Anti-inflammatory effects of luteolin and luteoloside from Taraxacum coreanum in RAW264.7 macrophage cells

Abstract

The effects of luteolin (LT) and luteoloside (LS) from Taraxacum coreanum, using lipopolysaccharide (LPS)/interferon-gamma (IFN-γ)-induced RAW264.7 macrophage cells, on anti-inflammation were investigated. Our study was focused on the ethyl acetate fraction from T. coreanum (ETC) and its active compounds and their protective role against inflammation. The ETC and its active compounds, LT and LS, showed dose-dependent inhibitory activity against the production of nitric oxide (NO) and reactive oxygen species (ROS) in LPS/IFN-γ-stimulated RAW264.7 cells. In addition, ETC and its active compounds inactivated nuclear factor-kappa B and down-regulated inflammatory mediators. The results also showed that treatment with ETC, LT, and LS decrease pro-inflammatory cytokines, tumor necrosis factor-alpha, and interleukin-6. In conclusion, our studies indicated that ETC has anti-inflammatory activity owing to inhibition of NO/ROS generation and down-regulation of inflammatory mediators and cytokines. Moreover, LT and LS are bioactive compounds of ETC with protective effects against inflammation.

Introduction

Inflammation is the biological response against pathogens that cause cell injury in the human body. Normal inflammation is immune response against harmful stimuli or damage by up-regulation of anti-inflammatory cytokines/mediators (Lawrence et al. 2002). However, chronic inflammation is associated with pro-inflammatory cytokines production and increased risk of tissue damage and other degenerative disorders (Allavena et al. 2008). The stimulation of macrophages with lipopolysaccharide (LPS) leads to the secretion of inflammatory cytokines. Nuclear factor-kappa B (NF-κB) is activated by inflammatory stimuli, such as LPS; activated NF-κB modulates the expressions of pro-inflammatory enzymes and cytokines (Karin and Ben-Neriah 2000). Nitric oxide (NO) is a reactive radical which is produced by inducible nitric oxide synthase (iNOS), and regulates physiological and pathological conditions (Sacco et al. 2006). However, the over-expression of NO induces various harmful responses such as tissue damage and acute or chronic inflammatory diseases (Kaplanski et al. 2003). Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) can induce inflammatory progression through mediators, iNOS and cyclooxygenase-2 (COX-2) (Warren 1990). NF-κB-targeted treatments might be effective against diseases, because of its role in the pathogenesis of inflammatory gene expression.

Taraxacum coreanum (TC) is a plant native to Korea and Japan. TC is used as a medicine for the treatment of diuretic disorder or inflammation (Koo et al. 2004; Lee et al. 2013). Recent studies also demonstrated that it has hypolipidemic and anti-oxidant activity against oxidative stress (Chiou et al. 1997). Flavonoids are polyphenol compounds that are important to human health. They possess a wide range of biological effects on inflammation, allergy, and asthma (Middleton and Kandaswami 1992). Wolbis et al. (1993) identified that flavonoids, quercetin, luteolin (LT), luteolin-7-glucoside (LS), and quercetin-7-glucoside in dandelion leaves and flower extracts. In particular, the total content of LT and LS in TC was contained more than that in T. ohwianum and T. officinale (Lee et al. 2011). However, the anti-inflammatory activity of the active compounds in TC has not been studied yet.

To investigate the anti-inflammatory effects of ethyl acetate (EtOAc) fraction of TC (ETC) and its active compounds, we used LPS- and interferon-gamma (IFN-γ)-stimulated RAW264.7 cells to induce inflammation by increased NO production. In addition, the regulatory mechanisms against the inflammatory process were also studied by measurement of the levels of inflammatory cytokines and mediators induced by NF-κB activation in LPS/IFN-γ-stimulated RAW264.7 cells.

Materials and methods

Plant materials

Aerial parts of TC collected at sides of the West Coast Express Highway (geographic coordinates: 36°53′35″N 126°37′41″E) in the Republic of Korea by permission of Korea Expressway Corporation were used.

Instruments and reagents

RAW264.7 macrophage cells from Korea Cell Line Bank (KCLB, Seoul, Korea) were used. To culture cells, fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), and penicillin/streptomycin were supplied from Welgene (Daegu, Korea). IFN-γ was from Pepro Tech (Rocky Hill, NJ, USA). The LPS, Griess reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were supplied from Sigma Chemical Co. (St Louis, MO, USA).

Preparation of ETC and its active compounds

Freeze-dried TC was extracted with methanol (MeOH) for 3 h; the process of MeOH extraction was repeated 8 times. The extract was concentrated by a rotary evaporator and suspended in water. The combined extract was partitioned with n-hexane, chloroform (CHCl₃), EtOAc, and n-butanol (n-BuOH), successively. A portion of the EtOAc fraction was subjected to a silica gel column chromatography using a gradient system of n-hexane–EtOAc and EtOAc–MeOH to yield LT and LS (Fig. 1).

Fig. 1

Chemical structures of LT and LS

Cell culture

The RAW264.7 cells maintained at 37 °C in a CO₂ (5 %) incubator with DMEM containing penicillin/streptomycin (1 %) and FBS (10 %) were sub-cultured weekly with 0.05 % trypsin-ethylenediaminetetraacetic acid (EDTA) in PBS.

Cell viability

After the cells reached confluence, the cells were seeded at 5 × 10⁴ cells/well into 24-well plates, for 2 h incubation, and then treated with samples for 24 h. RAW264.7 cells were then stimulated with LPS (1 μg/mL)/IFN-γ (10 ng/mL) for 24 h. The cells were incubated with 1 mL of MTT solution (5 mg/mL) for 4 h at 37 °C, and the medium containing MTT was removed. And then, the formazan crystals were dissolved with 1 mL of DMSO, and viable cells were detected to measure absorbance at 540 nm (Mosmann 1983).

Measurement of NO production

The NO production was investigated as the nitrite accumulation in the medium treated with the Griess reagent. To measure nitrite, the cell supernatants were added with a same volume of the Griess reagent. And then, the nitrite concentration was measured using microplate spectrophotometer at a wave length of 540 nm.

Measurement of ROS production

The ROS scavenging activity of ETC and its active compounds was measured using dichlorodihydrofluorescein diacetate (DCFH-DA) (Cathcart et al. 1983). RAW264.7 cells were incubated with LPS/IFN-γ for 24 h. After that, ETC (5, 25, 50, and 100 μg/mL) and its active compounds, LT and LS (0.5, 2.5, 5, and 10 μg/mL), were added for 24 h at 37 °C. Then florescence was read for 60 min, at wavelengths of 480 nm for excitation and 535 nm for emission, using a florescence plate reader (BMG LABTECH, Ortenberg, Germany).

RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)

According to the manufacturer’s instruction, total RNA was isolated using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). Cells were lysed by the Trizol reagent and RT-PCR was performed using TOP script One-step RT-PCR (Enzynomics, Daejeon, Korea). The RNA was reverse-transcribed into cDNA which is used as a template for amplification of RT-PCR (Table 1). The products of PCR were analyzed on 1 % agarose gels; the expression was visualized under LED slider imager (Maestrogen, NV, USA).

Table 1 Primers and conditions used in PCR

Statistical analysis

Data are expressed as the mean ± SD. Statistical significance was measured by one-way ANOVA, followed by Duncan’s post hoc tests (P < 0.05).

Results

Effect of ETC and its active compounds on NO production

LPS/IFN-γ treatments induced NO formation in RAW264.7 cells (Fig. 2). Exposure of LPS/IFN-γ-stimulated RAW264.7 cells to ETC (5, 25, 50, and 100 μg/mL) and its active compounds, LT and LS (0.5, 2.5, 5, and 10 μg/mL), for 24 h did not have any cytotoxic effects at a concentration lower than 100 μg/mL ETC and 10 μg/mL active compounds (data not shown). LPS significantly increased NO levels, whereas the treatment with various concentrations of ETC remarkably inhibited NO production in LPS/IFN-γ-activated RAW264.7 cells dose-dependently. In addition, we checked the effects of LT and LS on NO production. The results showed that NO production was decreased by LT and LS treatments, in a dose-dependent manner. Especially, treatments with LT at 2.5, 5, and 10 μg/mL suppressed NO production by 46.07, 43.98, and 42.18 %, respectively, compared to LPS/IFN-γ-stimulated control group (100 %). These results suggested that both ETC and its active compounds have inhibitory effects on NO production in LPS/IFN-γ-stimulated RAW264.7 macrophage cells.

Fig. 2

Effects of ETC (A), LT, and LS (B) on NO production in RAW264.7 cells treated with LPS/IFN-γ. Values are the mean ± SD. a–e Means with different letters are significantly different (P < 0.05) as determined by Duncan’s multiple range test

Effect of ETC and its active compounds on ROS production

Exposure of RAW 264.7 cells to LPS/IFN-γ for 24 h increased intracellular ROS levels (Fig. 3). In contrast, ETC treatments prevented the increase of LPS/IFN-γ-induced ROS production. When the cells were treated with ETC at 100 μg/mL, ROS production decreased by 71.27 % compared to the control group (100 %). In addition, the active compounds markedly decreased ROS formation in the presence of LPS/IFN-γ (Fig. 3). In particular, LT had a stronger ROS scavenging activity than LS.

Fig. 3

Effect of ETC (A), LT, and LS (B) on levels of ROS in RAW264.7 cells treated with LPS/IFN-γ. Values are the mean ± SD. a–f Means with different letters are significantly different (P < 0.05) as determined by Duncan’s multiple range test

Effect of ETC and its active compounds on TNF-α and IL-6 release

Figure 4 shows the effect of ETC and its active compounds on production of TNF-α and IL-6 in LPS/IFN-γ-stimulated RAW264.7 cells. As a result, treatments with ETC and its active compounds inhibited the production of TNF-α and IL-6 dose-dependently. LT inhibited TNF-α and IL-6 production more significantly than LS, suggesting that LT inhibited TNF-α and IL-6 production more significantly than its glycoside LS in LPS/IFN-γ-stimulated RAW264.7 cells.

Fig. 4

Effect of ETC (A), LT, and LS (B) on mRNA expressions of NF-κB, iNOS, and COX-2 in RAW264.7 cells treated with LPS/IFN-γ. Values are the mean ± SD. a–f Means with different letters are significantly different (P < 0.05) as determined by Duncan’s multiple range test

Effect of ETC and its active compounds on mRNA expression of NF-κB, iNOS, and COX-2

As shown in Fig. 5, LPS/IFN-γ significantly increased mRNA expressions of pro-inflammatory mediators, NF-κB, iNOS, and COX-2, in the control group compared to untreated group. However, treatments with ETC and its active compounds, LT and LS, suppressed NF-κB, iNOS, and COX-2 expressions dose-dependently. These results indicated that treatments with ETC and its active compounds block the degradation of NF-κB activation, and therefore, iNOS and COX-2 expressions were attenuated in LPS/IFN-γ-stimulated RAW264.7 cells.

Fig. 5

Effect of ETC, LT, and LS on TNF-α (A) and IL-6 production (B) in RAW264.7 cells treated with LPS/IFN-γ. Values are the mean ± SD. a–e Means with different letters are significantly different (P < 0.05) as determined by Duncan’s multiple range test

Discussion

Chronic inflammatory diseases were accompanied by increased ROS production and inflammatory gene expressions (Wang et al. 1994; Wiseman and Halliwell 1996). TC, a member of Asteraceae, has been used as a medicinal herb to treat diuretic and liver disease. Several reports suggested that flavonoids, one of the large families of plant constituents, have inhibitory activity against inflammation (Havsteen 1983; Kim et al. 2004). According to Hu and Kitts, LT and LS, which are flavones isolated from dandelion flower extract, alleviated oxidative reaction (Hu and Kitts 2003). In our previous study, the EtOAc fraction had the strongest anti-oxidant property compared to the other fractions (Lee et al. 2012). Therefore, we focused on the anti-inflammatory effects of ETC (5, 25, 50, and 100 μg/mL) and the active compounds (1, 2.5, 5, and 10 μg/mL) isolated from ETC. The concentration of active compounds, LT and LS (1, 2.5, 5, and 10 μg/mL), is equivalent to the 1.7, 8.7, 17.5, and 35 μM of LT and 1.1, 5.6, 11.1, and 22.3 μM of LS, respectively. We hypothesized that ETC and its active compounds regulate ROS generation and inflammatory gene expression.

Macrophage cells are important to inflammation reaction induced by cytotoxicity and multiple inflammatory diseases through cellular response (MacMiking et al. 1997). RAW 264.7 macrophage cells stimulated by LPS/IFN-γ enhanced the expressions of inflammatory cytokines and mediators (Xie and Nathan 1994). The over-production of ROS by macrophage cells is one of the most important hallmarks of inflammatory reaction. D’Acquisto et al. (2002) reported that ROS are involved in cellular stress and modulation of NF-κB activation. Therefore, inhibition of ROS production is considered a therapeutic target for preventing inflammatory diseases. ETC, LT, and LS showed the most potent anti-oxidant and anti-inflammatory effects through inhibition of ROS generation (Fig. 3). These results suggested that the anti-oxidative and anti-inflammatory activities of ETC are attributed to its active compounds, LT and LS.

NO, a reactive free radical, is produced by l-arginine and is central role in the inflammatory process or infection (Marletta 1993). At adequate concentrations, NO can generate and regulate intracellular signals, thereby affecting the immune cells. However, uncontrolled production of NO is associated with inflammation. Our results demonstrated that ETC and its active compounds have concentration-dependent inhibition of NO production. Moreover, treatments with LT and LS on RAW264.7 cells stimulated with LPS/IFN-γ significantly reduced NO production. The previous reports demonstrated that cytotoxicity of LS was lower than its aglycone form, LT. Other reports confirmed that glycosylation may contribute to reduced toxicity and that LS had stronger NO suppression activity than LT (Wang and Mazza 2002a; Hu and Kitts 2004).

To investigate the inhibitory mechanisms of NO production, the effects of ETC and its active compounds on mRNA expressions in LPS/IFN-γ-induced RAW264.7 cells were determined. NF-κB is a transcriptional activation factor that plays a crucial role in the inflammatory response, including the regulation of iNOS and COX-2 (Spitzer et al. 2002). Therefore, iNOS and COX-2 expressions, and NF-κB activation have been used as biomarkers for anti-inflammatory activity. The present results indicated that ETC, LT, and LS suppressed NF-κB activation as well as LPS/IFN-γ-induced iNOS and COX-2 mRNA expression. Previous study mentioned that phenolic compounds inhibited inflammatory mediators and suppressed inflammatory responses through NF-κB pathway (Rao et al. 2005; Jung et al. 2007). Therefore, it is strongly suggested that the inhibition of NO production may be owing to decreased iNOS and COX-2 expressions.

TNF-α and IL-6 are multifunctional inflammatory cytokines. These cytokines contribute to tissue damage and multiple organ failure (Akira et al. 1990; Hirano 1992). IL-6 is a pro-inflammatory cytokine involved in the regulation of immune responses, which are induced by macrophages (Tilg et al. 1994). Moreover, a large amount of TNF-α secretion produced by LPS leads to NO generation (Aggarwal and Natarajan 1996). Thus, the inhibition of cytokine production is important for potential anti-inflammatory activity. Several studies demonstrated that phenolic compounds possessed TNF-α and IL-6 inhibitory activities (Xagorari et al. 2001; Wang and Mazza 2002b). In addition, dandelion leaf extract suppressed the TNF-α production by inhibiting IL-1 production in LPS-stimulated rat astrocytes (Kim et al. 2000). These results indicated that the production of TNF-α and IL-6 significantly increased in LPS/IFN-γ-stimulated RAW264.7 cells. However, ETC, LT, and LS possess inhibitory efficacy against the production of TNF-α and IL-6. In particular, ETC and its active compounds showed stronger inhibitory effects against IL-6 than TNF-α production. These results suggested that down-regulation of IL-6 might be related to its production of TNF-α. In addition, compared to the aglycone form, the glycoside form had stronger inhibitory effects against TNF-α and IL-6 production in RAW264.7 macrophage cells.

To investigate the anti-inflammatory effect of LT aglycone and its glycosides, the production of NO and cytokines, which have crucial role during the inflammatory process by defensing against foreign agents, was measured. Our study showed that LT and LS significantly inhibited production of NO dose-dependently. In particular, LS had a strong NO scavenging effect rather than LT by down-regulating iNOS and COX-2 gene expression. In contrast to inhibitory effect of LS on NO generation, treatment of LT resulted in a more considerable decrease in TNF-α and IL-6 secretion in LPS/IFN-γ-stimulated RAW264.7 macrophage cells. Previous study has also reported that stronger suppression of NO production was observed in LS-treated group than LT-treated group (Hu and kitts 2004). However, Park and Song (2013) found that LT was more effective in inhibition of LPS-induced prostaglandin E₂ (PGE₂) production and up-regulation of NF-κB and activator protein (AP)-1 expression. These findings suggest that LT and LS display a selective activity in secretion of pro-inflammatory cytokines, NO production, and regulation of inflammation-related mRNA expression. Taken together, the inhibition of NO production by LS was attributed to the suppression of iNOS and COX-2 expression, rather than a cytokines. On the other hand, LT plays anti-inflammatory role by suppression of TNF-α and IL-6 secretion.

In conclusion, the present study revealed that ETC and its active compounds, LT and LS, had anti-inflammatory effects on LPS/IFN-γ-induced RAW264.7 cells. They inhibited the generation of ROS and NO induced by LPS/IFN-γ. In addition, activation of NF-κB was inhibited by the expressions of iNOS and COX-2, and the production of TNF-α and IL-6 was inhibited by the treatment with LT and LS. These results suggest that ETC would play a beneficial role in alleviating the inflammatory process and that LS and LT are attributed to the protective role of ETC.