Introduction

Inflammation can be divided principally into acute and chronic inflammation. Acute inflammation is the body’s normal protective response to an injury, irritation, or surgery [1]. However, chronic inflammation induces various chronic diseases including cancer, cardiovascular diseases, Alzheimer’s disease, type II diabetes, arthritis, autoimmune diseases, neurological diseases, and pulmonary diseases [2, 3]. During the inflammatory process, interleukins or growth factors induce the proliferation and activation of leukocytes [4]. Activated leukocytes then mediate inflammation and programmed cell death [5, 6]. Understanding these mechanisms could therefore provide therapeutic clues to modulate the activity of leukocytes during acute or chronic inflammation.

During the inflammatory process, various inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), pro-inflammatory cytokines, and adhesion molecules are closely associated with the classical symptoms of inflammation such as pain, heat, redness, swelling, and loss of function. NO is generated from amino acid l-arginine by the enzymatic action of inducible NO synthase (iNOS) which is stimulated during inflammation by bacterial endotoxins (e.g., lipopolysaccharide) and cytokines [7, 8]. The expression of pro-inflammatory cytokine and adhesion molecules is regulated by nuclear factor-κB (NF-κB), which is composed of p50 and p65 subunits [9, 10]. Under normal conditions, it is sequestered in the cytosol where it is bound to the inhibitor IκBα. When inflammation is induced by injury, irritation, or surgery, IκB undergoes ubiquitin-mediated degradation by proteasomes. The removal of IκB unmasks the nuclear-localization signals in both subunits of NF-κB, allowing their translocation to the nucleus. In the nucleus, NF-κB activates transcription of numerous target genes such as pro-inflammatory cytokine and adhesion molecules [11, 12]. Thus, inhibitors acting on the NF-κB pathway can be candidates for therapeutic agents against acute or chronic inflammation.

Chlorogenic acid (Fig. 1a) is an ester formed from cinnamic acids and quinic acid and is also known as 5-O-caffeoylquinic acid (5-CQA) (IUPAC nomenclature) or 3-CQA (pre-IUPAC nomenclature) [13]. It is synthesized in the process of aerobic respiration and is abundant in coffee beans, potatoes, and apples [14]. It is reported that chlorogenic acid has various functions such as anti-oxidant and hypotensive activity [15, 16]. Furthermore, chlorogenic acid prevents cardiovascular disease by increasing high-density lipoprotein [17]. Most natural products containing chlorogenic acid showed anti-inflammatory effects, suggesting that chlorogenic acid may be a potential anti-inflammatory agent [9, 18]. To date, however, it remains largely unknown, and so we need to study the anti-inflammatory effects of chlorogenic acid.

Fig. 1
figure 1

Effects of chlorogenic acid on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV2 microglia and RAW 264.7 macrophages. a Chemical structure of chlorogenic acid. b, c NO assay using the Griess reagent. Cells were treated with LPS (1 μg/ml) in the absence or presence of chlorogenic acid at different concentrations (0, 5, and 20 μM, respectively) for 24 h. Amounts of NO were determined using the Griess reagent and a standard curve created using NaNO2 in BV2 (b) and RAW 264.7 cells (c); **P < 0.01, vehicle vs. LPS-treated group; ++ P < 0.01, LPS vs. LPS plus chlorogenic acid-treated group. d Cell viability assays using the MTT assay were then performed for 24 h following treatment with chlorogenic acid (0, 1.25, 2.5, 5, 10, and 20 μM). Values are expressed relative to that of vehicle-treated cells, normalized to 100 %. Values shown in the graphs are mean ± standard deviation (SD) and were obtained from three independent experiments

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Here, we investigated the anti-inflammatory effects of chlorogenic acid and how it induces an anti-inflammatory effect in lipopolysaccharide (LPS)-inflamed murine RAW 264.7 macrophage cells. Our findings suggest that chlorogenic acid may be a candidate for use in the treatment of a variety of inflammatory diseases.

Materials and methods

Reagents and cells

RAW 264.7 and BV2, widely used murine microglia/macrophage cell lines, were obtained from the Korean Cell Line Bank (KCLB) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL) supplemented with 10 % fetal bovine serum (FBS; Gibco BRL), penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco BRL). LPS, chlorogenic acid, and Griess reagent were purchased from Sigma-Aldrich.

Nitric oxide assay

Nitric oxide assay was performed as described previously [19]. RAW 264.7 and BV2 cells at 5 × 104 cells/well were cultured in flat-bottom 96-well plates in triplicate for 24 h and then incubated with LPS (1 μg/ml) with or without pretreatment with chlorogenic acid for 1 h. After 24 h of culture, the culture supernatant was collected; this was used as a measure of NO production. The culture supernatant (50 μl) was mixed with an equal volume of Griess reagent and the absorbance was measured at 550 nm. Finally, the concentration of nitrite was calculated from a standard curve drawn with known concentrations of sodium nitrite dissolved in DMEM. The relative cell viability of each parallel experimental group (n = 3) was expressed in percentage (%) based on a no-additions (NA) control, while untreated controls were considered as 100 % viable.

Reverse transcriptase–polymerase chain reaction (RT-PCR) and real-time PCR

RT-PCR was performed as described previously [20]. RAW 264.7 cells were cultured in the presence of each sample alone or in combination with LPS in a 6-well plate (1 × 106 cells/ml) for 6 h. Total cellular RNA was isolated using the TRIzol (Invitrogen) following the manufacturer’s instructions. Total RNA (3 μg) was reverse-transcribed into cDNA using M-MLV Reverse Transcriptase (Promega). The PCR primers used in this study are listed below and were purchased from Bioneer: forward strand iNOS 5′-cagctgggctgtacaaacctt-3′, reverse strand iNOS 5′-cattggaagtgaagcgtttcg-3′; forward strand IL-1β 5′-aagggctgcttccaaacctttgac-3′, reverse strand IL-1β 5′-tgcctgaagctcttgttgatgtgc-3′; forward strand TNF-α 5′-catcttctcaaaattcgagtgacaa-3′, reverse strand TNF-α 5′-tgggagtagacaaggtacaaccc-3′; forward strand COX-2 5′-ttcaaaagaagtgctggaaaaggt-3′, reverse strand COX-2 5′-gatcatctctacctgagtgtcttt-3′; forward strand IL-6 5′-gaggataccactcccaacagacc-3′, reverse strand IL-6 5′-aagtgcatcatcgttgttcataca-3′; forward strand CXCL1 5′-tggggacaccttttagcatc-3′, reverse strand CXCL1 5′-cttgaaggtgttgccctc-3′; forward strand β-actin 5′-agagggaaatcgtgcgtgac-3′, reverse strand β-actin 5′-ggccgtcaggcagctcatag-3′. A variable number of cycles were used to ensure that amplification occurred in the linear phase. PCR amplification employed β-actin as the internal control, and PCR products were separated on a 2.0 % agarose gel and visualized by RedSafe nucleic acid staining (Intron, Korea), and UV irradiation. Real-time PCR was done with the same primers of RT-PCR and performed on a Rotor-Gene Q real-time PCR cycler (Qiagen) using Rotor-Gene SYBR Green RT-PCR kit (Qiagen) according to the manufacturer’s instructions.

Western blot analysis

RAW 264.7 cells were cultured in the presence of LPS or in combination with each sample in a 6-well plate (1 × 106 cells/well). After removal of the supernatants, extracts of RAW 264.7 cells were directly prepared in lysis buffer (0.5 % Triton, 50 mM β-glycerophosphate pH 7.2, 0.1 mM sodium vanadate, 2 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl urea, 2 μg/ml of leupeptin, and 4 μg/ml of aprotinin). The lysates were resolved by 10 % SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in Tris-buffered saline (10 mM Tris–Cl pH 7.4) containing 0.5 % Tween 20 and 5 % nonfat dry milk, incubated with the first specific antibody in blocking solution for 5 h at room temperature, washed, and incubated with the second antibody for 1 h at room temperature. The protein bands were detected by chemiluminescence (FUSION-SL4, Vilber). Monoclonal antibodies against iNOS, β-actin, and peroxidase-conjugated secondary antibody were from Santa Cruz Biotechnology. In a parallel experiment, cytosolic protein was prepared using buffer A [20 mM HEPES (pH 8.0), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 mm benzamidine, and 0.2 mm sodium orthovanadate]. The cell lysates were collected in homogenization tubes and were cleared by centrifugation twice at 2,500 rpm for 5 min at 4 °C. The cell pellets were discarded, and the supernatant was subjected to another centrifugation at 13,000 rpm for 30 min at 4 °C. At this stage, the supernatant was further centrifuged at 40,000 rpm for 1 h at 4 °C. The resulting supernatant was labeled the cytosolic fraction and used. Polyclonal antibodies against IκB and GAPDH were purchased from Santa Cruz Biotechnology.

Cell viability assay

RAW 264.7 cells were plated into 96-well plates at 5 × 104 cells/well and allowed to attach for 24 h. Prior to chlorogenic acid treatment, media was removed and replaced with 0.1 ml of fresh media. Confluent cells were stimulated with different concentrations of chlorogenic acid. After 24 h, the media was removed and replaced with 0.1 ml fresh media. Next, 20 μl of CellTiter 96® Aqueous One Solution Reagent (Promega, Madison, WI, USA) was added to each well, incubated at 37 °C for 2 h and then read at 490 nm.

Cell adhesion assays

In the cell–matrix adhesion assay, RAW 264.7 cells were added to each well coated with EMCs, which are fibronectin (Invitrogen), type I collagen (BD), laminin (BD), and gelatin (Sigma). After incubation for 15 min, RAW 264.7 cells were washed twice with PBS. Attached cells were stained with Crystal violet and washed twice. After lysis with 0.2 % NP-40, absorbance of lysates was analysed with ELISA at 590 nm.

Induction of endotoxin shock

ICR mice were injected three times intraperitoneally (i.p.) with a dose of 6.6 μg of LPS (330 μg/kg of body weight) and/or with a dose of 0.1 mg of chlorogenic acid (5 mg/kg body weight) for 3 days. Animals were killed at 24 h after LPS administration.

Immunofluorescent microscopy and quantification

RAW 264.7 or BV2 cells were plated at 1 × 105 cells/well in 4-well chamber slides (Falcon). For immunostaining, cells were washed twice with cold phosphate-buffered saline, fixed in 4 % paraformaldehyde for 10 min, permeabilized with 0.1 % Triton X-100 for 15 min, and blocked with 10 % normal goat serum for 30 min. Slides were incubated for 18 h at 4 °C with primary antibody at a 1:500 dilution, washed, and then incubated for 50 min with Alexa546-conjugated IgG (Molecular Probes) at a 1:1,000 dilution as secondary antibodies. Antibodies used for immunostaining were: ninjurin1 (Ninj1; 1:500, a kind gift from Dr. J. Milbrandt), iNOS (1:500, BD Biosciences), and p65 (1:200, Cell Signaling). Nuclei were stained using 4′-6-diamidino-2-phenylindole (DAPI, Invitrogen). Images were obtained with an Axiovert M200 microscope (Zeiss). Pixel intensities of iNOS, Ninj1, and DAPI were measured as percent area of immunoreactivity, using ImageJ, then recorded and compared statistically. All pixel intensities were measured and compared using images at ×40 magnification.

Data analysis and statistics

Quantification of band intensity was analyzed using ImageJ (http://rsb.info.nih.gov/ij/) and normalized to the density of the GAPDH, tubulin, or Ponceau S staining band. All data are presented as mean ± SD and changed into relative percentage. Statistical comparisons between groups were done using Student’s t test. P < 0.05 was considered statistically significant.

Results

Chlorogenic acid inhibits NO production in LPS-stimulated murine microglia/macrophages without cell toxicity

First, we investigated the effects of chlorogenic acid on LPS-induced NO production in mouse BV2 microglia and RAW 264.7 macrophages. Chlorogenic acid significantly inhibited LPS-induced NO production in a dose-dependent manner (Fig. 1b, c). To examine whether the inhibitory effect of chlorogenic acid on NO production resulted from cellular toxicity, cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The data showed that chlorogenic acid had no effect on cell viability up to a concentration of 20 μM (Fig. 1d). These results suggest that chlorogenic acid has an inhibitory effect on NO production without cell toxicity in microglial cells.

Chlorogenic acid inhibits iNOS expression in LPS-stimulated RAW 264.7 cells

Next, we examined the effects of chlorogenic acid on LPS-induced iNOS gene expression in RAW 264.7 cells. LPS-induced iNOS mRNA (Fig. 2a, b; upper) and protein (Fig. 2b; lower) expression were also significantly attenuated in a dose-dependent manner by chlorogenic acid. Moreover, immunofluorescence staining and quantification of iNOS pixel intensity (% area of immunoreactivity) showed that LPS-induced iNOS up-regulation is abolished by chlorogenic acid treatment (Fig. 2c, d). These results indicate that chlorogenic acid has an inhibitory effect on iNOS induction in RAW 264.7 cells.

Fig. 2
figure 2

Effects of chlorogenic acid on inducible NO synthase (iNOS) expression in LPS-stimulated RAW 264.7 cells. a, b Cells were treated with LPS (1 μg/mL) in the absence or presence of chlorogenic acid at different concentrations (0, 2, 5, and 20 μM, respectively) for 24 h. The iNOS mRNA level at 24 h was analyzed using real-time PCR (a) and conventional RT-PCR (b, upper) and then the iNOS protein level at 24 h (b, lower) was determined using Western blotting after LPS stimulation; **P < 0.01, vehicle vs. LPS-treated group; ++ P < 0.01, LPS vs. LPS plus chlorogenic acid-treated group. c After LPS (1 μg/ml) stimulation either with or without chlorogenic acid treatment for 24 h, cells were fixed and immunostained using an iNOS antibody and a goat anti-rabbit IgG conjugated with Alexa Fluor 488 (green). Cells were also stained with DAPI (4′,6-diamidino-2-phenylindole) to visualize nuclei (blue). d Immunostaining of iNOS was quantified using ImageJ (NIH Image). Values were normalized to 100 % for the immunoreactivity of the untreated control group. Error values are mean ± SD for each group (n = 1); *P < 0.05, vehicle vs. LPS-treated group; + P < 0.05, LPS vs. LPS plus chlorogenic acid-treated group. Images are 40× magnification (color figure online)

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Chlorogenic acid inhibits expression of pro-inflammatory cytokines in LPS-stimulated RAW 264.7 cells

We examined the effects of chlorogenic acid on the expression of pro-inflammatory cytokines such as IL-1β and TNF-α. LPS-induced expressions of IL-1β and TNF-α were significantly inhibited by chlorogenic acid (20 μM) at 24 h (Fig. 3a). To confirm the anti-inflammatory effect of chlorogenic acid, we further investigated the effect of chlorogenic acid on the expression of other inflammation-related genes. As shown in Fig. 3b, LPS caused mRNA induction of COX-2, IL-6, and CXCL1 and chlorogenic acid significantly attenuated LPS-induced COX-2, IL-6, and CXCL1 mRNA levels. Taken together, these results suggest that chlorogenic acid may reduce the expression of pro-inflammatory cytokines and chemokines.

Fig. 3
figure 3

Effects of chlorogenic acid on the mRNA expression of pro-inflammatory cytokines and other inflammatory-related genes in LPS-stimulated RAW 264.7 cells. a, b Cells were treated with LPS (1 μg/mL) in the absence or presence of chlorogenic acid at different concentrations (0, 2, 5, and 20 μM, respectively) for 24 h. The mRNA expression levels of pro-inflammatory cytokine genes (interleukin-1β and tumor necrosis factor-α) (a) and other inflammation-related genes (cyclooxygenase-2, chemokine (C-X-C motif) ligand 1 (CXCL1), and interleukin-6) at 24 h (b) were then determined after LPS (1 μg/ml) stimulation. Expression levels of mRNA were represented as relative to that of control and normalized to 1

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Chlorogenic acid inhibits Ninj1 expression and cell-to-matrix adhesion in LPS-stimulated RAW 264.7 cells

Adhesion molecules are important for a recognition system between leukocytes and other cells or cellular matrix proteins during inflammation. Among them, ninjurin1 (Ninj1) is known to increase movement to the site of the inflammation and the activity of leukocytes in both developmental processes and inflammatory responses [21]. Thus, we further investigated the effect of chlorogenic acid on the expression of Ninj1 both in vitro and in vivo and the adhesive activity of microglia/macrophages. LPS-induced expression of Ninj1 mRNA and protein was also significantly attenuated in a dose-dependent manner by chlorogenic acid (Fig. 4a, b). Moreover, immunofluorescence staining and quantification analysis of Ninj1 pixel intensity (% area of immunoreactivity) showed that chlorogenic acid abolishes Ninj1 up-regulation mediated by LPS treatment (Fig. 4c, d). Next, we examined the effect of chlorogenic acid on cell-to-matrix adhesion of RAW 264.7 cells. Under LPS-induced inflammatory conditions, the adhesion of RAW 264.7 cells on gelatin, type I collagen, laminin, and fibronectin matrix was increased compared with the control (Fig. 5). However, chlorogenic acid abolished the LPS-induced adhesion activity of RAW 264.7 cells in a dose-dependent manner (Fig. 5). Thus, these results showed that chlorogenic acid down-regulates Ninj1 expression and has an inhibitory effect on the adhesion activity of RAW 264.7 cells.

Fig. 4
figure 4

Effects of chlorogenic acid on Ninj1 expression in LPS-stimulated RAW 264.7 cells. a, b Cells were treated with LPS (1 μg/mL) in the absence or presence of chlorogenic acid at different concentrations (0, 2, 5, and 20 μM, respectively) for 24 h. Ninj1 mRNA level at 24 h was analyzed using real-time PCR (a) and conventional RT-PCR (b, upper) and then the iNOS protein level at 24 h (b, lower) was determined using Western blotting after LPS stimulation; **P < 0.01, vehicle vs. LPS-treated group; ++ P < 0.01, LPS vs. LPS plus chlorogenic acid-treated group. c Cells were treated with LPS (1 μg/ml) in the absence or presence of chlorogenic acid at 20 μM concentration. Immunofluorescent staining of Ninj1 (green). Nuclei were counterstained using DAPI (blue). d Immunostaining of Ninj1 was quantified using ImageJ (NIH Image). Values were normalized to 100 % for the immunoreactivity of the untreated control group. Error values are mean ± SD for each group (n = 1); **P < 0.01, vehicle vs. LPS-treated group; ++ P < 0.01, LPS vs. LPS plus chlorogenic acid-treated group. Images are 40× magnification (color figure online)

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Fig. 5
figure 5

Effects of chlorogenic acid on the cell-to-matrix adhesion activity in LPS-stimulated RAW 264.7 cells. a, b RAW 264.7 cells suspended in serum-free media were added to each well coated with extracellular matrix such as gelatin, type I collagen, laminin, and fibronectin. Non-adherent cells were removed by washing and the remaining adherent cells were quantified as described in “Materials and methods”. Cell-to-matrix adhesion assays used RAW 264.7 cells with either LPS (1 μg/ml) alone or chlorogenic acid. Adhesion values are expressed relative to the adhesion of vehicle-treated cells, normalized to 100 %. Values shown in the graphs are mean ± SD and were obtained from three independent experiments; *P < 0.05, vehicle vs. LPS-treated group; + P<0.05, LPS vs. LPS plus chlorogenic acid-treated group

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Inhibitory effects of chlorogenic acid are mediated by NF-κB suppression in LPS-stimulated murine microglia/macrophages

Since the activation of NF-κB by LPS can induce the expression of pro-inflammatory mediators, we checked the effect of chlorogenic acid on the NF-κB signaling pathway under LPS-induced inflammation. To evaluate whether chlorogenic acid could influence the turnover and subcellular distribution of IκB proteins, and therefore NF-κB activation, the amounts of IκBα in the cytosol were determined by immunoblot analysis. As Fig. 6a shows, the marked decrease of IκB proteins elicited by LPS was markedly impaired in the presence of chlorogenic acid. Moreover, an important nuclear accumulation of p65 (NF-κB) was observed in cells treated with LPS, and chlorogenic acid treatment significantly attenuated the observed nuclear translocation (Fig. 6b, c). Taken together, these results indicated that chlorogenic acid’s inhibition of the NF-κB signaling pathway may be the mechanism responsible for the suppression of NO and pro-inflammatory cytokines in LPS-stimulated murine microglia/macrophages.

Fig. 6
figure 6

Effects of chlorogenic acid on NF-κB signaling pathway in LPS-stimulated RAW 264.7 cells. a Cells were treated with LPS (1 μg/ml) in the absence or presence of chlorogenic acid at different concentrations (0, 2, 5, and 20 μM, respectively) for 24 h. Total cytosolic protein was subjected to 15 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western blotting using anti-IκBα. b Cells were treated with LPS (1 μg/ml) in the absence or presence of chlorogenic acid at 20 μM for 24 h. Total nuclear protein was subjected to 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western blotting using anti-NF-κB. NS indicates a non-specific immunoreactive band. Ponceau staining was used as a standard. c Cells were treated with LPS (1 μg/ml) in the absence or presence of chlorogenic acid at 20 μM concentration. Immunofluorescent staining of NF-κB (green); nuclei were counterstained using DAPI (blue). Images are 40× magnification (color figure online)

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Chlorogenic acid inhibits the entry of Ninj1-expressing macrophages into mouse retina under endotoxin treatment

To investigate the effect of chlorogenic acid on the activation of macrophages in vivo, we used the LPS-inflamed mouse model and examined the entry of activating Ninj1-expressing macrophages into the retina. In untreated mice, Ninj1-expressing macrophages were not seen around retinal vessels (Fig. 7a). When the mice were treated with LPS, the number of Ninj1-expressing macrophages was increased and this was blocked by chlorogenic acid treatment. We confirmed this with Western blot analysis. LPS treatment enhanced the expression level of Ninj1 in retina lysates (Fig. 7b) and chlorogenic acid treatment led to the reduction of Ninj1 expression when compared with the LPS-treated group. These results suggest that chlorogenic acid may inhibit the migration and activation of macrophages during retinal inflammation.

Fig. 7
figure 7

Effects of chlorogenic acid on Ninj1 expression in LPS-induced retinal inflammation model. a Immunostaining of adult whole-mounted retinas with antibodies for Ninj1 (red) and GS-lectin (green). Ninj1-positive cells appeared only after intraperitoneal injection of LPS in rat adult retina (7 weeks). Nuclei were counterstained using DAPI (blue). b Ninj1 expression in eye lysates after 0–330 μg/kg of LPS injection in adult rat (7 weeks) in vivo. Mice were injected three times intraperitoneally (IP) with a dose of 6.6 μg of LPS (330 μg/kg of body weight) and/or with a dose of 0.1 mg of chlorogenic acid (5 mg/kg body weight) over 3 days. Animals were killed at 24 h after last LPS administration (color figure online)

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Discussion

In this study, we demonstrated the inhibitory activities of chlorogenic acid in RAW 264.7 cells and mouse retina under LPS-induced inflammation, at least in part owing to its regulation of NF-kB and Ninj1. Those results include: (1) decreased NO production mediated by down-regulation of iNOS; (2) suppression of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6, as well as the chemokine CXCL1 through down-regulation of NF-κB; (3) inhibition of Ninj1, which is important for leukocyte infiltration. This was consistent with previous findings, in which chlorogenic acid reduces inflammation and fibrosis through inhibition of the TLR4 signaling pathway in carbon tetrachloride (CCl4)-induced liver fibrosis [22] and LPS-inflamed keratinocytes [23]. Although further studies are needed for the role of chlorogenic acid in inflammation, our present study provides an important proof-of-principle for the development of chlorogenic acid as an anti-inflammatory agent.

LPS is a cell wall component of Gram-negative bacteria, and plays a central role in the pathogenesis of septic shock [24]. When macrophages are exposed to bacterial products such as LPS, the LPS binds to Toll-like receptor 4 (TLR4) which activates two major signaling pathways, myeloid differentiation factor 88 (MyD88) and TIR-domain-containing adaptor inducing IFN-β (TRIF), which result in activation of NF-κB. When the NF-κB signaling pathway is activated, macrophages secrete NO and pro-inflammatory cytokines, and express adhesion molecules [25–27]. Therefore, putative agents that can regulate the NF-κB activation and adhesion molecules have the potential to improve many inflammation-related symptoms in patients. In particular, the activation, adhesion, and homing of leukocytes are regarded as a mostly effective therapeutic target for inflammation-related diseases [21, 27]. Ninj1 is an important adhesion molecule whose functions include immune surveillance, cell interaction, cell differentiation, and trafficking of leukocytes [28, 29]. Furthermore, Ninj1 is associated with multiple sclerosis, and regulates the migration of myeloid cells [21, 28]. In the present study, we observed that chlorogenic acid attenuates Ninj1 expression and decreases the cell-to-matrix adhesion ability of RAW 264.7 cells. Thus, chlorogenic acid will be a putative anti-inflammatory drug for regulating the adhesion and trafficking of leukocytes in leukocyte-mediated inflammatory diseases.

Various natural products including polyphenols such as resveratrol, flavonoids, and chlorogenic acid have various advantages for health [30, 31]. It has been widely accepted that chlorogenic acid has many health benefits as an anti-aging, anticancer, and anti-hypertension agent [15, 17, 18]. However, there is some controversy over whether chlorogenic acid prevents diabetes and allergy. For example, it is reported that chlorogenic acid reduced insulin responses and early fasting glucose [32]. In contrast, other studies show that chlorogenic acid enhances glucose uptake in skeletal muscle cells via AMPK activation, contributing a beneficial effect on type 2 diabetes mellitus [16, 33, 34]. Therefore, considering the controversial effect of chlorogenic acid in some diseases, extensive efforts are required to develop chlorogenic acid as a therapeutic agent.

In this study, we showed the inhibitory effects of chlorogenic acid on expression of NO, pro-inflammatory cytokines, and an important adhesion molecule Ninj1 regulated by the NF-κB pathway in LPS-stimulates RAW 264.7 cells. In conclusion, chlorogenic acid is a potential therapeutic drug for treating inflammatory diseases such as sepsis.