SPENT HOPS (HUMULUS LUPULUS L.) EXTRACT AS MODULATOR
OF THE INFLAMMATORY RESPONSE IN LIPOPOLYSACCHARIDE
STIMULATED RAW 264.7 MACROPHAGES

Inflammation is an ordered reaction of tissue to a damaging factor. It is characterized by a typical yet variable combination of processes and features, including pain, swelling, redness, fever and loss of function. An inflammatory response can be triggered by many factors (1). In terms of their origin and nature these can be categorized into infectious and non-infectious causes. Infectious inflammatory processes are caused by biological factors including, infectious agents - bacteria, viruses, fungi, protozoa or components of individual microorganisms. The non-infectious inflammation can be induced by chemical agents causing tissue damage or physical factors including radiation or mechanical injuries. Furthermore, sterile inflammatory processes can be triggered by endogenous factors such as those arising from adipose tissue in overweight individuals. Another example of non-infectious inflammation is allergic inflammation (2). As a result of the action of an inflammatory factor, the body’s response is a process of defense, protection and repair. Inflammation can occur at three levels as a cellular response, humoral response or hemostatic response (3). The first phase of the inflammatory response is the cellular response. During this phase, the number of leukocytes increases dramatically. When a pathogen overcomes natural barriers or breaks them, a stimulation of phagocytic cells is initiated, the task of which is phagocytosis and the release of mediators of inflammatory reactions. Macrophages constitute a major class of cells with phagocytic properties. They are derived from monocytes arising in the bone marrow during haematopoiesis. Macrophages are important elements of the innate immunity. Upon their stimulation numerous substances are released and secreted, including cytokines, enzymes and other compounds (4).

A major group of pro-inflammatory cytokines are the interleukins. They are mediators which enable communication between macrophages and other immune cells. The two major interleukins: 1 (IL-1) and 6 (IL-6) play a central role in inflammatory processes (5). There are two main forms of interleukin 1: IL-1α and IL-1β. IL-1β is involved in the synthesis of acute phase proteins and stimulates the formation of another important interleukin in the inflammatory process such as IL-6 (6). The importance of IL-6 in the development of cancer or rheumatoid arthritis is also crucial (7, 8). Another important pro-inflammatory cytokine is tumor necrosis factor (TNF). There are two main forms of this protein: TNF-α and TNF-β. The TNF-α is among others responsible for an increase of the amount of free radicals in the cell, and consequently to its damage and death. Excessive secretion of this factor or the increased activity are observed in cancer and autoimmune diseases, among others: in Crohn’s disease (9, 10).

Next to inflammatory cytokines, other molecules such as enzymes which are responsible for the formation of mediators of inflammatory reaction have an important role in inflammatory processes. A key enzyme is cyclooxygenase-2 (COX-2), i.e. induced cyclooxygenase. It is one of the two isoforms of the cyclooxygenase enzyme, next to the constitutive isoform 1 (COX-1). COX-2, which is activated via inflammatory agents, is responsible for catalysing the formation of prostanoids from arachidonic acid (11). Another important enzyme contributing to the development of inflammation is nitric oxide synthase. In the course of the inflammatory process, one of the isoforms of this enzyme - the inducible nitric oxide synthase (iNOS) - is produced. This production is enhanced by the secretion of proinflammatory cytokines like IL-1 and TNF-α (12). In the course of inflammation, there is also an increase of transcription factors activity such as nuclear factor kappa B (NF-κB) and its phosporylated form (p-NF-κB), which control the expression of many genes involved in inflammatory processes. Disorders in the regulation of NF-κB are associated with cancer diseases and several inflammatory disorders. The inhibitor of kappa B (IκB-α) and its phosporylated form (p-IκB-α) have significant role in the activity of NF-κB (13).

In the treatment of inflammation, non-steroidal anti-inflammatory drugs, steroids or monoclonal antibodies are used depending on the symptoms, the place of the course of the inflammatory reaction, ethiology or concomitant diseases (14). Polyphenols constitute a group of plant-derived compounds that have attracted the attention because of their potential as protective and anti-inflammatory agents. Compared to medicinal compounds they might act in a more subtle way and possess fewer side effects compared to drugs (15-17). Numerous studies show the effectiveness of polyphenolic compounds in reducing inflammation or in inhibiting the formation of inflammatory mediators (18-20). The main sources of these compounds are fruits, green tea, beer, spices, herbs or plant extracts (21-23). Another interesting plant extract, for which still limited data on its biological activities are available, is extract obtained from the spent hops (Humulus Lupulus L.). Humulus Lupulus Linnaeus belongs to the Cannabaceae family and is widely cultivated in Europe, and in the USA. Its female inflorescences (hop cones) are used in the manufacturing of beer (24, 25). Compounds contained in spent hops extract are assumed to have anticoagulant properties (26), antibacterial properties (27), anti-hyperlipidemic (28), and hypoglycemic activity (29). It was reported that spent hops extract contains diversified phenolic compounds such as hydroxycinnamic acids, proanthocyanidin oligomers, flavan-3-ol monomers, and flavonol glycosides (26). According to a paper by Weber et al. hop extract with 50% humulone and lupulone exhibited antibacterial activity against Propionibacterium acnes and Staphylococcus aureus (30). Luzak at al. showed that spent hops extract inhibited ADP-induced platelet aggregation and ameliorated anticoagulant activity of human endothelial cells (26). In our previous study, a spent hops extract (SHE) demonstrated cytotoxic effects on human colon cancer cells (SW-480 and HT-29). In addition, SHE inhibited the viability of two colon cancer cell lines (SW-480 and HT-29) much more than the viability of normal epithelial colon cells CCD 841 CoN (31).

The aim of the present study was to investigate the impact of spent hops extract (a rich source of flavanols) on the expression of a set of inflammatory markers from activated macrophages. We hypothesized that the examined SHE may reduce the expression of cytokines and inflammatory mediators at the level of both mRNA and protein.

MATERIALS AND METHODS

Spent hops extract

Dried spent hops - a residue from the extraction of hop cones (Humulus lupulus L.) by supercritical CO2, was supplied by the Fertilizer Research Institute Pulawy (Poland). This by-product was ground to fine powder in commercial coffee grinder and then extracted with acetone-water (70:30, v/v) as described previously (32). Phenolic profile of spent hops extract (SHE) was carried out using an Acquity Ultra Performance LCTM system (UPLCTM) (Waters Corporation, Milford, USA) coupled with a Micromass Q-Tof Micro mass spectrometer (Waters, Manchester, U.K.) according to the procedure described by Labieniec-Watala et al. (33).

Cell culture

The murine macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (ATCC; ref: TIB-71) (LGC Standards, Poland). RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco®, USA) supplemented with 10% bovine calf serum (BCS), 2 mM L-glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin. The cells were grown in a humidified atmosphere of 5% CO2 at 37°C. All experiments were carried out between passages 6 and 10. The cells were seeded in such quantity that the confluence at the end of the experiment did not exceed 80% in control wells. In the present work, SHE was tested within the concentration range from 5 to 50 µg/mL, budesonide was tested at concentration 1 µM and Bay 11-7082 was tested at concentration 10 µM (as positive controls instead of the SHE). For all bioassays. SHE was dissolved in serum-free medium (no other solvent was used).

Cell viability assay

The effect of SHE on the cell viability of RAW264.7 macrophages was evaluated by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, St. Louis, MO, USA) reduction assay. Briefly, the cells were harvested, suspended in the growth medium supplemented with 10% BCS, and seeded at a density of 2 × 104 cells per well in a 96-well plate. After 24 h of incubation, the medium was removed, and then the cells were suspended in a medium with 3% BCS with or without SHE (5, 10, 25, 50 µg/mL). At the same time, RAW264.7 were stimulated with 1 µg/mL of lipopolysaccharide (LPS). After 24 h of incubation, 20 µL of MTT solution (5 mg/mL) was added to each well and incubated for 2 hours. Next, the medium with MTT was precisely removed, and 100 µL of 100% dimethyl sulfoxide (DMSO) was added to each well. Then, the plate was shaken for 15 min at room temperature by using a plate shaker (Lab-Line Instruments, Inc., Melrose Park, Illinois, USA). The viable cell number was correlated with the production of formazan, which was dissolved in DMSO. Optical density was measured by microplate reader (iMarkTM, BioRad Laboratories) at a wavelength of 595 nm. The analysis was based on three replicants from three independent experiments. The percentage of cell viability was calculated against untreated cells.

Quantitative real time PCR (Q-PCR)

RNA isolation, cDNA synthesis and quantitative polymerase chain reaction (Q-PCR), experiments were performed as described previously (15). Briefly, cells were harvested, suspended in growth medium (10% BCS) and seeded at a density of 1.5 × 106 cells in 25 cm2 bottles. Cells were stimulated with LPS (1 µg/mL) and incubated with or without SHE (5, 10, 25 µg/mL) for 6 hours. Positive control samples were treated with budesonide (1 µM) or Bay 11-7082 (10 µM), instead of SHE. Negative control samples were incubated without LPS and SHE. Total RNA was extracted using TRIzol® reagent (Invitrogen™, Carlsbad, CA) according to the manufacturer’s guidelines. The concentration and purity of isolated RNA were determined spectrophotometrically at 260 and 280 nm. By means of quantitative polymerase chain reaction (Q-PCR), we determined the influence of SHE on mRNA expression levels in RAW264.7 macrophages of the following genes: IL-1β: (Mm00434228_m1), IL-6: (Mm00446190_m1), TNF-α: (Mm00443258_m1), Ptgs2: (Mm00478374_m1), NOS2: (Mm00440502_m1), NFκB1: (Mm00501346) (Applied Biosystems, Foster City, CA). The synthesis of cDNA was performed from 12 µg of total RNA in the total volume of 60 µ l using Maxima® First Strand cDNA Synthesis Kit for RT-qPCR. Subsequently, 10 µl obtained cDNA were taken and diluted forty times with RNase-free water. Finally, volumes of 4.5 µl (corresponding to 0,0225 µg of total RNA) were used for Q-PCR which was carried out using the LightCycler®96 System thermocycler (Roche, Diagnostics, Mannheim, Germany). The detection of Q-PCR products was done in a total volume of 10 µl using dyes FAM with the LightCycler® 96 Instrument. For Q-PCR Master Mix (FastStart Essential DNA Probes Master, Roche) and probes (TaqMan® Gene Expression Assays). The Q-PCR amplification procedure was comprised of the following stages: initial denaturation step at 95°C for 6 min, followed by 45 cycles of 95°C for 10 s (melting) and 60°C for 30 s (annealing and extension). Gene expression levels were normalized using endogenous control: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1) (Applied Biosystems, Foster City, CA). The Ct (threshold cycle) values for studied genes were normalized to Ct values obtained for a housekeeping gene GAPDH. The relative amount of each gene was calculated using the 2–ΔΔCt method (34).

Western blot analysis

RAW264.7 cells were harvested, suspended in growth medium with 10% BCS, and seeded at a density of 6 × 106 cells in 75 cm2 bottles. After 24 h, the cells were washed twice with PBS and then suspended in medium with 3% BCS with or without SHE (5, 10, 25 µg/mL). At the same time, cells were stimulated with 1 µg/mL of LPS. RAW264.7 were treated with SHE for 1 hour or 24 hours. Positive control samples were treated with budesonide (1 µM) or Bay 11-7082 (10 µM), instead of SHE. Negative control samples were incubated without LPS and SHE. Cell lysates were prepared using a Mammalian Cell Lysis Kit (Sigma-Aldrich Co. LLC, St. Louis, MO, USA). The protein content of the cell lysates was then determined by using the Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein cell lysates (40 µg protein/well) were subjected to Mini-PROTEAN® TGX™ Gels (Bio-Rad, Hercules, CA, USA). Separated proteins were transferred using a semi-dry system onto PVDF membranes (pore size: 0.45 µm; Life Technologies, Carlsbad, CA, USA) in transfer buffer containing 20% (v/v) methanol, 192 mM glycine, and 25 mM Tris, pH 8.3. The PVDF membranes were incubated at room temperature for 1 hour in 5% non-fat dry milk in Tris-buffered saline with Tween 20 (PBST, 150 mM NaCl, 0.05% Tween 20, 100 mM Tris-HCl, pH 7.4) to saturate non-specific protein-binding sites. Subsequently, the membranes were incubated with specific primary antibodies diluted in PBST overnight at 4°C for immunodetection of the studied proteins. The primary mouse monoclonal antibodies were as follows: β-actin (sc-47778), Cox-2 (sc-19999), IκB-α (sc-164), IL-1β (sc-515598), NOS2 (sc-7271), NF-κB p65 (sc-8008), p-NF-κB p65 (sc-135769), TNF-α (sc-52746; all antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA). After wash steps (five times, 10 min) using PBST, membranes were incubated with the secondary antibody (m-IgGκ BP-HRP; sc-516102, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature. The detection of bands was performed using the enhanced chemiluminescence blotting detection system (SuperSignal West Femto Maximum Sensitivity Substrate, ThermoFisher Scientific, Rockford, USA). The intensity of the bands was quantified by densitometric analysis using the GelDoc™ EQ system with Image Lab Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunoblots were normalized to the β-actin content.

Enzyme-linked immunosorbent assay (ELISA)

This method was used for the assessment of IL-6 protein expression. RAW264.7 macrophages were seeded in 96-well plates (2 × 106 cells/well) in growth medium with 10% BCS for 24 hours, then the medium was removed and cells were suspended in medium with 3% BCS with or without SHE (5, 10, 25 µg/mL). At the same time, macrophages were stimulated with 1 µg/mL of LPS. Positive control samples were treated with budesonide (1 µM). Negative control samples were incubated without LPS and SHE. Cells were treated with SHE for 24 hours. Protein level of IL-6 was determined with a murine IL-6 ELISA Kit (Diaclone, Besancon, Cedex, France) according to the manufacturer’s protocol.

Nitric oxide determination

RAW 264.7 macrophage cells were suspended in the growth medium supplemented with 10% BCS on a 96-well plate at a density of 2 × 104 cells/well. After 24 hours, the medium was removed and the cells were suspended in a medium with 3% BCS with or without LPS, budesonide and varying concentrations of SHE for 24 hours. Then, accumulation of NO metabolite in the cell culture supernatant was measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride; Sigma Aldrich). An equal volume (100 µl) of the supernatant was mixed with Griess reagent (100 µl) in a 96-well plate. After incubation at room temperature for 15 min, the absorbance was measured by microplate reader (iMarkTM, BioRad Laboratories) at a wavelength of 540 nm. The concentration of NO was determined using an NO standard curve.

Statistical analysis

Statistical analyses were performed using PRISM 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Data is presented as mean ± SEM, as indicated in the figure legends. The number of independent experiments is given in the figure legends. The statistical significance of differences between means was determined by a one-way ANOVA followed by a post hoc multiple comparison Newman-Keuls test. P values of < 0.05 were considered to be statistically significant.

RESULTS

Characterization of spent hops extract composition

The phenolics composition of SHE examined in the present work was described earlier by Luzak et al. (26). The content of total phenolic compounds in the SHE was 62.29 g/100 g of the extract. Twenty-six phenolic compounds including twelve flavonols, eight proanthocyanidins, four hydroxycinnamic acids, and two flavanol monomers were identified in SHE. Flavonols and proanthocyanidins were the dominant phenolic compounds in extract and constituted 39.24 and 37.42% of the total phenolics, respectively. Fig. 1 shows a chromatogram of phenolic compounds detected at 280 nm in which compounds with a content of more than 2% by weight are indicated according to Luzak et al. (26).

Fig. 1. Ultra performance liquid chromatography-ultraviolet (UPLC-UV) chromatogram of phenolic compounds of spent hops extract (SHE) monitored at 280 nm. (1) - neochlorogenic acid; (2) - proanthocyanidin trimer; (3) - B3 dimer; (4) - (+) catechin; (5) - chlorogenic acid; (6) - (–) epicatechin; (7) - quercetin-3-hexoside; (8) - quercetin-3-acetylhexoside; (9) - kaempferol-3-hexoside.

Effect of spent hops extract on viability of RAW 264.7 macrophage cells

The LPS-stimulated cells were treated for 24 hours with various concentrations of SHE: 5, 10, 25, 50 µg/mL (Fig. 2). Budesonide (a corticosteroid with anti-inflammatory properties) was tested at a concentration of 1 µM. Control samples were incubated without LPS and SHE. The slight increase in the cell viability was detected at concentrations: 10, 25 µg/mL, unlike the concentration 50 µg/mL, for which the viability increased significantly. For the first three concentrations, the results were not statistically significant for the LPS-stimulated macrophages, while for the highest concentration (50 µg/mL), we observed a statistically significant increase. Applied MTT test helped to choose the range of tested SHE (5, 10, and 25 µg/mL) for another experiments. However, due to the fact that the concentration of 50 µg/mL caused the increase of the viability of cells, we didn’t take it into account. In addition, the cell viability for the LPS-stimulated RAW 264.7 cells with budesonide was at a similar level to that of the LPS-stimulated cells alone.

Fig. 2. Effects of spent hops extract (SHE) on cell viability in lipopolysaccharide (LPS)-activated macrophage RAW 264.7 cells. The viability of RAW 264.7 cells was determined by MTT assay after incubated with indicated concentrations of SHE for 24 hours supplemented with or without 1 µg/mL of LPS. Each value represents the mean value ± SEM, n = 3 independent experiments (each experiment was carried out in triplicate). Significance of differences between means: #P < 0.05, ##P < 0.01, ###P < 0.001 versus control (untreated cells); *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells.

Effect of spent hops extract on expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α)

To elucidate of the potential anti-inflammatory action of SHE, we tried to determine its influence on expression of selected genes involved in the development of inflammation, inter alia genes coding for pro-inflammatory cytokines: IL-1β, IL-6, TNF-α. As shown in Fig. 3A, the mRNA of IL-1β was reduced, significantly to 66% and 65%, respectively for concentration of 10 and 25 µg/mL. The used positive control (budesonide) decreased the expression of IL-1β mRNA by 54%. The results of Western blot analysis confirmed the ability of SHE to inhibit IL-1β expression also on the protein level, as shown in Fig. 4A and 4C. At the concentrations of 5, 10, and 25 µg/mL of SHE the expression level of IL-1β protein decreased to 84%, 63%, and 64%, respectively, versus activated macrophage that were not exposed to SHE.

Fig. 3. Effects of spent hops extract (SHE) on LPS-induced pro-inflammatory cytokine mRNA expression in RAW 264.7 cells. LPS-stimulated (1 µg/mL) RAW264.7 were treated with SHE (5, 10, 25 µg/mL) for 6 hours. The mRNA levels IL-1β, IL-6 and TNF-α were measured by quantitative polymerase chain reaction (Q-PCR) (A, B and C). Data are representative of three independent experiments, each value represents the mean value ± SEM. Significance of differences between means: #P < 0.05, ##P < 0.01, ###P < 0.001 versus control (untreated cells), *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells.

Fig. 4. Effects of spent hops extract (SHE) on LPS-induced pro-inflammatory cytokine protein expression in RAW 264.7 cells. Protein expression of IL-1β and TNF-α in LPS-stimulated (1 µg/mL) RAW264.7 cells incubated with different concentrations of SHE (5, 10, 25 µg/mL) for 24 hours was examined by Western blot assay (A, B and C). Quantification of protein bands was performed after densitometric analysis scanning of immunoblots and normalized to β-actin level. The normalized amount of protein in LPS-stimulated cells was taken as 100%. Protein expression of IL-6 was examined by an enzyme-linked immunosorbent assay (D). Each value on the graphs represents the mean value ± SEM, n = 3 independent experiments. Significance of differences between means: #P < 0.05, ##P < 0.01, ###P < 0.001 versus control cells, *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells.

The tested extract showed stronger inhibitory properties with another cytokine - IL-6 (Fig. 3B and 4D). For the lowest concentration of SHE, we found an increase in the expression of mRNA by 22% relative to the LPS-stimulated RAW 264.7. At a concentration of 10 µg/mL, the level of mRNA expression decreased by 62%, while for the highest concentration (25 µg/mL), the decrease was 68% (Fig. 3B). At these concentrations, SHE showed stronger inhibitory properties than budesonide, which caused a decrease to 69%. Moreover, SHE treatment caused a concentration-dependent reduction in IL-6 protein expression after 24 hours of incubation, as determined by ELISA test (Fig. 4D). The suppression of IL-6 expression reached 25% and 39% at the concentrations of 10 and 25 µg/mL of SHE, respectively, versus LPS-activated RAW 264.7.

For another pro-inflammatory cytokine - TNF-α, we observed a decrease for each tested concentrations (Fig. 3C). At concentration of 10 and 25 µg/mL, the TNF-α mRNA expression was strongly inhibited by SHE. For the concentration of 10 µg/ mL, we observed a decrease to 58% against the LPS-stimulated macrophages, whereas for the concentration of 25 µg/ mL, the expression dropped to 36%. However, the SHE did not cause any significant changes in protein expression of TNF-α (Fig. 4B and 4C).

Effect of spent hops extract on expression of pro-inflammatory enzymes (COX-2, iNOS)

As demonstrated in Fig. 5A, SHE strongly inhibited the expression of COX-2 mRNA (by 48% compared to control) at concentration of 5 µg/mL. For the remaining concentrations of SHE (10 and 25 µg/mL), the expression of COX-2 mRNA was decreased by 43% and 53%, respectively. Moreover, for all concentrations of tested extract, the suppression of COX-2 mRNA expression was greater than for the used corticosteroid. The assessment of COX-2 protein expression confirmed the impact of SHE on the mRNA expression (Fig. 6A and 6C). A marked decrease in COX-2 expression was observed after a 24-hour incubation with SHE. At the concentrations of 10 and 25 µg/mL, the inhibition reached 33% and 68%, respectively. In addition, for the highest concentration, the effect of SHE was stronger than the positive control (budesonide). As shown in Fig. 5B, the tested SHE did not cause any significant changes in iNOS mRNA expression. In contrast, the SHE treatment caused a statistically significant and concentration-dependent reduction in protein expression of iNOS, as shown in Fig. 6B and 6C. At the concentrations of 5, 10, and 25 µg/mL of SHE the expression level of iNOS protein decreased to 53%, 14% and 2%, respectively, versus activated macrophages. What is more, the reduction of iNOS protein expression by tested SHE (for all concentrations) was stronger than by budesonide.

Fig. 5. Effect of spent hops extract (SHE) on mRNA expression of pro-inflammatory enzymes (COX-2, iNOS) and nuclear factor (NF-κB) in LPS-stimulated RAW 264.7 cells. LPS-stimulated (1 µg/mL) RAW264.7 were treated with SHE (5, 10, 25 µg/mL) for 6 hours. The mRNA levels COX-2, iNOS and NF-κB were measured by quantitative polymerase chain reaction (Q-PCR) (A, B and C). Data are representative of three independent experiments, each value represents the mean value ± SEM. Significance of differences between means: #P < 0.05, ##P < 0.01, ###P < 0.001 versus control (untreated cells), *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells.

Fig. 6. Effect of spent hops extract (SHE) on protein expression of pro-inflammatory enzymes and nitric oxide production in LPS-stimulated RAW 264.7 cells. Protein expression of COX-2 and iNOS in LPS-stimulated (1 µg/mL) RAW264.7 cells incubated with different concentrations of SHE (5, 10, 25 µg/mL) for 24 hours was examined by Western blot assay. Quantification of protein bands was performed after densitometric analysis scanning of immunoblots and normalized to β-actin level. The normalized amount of protein in LPS-stimulated cells was taken as 100% (A, B and C). Effect of SHE on LPS-induced NO production in macrophage RAW 264.7 cells (D). Cells (2 × 104 cell/well in 96-well pate) were treated with SHE (0, 5, 10, 25 µg/mL) for 24 hours. The amount of nitrite oxide in the medium was measured using Griess assays. Each value on the graphs represents the mean value ± SEM, n = 3 independent experiments. Significance of differences between means: #P < 0.05, ##P < 0.01, ###P < 0.001 versus control cells; *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells.

Effect of spent hops extract on lipopolysaccharide-activated nitric oxide production

Nitric oxide production was assessed to examine the downstream product of iNOS. As demonstrated in Fig. 6D, SHE inhibited significantly the NO production for all tested concentrations. At the concentrations of 5, 10, and 25 µg/mL of SHE the NO levels decreased from 23.05 µM (LPS-activated RAW 264.7) to 12.26, 12.70 and 13.69 µM, respectively.

Effect of spent hops extract on lipopolysaccharide-activated nuclear factor-κB activity

Due to the fact that NF-κB is an important transcription factor complex that controls the expression of inflammation-associated enzymes and cytokines, the next stage assessed the impact of SHE on both mRNA and protein expression of NF-κB. The expression of NF-κB mRNA was significantly inhibited for all examined concentrations of tested extract (Fig. 5C). The suppression of NF-κB mRNA expression reached 56%, 52 %, and 62% versus LPS stimulated macrophages at the concentrations of 5, 10, and 25 µg/mL of SHE respectively. The SHE reduced the expression of mRNA more strongly than the used inhibitor of NF-κB activation (Bay 11-7082; expression reduction to 66%). The protein expression was measured by Western blot after 1 hour treatment of the cells with the SHE. At the lowest concentration, the SHE caused a decrease of expression of NF-κB protein by 45%. For other concentrations of 10 and 25 µg/mL, protein levels were suppressed by 54% and 58%. The expression of the phosphorylated form of this transcription factor p-NF-κB, was also strongly reduced. At the concentration of 10 µg/mL, the protein level was decreased by 63%. In addition, this change was identical as in the case of Bay 11-7082. After the SHE treatment, the protein level of the IkB molecule increased for all concentration values. The highest increase was with a concentration of 25 µg/ml accounting for 74%. All obtained results of SHE effect were statistically significant (Fig. 7). The SHE did not cause any significant changes in p-IκB expression (data not shown).

Fig. 7. Effects of spent hops extract (SHE) on the protein expression of NF-κB, p-NF-κB and IκB-a in LPS-stimulated RAW 264.7 cells. Protein expression of NF-κB, p-NF-κB and IkB-α in LPS-stimulated (1 µg/mL) RAW264.7 cells incubated with different concentrations of SHE (5, 10, 25 µg/mL) for 1 hour was examined by Western blot assay (A, B, C and D). Quantification of protein bands was performed after densitometric analysis scanning of immunoblots and normalized to β-actin level. The normalized amount of protein in LPS-stimulated cells was taken as 100%. Each value on the graphs represents the mean value ± SEM, n = 3 independent experiments. Significance of differences between means: #P < 0.05, ##P < 0.01, ###P < 0.001 versus control cells, *P < 0.05, ** P < 0.01, *** P < 0.001 versus LPS-stimulated cells.

DISCUSSION

Female cones of the hop plant (Humulus lupulus L.) are used in the production of beer and they are responsible for its bitterness and characteristic aroma. Hop cones contain many biologically active phenolic compounds such as flavonols, flavan-3-ols, phenolic carboxylic acids, and prenylflavonoids. One of the most investigated compound of hop prenylflavonoids is xanthohumol, which exhibits strong biological activities, especially antioxidant, antibacterial and anti-inflammatory effects (24, 35-37). Recently, we demonstrated that spent hops extract (SHE), which is a rich source of various polyphenols and is obtained in large amounts as a waste product of beer industry, had cytotoxic activity on colon cancer cells. Using two colon cancer cell lines, our previous study revealed that SHE had a more pronounced effect on the SW-480 cell line than on the HT-29 cell line. In addition, SHE had the lowest toxicity on CCD841CoN normal epithelial colon cells, which are an important component in the maintenance of the intestinal mucosa and play an important role in the immune response of the intestine. The IC50 value for SW-480 cell line was obtained at the concentration 400 µg/mL after 48-hours incubation, for HT-29 cell line at the concentration 200 µg/mL after 72-hours incubation while for CCD841CoN cell line the IC50 value was not received (31). In this study, we investigated the anti-inflammatory activity of SHE in murine RAW 264.7 macrophage cells. Macrophages play central roles in inflammation and they secrete several pro- and anti-inflammatory mediators and enzymes (38).

Studies suggest that several components of Humulus Lupulus have anti-inflammatory properties (24, 30, 39, 40). These compounds can affect gene expression of such as TNF-α, prostaglandin-endoperoxide synthase 2 (Ptgs2) and nitric oxide synthase-2 (NOS-2), and they can modulate gene expression of transcription factors which are indirectly responsible for inflammation for example NF-κB (30, 37, 39, 41, 42).

In the present study, the tested extract decreased IL-1β expression both at mRNA and protein level (Figs. 3A and 4A). It is well known that IL-1β activates leukocytes and other cells of connective tissue and takes part in remodeling of tissues (5). Another group previously reported a suppressive action of Humulus Lupulus on the expression of IL-1β. Cho et al. investigated the influence of xanthohumol, the main prenylated chalcone of Humulus Lupulus, on the synthesis of IL-1β. They showed a decrease in the expression of IL-1β both at mRNA and protein level in activated RAW264.7 cells (43). Similarly, previous studies noted that quercetin, neochlorogenic and chlorogenic acids suppressed the production of IL-1β in LPS-activated macrophages (44-46). It should be noted, however, that above-mentioned phenolics in contrast to xanthohumol are the bioactive components of SHE. In an in vivo study, Sevastre-Berghian et al. investigated the biological effects of quercetin and Hypericum dry extracts (HpE) on brain oxidative stress biomarkers and inflammatory cytokines in rats with experimentally-induced anxiety (47). They observed that quercetin and HpE administration in rats treated with anxiogenic drug down-regulated both IL-1α and IL-1β protein expression in hippocampus as compared to control group. Additionally, the quercetin administration in rats increased catalase (CAT) and superoxide dismutase (SOD) activities, which are crucial anti-oxidant enzymes. Their inhibition may cause accumulation of cellular superoxide radicals leading to free radical-mediated damage, which in result may often contribute to the development of inflammation. However, it is worth noting that both tested extracts (SHE and HpE) revealed a high content of quercetin (including its derivatives), which is potent scavenger of reactive oxygen species (ROS). A study by Mazur-Bialy et al. investigated the impact of irisin, a myokine released by exercising skeletal muscles, on the ROS scavenging potential as well as on the gene expression of crucial anti-oxidant enzymes in non-stimulated and LPS-stimulated murine macrophages (RAW 264.7) (48). Interestingly, the LPS-stimulated macrophages showed significantly more efficient H2O2 neutralization than quiescent cells, and the level of ROS was similar regardless of the irisin concentration after the incubation period applied in this study. An apparent effect of irisin on ROS neutralization was observed also in non-stimulated macrophages. Moreover, in LPS-stimulated cells irisin increased mRNA levels of enzymes involved with antioxidative stress pathways, namely, SOD, CAT-9, and glutathione peroxidase (GSH-Px). The results suggest that irisin acts directly on the target cells linked with inflammatory conditions such as macrophages and efficiently activates the protective mechanisms leading to the ROS scavenging. Importantly, the status of macrophage activation can have an impact on their capacity for ROS neutralization.

Another interleukin which exacerbates inflammatory processes, while among others promoting differentiation of lymphocytes and stimulating acute-phase proteins production is IL-6. Therefore, the inhibition of this mediator is associated with mitigation of inflammation (49). Our results indicate that SHE down-regulated IL-6 mRNA and protein expression in LPS-activated macrophages (Figs. 3B and 4D). It was reported that humulone and lupulone (a-bitter acid and b-bitter acid), which are derivatives of hop (Humulus Lupulus L.) also inhibited IL-6 expression in acne in vitro model (30). Moreover, the studies by Hege at al. showed that isohumulone (iso-a-acid) inhibited IL-6 too. It decreased expression of IL-6 mRNA in LPS-stimulated J774.1 macrophages cells (40). Above research shows the effect of hop extracts rich in humulone, lupulone (50% humulone and lupulone) and isohumulone (30% isohumulone), while our extract contains mainly proanthocyanidin oligomers, flavan-3-ol and hydroxycinnamic acids. In addition, we showed significant impact of SHE on the mRNA expression of TNF-α, which is also a critical cytokine involved in inflammatory responses. No changes in TNF-α protein expression were found (Figs. 3C and 4B). Moreover, the inhibition of TNF-α is an important treatment strategy for inflammation and inflammation-related diseases (9). Earlier, another group showed a suppressive effect of Humulus Lupulus on the TNF-α production through hop crude extract treatment of RAW 264.7 macrophages (37). The impact evaluation of hop crude extract on the IL-6 expression was not investigated. The suppressive effect of Humulus Lupulus on TNF-α expression was confirmed by study using xanthohumol, which significantly decreased TNF-α protein production (50). In comparison to our result, this different effect could be caused by the examination of single compound, while in our work the effect was caused by a lot of compounds contained in extract and their action could be various. Our results are in line with reports on the suppression of IL-6 and TNF-α expression by hop (Humulus Lupulus) and its derivatives. For instance, Tang et al. found that quercetin, which was main flavonol in the SHE, significantly reduced both TNF-α and IL-6 expression in LPS-stimulated RAW264.7 cells (44). Likewise, previous studies showed the inhibition of the production pro-inflammatory cytokines (TNF-α and IL-6) by both neochlorogenic acid and chlorogenic acid in LPS-stimulated macrophages (45, 46, 51). It is worth noting that our tested extract contained mentioned above neochlorogenic (4.23 g/100 g) and chlorogenic acids (2.08 g/ 100 g) (26).

In inflammatory reaction, enzymes have important roles, because they are responsible for the synthesis of inflammatory mediators including prostaglandins and prostacyclins. In many of these reactions, COX-2 is playing a role. Eicosanoids are responsible for most of the basic symptoms of inflammation such as redness, oedema and pain and exacerbation of inflammatory response (52). SHE inhibited strongly the mRNA and protein expression of COX-2 (Figs. 5A, 6A and 6C). Moreover, the suppression of prostaglandins was revealed by the isomerized hops extract in both an in vitro and in vivo model. Feeding rats with isomerized hops reduced the PGE2 level in their colonic mucosa (53). The results of Nozawa’s team confirmed the inhibitory impact of Humulus Lupulus on COX-2 expression and emphasized our results. It was also found that chlorogenic acid significantly decreased LPS-induced up-regulation of COX-2 at protein and mRNA levels in RAW264.7 cells (54).

Nitric oxide is a modulator of various biological processes including inflammation and it is generated by iNOS (12). This enzyme is activated in LPS-stimulated macrophages and its inhibition leads limitation of inflammation. So far, several studies demonstrated a suppressive effect of Humulus Lupulus on iNOS expression, at mRNA and protein level. Nitric oxide production was reduced by xanthohumol and hop extract in in vitro models (43, 55). Moreover, iNOS-suppressive properties of xanthohumol were demonstrated in in vivo models (39, 56). In the present study, SHE significantly inhibited both the protein expression of iNOS and the production of NO in LPS-activated RAW 264.7 macrophages (Fig. 6B, 6C and 6D).

In the present study, SHE also exhibited anti-inflammatory effects via the inhibition of NF-κB activity. This transcription factor is responsible for gene expression and regulates multiple important physiological and pathological processes. Inhibitor of kappa-B (IκB) affects the action of NF-κB, and when increased it is responsible for the reduction of NF-κB activation (13). Currently, there is evidence that the reduction of activity of NF-κB and its phosphorylated form (p-NF-κB) causes the suppression of inflammation, cancer development and progression (57, 58). Our results showed that SHE inhibited NF-κB mRNA and protein expression of both NF-κB and p-NF-κB (Figs. 5C and 7A, 7B, 7D). Furthermore, increase of IκB-α was shown in the present study (Fig. 7C and 7D). The increased activity of IκB-α causes that SHE produced inhibitory properties on the NF-κB pathway suggesting a wide spectrum of activity SHE on NF-κB signaling pathways. Other researchers have shown that xanthohumol did not inhibit NF-κB protein production in human benign prostate hyperplasia epithelial cells and they observed the increase of NF-κB protein expression (59). This could be caused by the type of cells tested, because other research demonstrated that xanthohumol suppressed NF-κB protein production in breast tumor cells and they confirmed inhibition of NF-κB activity which was also observed in present study (42). There are also studies showing that catechin reduced LPS-stimulated inflammation in human dental pulp cells by inhibiting activation of the NF-κB pathway (60). A study by Ye et al. showed that chlorogenic acid significantly inhibited the LPS-induced AKI mice expression of phosphorylated NF-κB p65 and IκB (61). Taking above-mentioned results into account, along with the fact that phenolics act in an additive, synergistic, or antagonistic manner, in our opinion, the anti-inflammatory potential of SHE might result from the fact that it contains phenolics belonging to various groups (proanthocyanidins, flavan-3-ol monomers, flavonols, and hydroxycinnamic acids). Furthermore, we also think that mutually complementary biological and synergistic activites of poliphenolic compounds included in SHE might influence on its anti-inflammatory potential.

In summary, the review of literature did not reveal any studies using the spent hops extract as an preparation modulating inflammation. The present study examined anti-inflammatory properties of SHE through its modulatory activity of inflammatory mediators. The results showed that SHE down-regulated the activity of inflammatory mediators by inhibiting NF-κB pathway. Our results underline that SHE may have interesting properties as therapeutic option in inflammatory disorders. An additional advantage of the extract is that it can be obtained as a waste product at low costs.

In conclusion, the spent hops extract showed anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophage cells. The tested extract inhibited the expression of the genes of molecules involved in inflammation such as IL-6, IL-1β, TNF-α, iNOS, NO, COX-2, and NF-κB, which underlines its a wide spectrum of anti-inflammatory activity.

Abbreviations: COX, cyclooxygenase; IκB-α, inhibitor of kappa B; IL-1, interleukin-1; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NF-κB, nuclear factor-kappaB; NO, nitric oxide; SHE, spent hops extract; TNF-α, tumor necrosis factor alpha.

Acknowledgements: The study was supported by grants from the Medical University of Lodz (No. 502-03/1-156-04/502-14-362-18 and No. 503/1-156-04/503-11-001).

Conflict of interests: None declared.

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