE₂ production in LPS-stimulated RAW 264.7 cells, and explored the possible molecular mechanism involved.

MATERIALS AND METHODS

Preparation of the G. bicolor ether extract (GBEE)

Gynura bicolor (Roxb. and Willd.) DC. were purchased from Yuanshan Village (Ilan, Taiwan). The same plant growing in Department of Forestry, National Chung Hsieh University (NCHU) and identified by Dr. Yen Hsueh Tseng. A voucher specimen (TCF13549) has been deposited at NCHU. Leaves of G. bicolor were removed, cleaned, and blended in cold water (4°C, w/w: 1/1). The homogenates were extracted with ether (v/v:1/1) for 6 h at 4°C. Finally, extracts were stirred on a stirring plate for 4 h, and then were dried in a rotary vacuum dryer. The percent yield of the ether extract was 0.3% (w/w).

Chemical composition analysis of the GBEE

In this study, the β-carotene content was analyzed as described by Xu et al.[8] The gallic acid content was analyzed as described by Wang et al.[9] The quercetin content was evaluated as described by Wang and Morris.[10] The rutin content was determined as described by Krizman et al.[11] The chlorophyll content was determined using a procedure described by Witham et al.[12]

Cell culture and treatment

The mouse macrophage-like cell line, RAW 264.7, was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). RAW 264.7 cells (at passage 8–13) were maintained in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO₂. Cells were plated at a density of 8 × 10⁵ per 30-mm culture dish and were incubated until 90% confluence was reached. To determine changes in cell viability, nitrite and PGE₂ concentrations, and iNOS, COX-2, p65, and IκB protein levels, cells were treated with 15, 30, 60, or 120 μg/ml of the GBEE in the presence of 1 μg/ml lipopolysacchadides (LPS; Sigma Co., St. Louis, MO) for various time intervals as indicated. For the electrophoretic mobility shift assay (EMSA), cultures were preincubated with GBEE for 3 h and then treated with 1 μg/ml LPS for 90 min. All GBEEs were dissolved in methanol, and the final concentration of methanol added to the medium was 0.1% (v/v).

Cell viability assay

The mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan was used to measure cell respiration as an indicator of cell viability.[13] The absorbance in cultures treated with LPS alone was regarded as 100% cell viability.

Nitrite and PGE₂ determination

Nitrate in the media was measured by the Griess assay[14] and was used as an indicator of NO production in cells. The absorbance at 550 nm was measured and calibrated using a standard curve of sodium nitrite prepared in culture medium. PGE₂ secreted into the medium was measured by a competitive enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). Concentrations of the mediator in the samples were calculated according to reference calibration curves of the standards. The results are expressed in pg/μl.

Immuoblot analysis

Cells were washed twice with cold phosphate buffered saline (PBS) and then harvested in 200 μl of lysis buffer containing 10 mM Tris–HCl, 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 20 μg/mL aprotinin at pH 7.4. Cellular protein levels were determined by the method of Lowry et al.[15] Equal amounts of cell protein in each sample were applied to 10% sodium dodecylsulfate (SDS) polyacrylamide gels.[16] After electrophoresis, proteins separated on the gels were transferred to polyvinylidene difluoride membranes.[17] The membranes were then incubated with an anti-iNOS, COX-2, phosphorylated (p)-IκBa, or P65 antibody (Santa Cruz Biotechnology Co., Santa Cruz, CA) at 37°C for 1 h, followed by a peroxidase-conjugated secondary antibody. The immunoreactive bands were detected with an enhanced chemiluminescence kit (Amersham ECL Advance Western Blotting Detection Kit, Amersham Pharmacia Biotech, Buckinghamshire, UK). Band intensities were measured with an AlphaImager 2000 (Alpha Innoctech, San Leandro, CA).

Nuclear protein preparation and EMSA

After preincubation with GBEE or its active peinciples for 8 h then with or without LPS for 3 h, isolation of cytosolic and nuclear fraction were practiced by a Nuclear Extraction Kit (Cayman Chemical Company. Ann Arbor, Michigan, USA). The nuclear proteins was collected and stored at -70°C until the EMSA was performed. In this study, the NF-κB DNA binding activity was analyzed by EMSA as described previously.[18]

Statistical analysis

Data are expressed as the mean ±SD from at least four independent experiments. Differences among treatments were analyzed by a one-way analysis of variance (ANOVA) and Tukey's test using the Statistical Analysis System (SAS, Cary, NC). P values of < 0.05 were considered significant.

RESULTS

Chemical composition of the GBEE

To determine which major pigment compounds are rich in GBEE, there are three prime plant pigments that were detected: 11.5 μg chlorophyll/g GBEE, 25.3 μg quercetin/g GBEE, and 7460 μg b-carotene/g GBEE, respectively. Gallic acid and rutin were not detectable in the GBEE.

Cell viability

In this study, a MTT assay was used to test whether up to 120 μg/ml of the GBEE caused cell damage. As shown in [Figure 1], when RAW 264.7 cells were treated with 15, 30, 60, or 120 μg/ml GBEE in the absence or presence of 1 μg/ml LPS for 24 h, no changes on the growth of the cells were observed.

Figure 1.

Effect of the Gynura bicolor ether extract (GBEE) on the cell viability of RAW 264.7 cells. RAW 264.7 cells were treated with 1 μg/ ml lipopolysaccharide (LPS) alone or with 15–120 μg/ml of the GBEE for 24 h, and cell viability was measured by an MTT assay. Values are the mean±SD of five separate experiments and are expressed as a percentage of the DMSO vehicle control

NO and PGE₂ production

As shown in [Figure 2A], NO production was induced by LPS compared with the vehicle control group (P < 0.05). When RAW 264.7 cells were co-treated with the GBEE, LPS-induced NO production dose-dependently decreased. A 30% decrease was noted in cells treated with 120 μg/ml GBEE (P < 0.05). In addition, a dose-dependent decrease in LPS-induced PGE₂ production was found with GBEE treatment. In the presence of 30, 60, or 120 μg/ml GBEE treatments, PGE₂ levels were 72%, 56%, and 10%, respectively, of cells treated with LPS alone (P < 0.05) [Figure 2B].

Figure 2.

The Gynura bicolor ether extract (GBEE) suppressed lipopolysaccharide (LPS)-induced NO and PGE2 production. RAW 264.7 cells were treated with 1 μg/ml LPS alone or with various concentrations of the GBEE for 24 h. NO production in cells treated with LPS alone was denoted as 100%. Values are the mean±SD of five separate experiments. (a–c) Values not sharing the same letter significantly differ (P < 0.05)

iNOS and COX-2 protein expressions

Consistent with changes in NO and PGE₂ production, similar suppression of iNOS and COX-2 protein expressions was noted in cells treated with the GBEE. Immunoblots revealed that iNOS levels in RAW264.7 cells treated with 15, 30, 60, or 120 μg/ml of the GBEE were 90%, 86%, 75%, and 64% that of cells treated with LPS alone (P < 0.05) [Figure 3A]. A similar dose-dependent decrease in COX-2 expression by the GBEE was also noted. When RAW 264.7 cells were treated with 30, 60, or 120 μg/ml of the GBEE, COX-2 protein levels were significantly inhibited by 16%, 34%, and 41%, respectively, compared with the LPS-treated group (P < 0.05) [Figure 3B].

Figure 3.

Suppression of lipopolysaccharide (LPS)-induced iNOS and COX-2 protein expressions by the Gynura bicolor ether extract (GBEE). RAW 264.7 cells were treated with 1 μg/ml LPS alone or with 15–120 μg/ ml of the GBEE for 6 or 24 h, and cellular proteins were used for iNOS or COX-2 protein determinations. The iNOS and COX-2 protein levels of each sample were quantified by densitometry and are expressed as the percentage of those treated with LPS alone. Data are the mean±SD of at least five separate experiments. (a–e) Values not sharing the same letter significantly differ (P < 0.05)

NF-κB activation

To examine whether suppression of iNOS and COX-2 expressions by the GBEE is dependent on inhibition of LPS-induced NF-κB activation, both western blot and EMSA assays were performed. [Figure 4A] shows that the p-IκBα protein was significantly decreased by 47% after 120 μg/ml GBEE treatment (P < 0.05). As shown in [Figure 4B], nucleic p-65 protein levels significantly decreased after 30, 60, and 120 μg/ml GBEE treatment by 3–41% (P < 0.05). NF-κB levels, however, significantly increased in nuclear extracts of LPS-stimulated cells. Results of changes in p-65 protein levels in the nucleic fraction show that the GBEE significantly decreased the translocation of NF-κB from the cytosol to nuclei in RAW 264.7 cells. Furthermore, [Figure 5C] shows that the DNA-binding activity of the NF-κB nuclear protein significantly increased in the LPS control group. However, the DNA-binding activity of the NF-κB nuclear protein was significantly inhibited when macrophages were treated with 30, 60, or 120 μg/ml of the GBEE.

Figure 4.

Suppression of lipopolysaccharide (LPS)-induced nuclear factor (NF)-κB activation by the Gynura bicolor ether extract (GBEE). RAW 264.7 cells were pretreated with 0–120 μg/ml of the GBEE for 3 h, followed by adding 1 μg/ml LPS for an additional 60 min. Phosphorylated (p)-IκBα (A) and nuclear p65 (B) protein levels of each sample were measured by immunoblotting. Nuclear NF-κB binding to DNA was determined by an EMSA (C) as described in “Materials and Methods” section. Proteins in each immunoblot were quantified by densitometry and are expressed as the percentage of that treated with LPS alone. Data are the mean±SD of at least five separate experiments. (A–C)^abc Values not sharing the same letter significantly differ (P< 0.05)

Figure 5.

Proposed model for the molecular mechanisms of the regulation by the Gynura bicolor ether extract (GBEE) of inflammation induced by lipopolysaccharide (LPS) in RAW264.7 cells

DISCUSSION

G. bicolor is a common dietary vegetable in Taiwan and Far East. To date, in vitro and in vivo models of biological evidence of the efficacy of G. bicolor are still limited. Recently, Lu et al.[1] shows the major constituents of G. bicolor related to it pigment sources and physiological effects are possibly the rich flavonoids. Previous study shows that G. bicolor are rich in anthocyanin and there are three major anthocyanidins, peralgonidin, delphinidin, and malvidin, found in G. bicolor.[19,20] Recently, a higher contents of sesquiterpene compounds such as beta-caryophyllene, alpha-caryophyllene, and alpha-copaene were found in G. bicolor by GC-MS.[21] In addition to anthocyanin and sesquiterpene compounds, GBEE was found containing chlorophyll, quercetin, and bata-carotene in this study.

In unstimulated cells, NF-κB is sequestered in the cytoplasm by binding to the inhibitor protein, IκB.[22] Exposure to a variety of external stimuli, including inflammatory cytokines, oxidative stress, ultraviolet irradiation, or bacterial endotoxins, causes the phosphorylation of IκBα, which leads to the disassociation of NF-κB and IκBα.[23,24] Activated NF-κB is then translocated to nuclei, where it binds to the cis-acting κB enhancer element of target genes, including numerous inflammatory-, antioxidant-, and cell survival-related genes, and activates their transcription.[25,26] Previous studies showed that 0.5–200 mM β-carotene acts as an antitumor agent in colon carcinogenesis through decreasing COX-2 expression (P < 0.05) and PGE₂ production (P < 0.05) in human colon cancer cells by inhibiting IκB phosphorylation and degradation.[27] In A549 cells and Chang liver cells, 5–200 μM quercetin suppressed IκB phosphorylation by inhibiting IκB kinase activity and enhanced NF-κB nuclear translocation, which subsequently attenuated interleukin-1b, interferon-g, and tumor necrosis factor-a mixture (20 ng/ml of each)-induced iNOS and COX-2 gene transcription.[28,29] GBEE are rich in these three compounds, they may be involving antiinflammatory response of RAW264.7 cells induced by LPS.

In the present study, GBEE suppressed activation of NF-κB downregulated NO and PGE₂ production. These findings indicate the potent antichronic inflammatory activity of G. bicolor.