Selective Inhibition of COX-2 by a Standardized CO₂ Extract of Humulus lupulus in vitro and its Activity in a Mouse Model of Zymosan-Induced Arthritis

• Abstract
• Introduction
• Materials and Methods
• CO₂ extract of Humulus lupulus L.
• LPS-stimulated PBMC and metabolic activity assay
• COX-1 and COX-2 whole blood assay (WBA)
• Zymosan-induced acute arthritis in mice
• ^99mTechnetium accumulation in the knee joint as a measure for joint swelling
• ³⁵SO₄ incorporation as a measure for cartilage proteoglycan synthesis
• Ex vivo whole blood assay
• PGE₂ measurement
• Statistical and data analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

A standardized CO₂ extract from Humulus lupulus L. (hop extract) was investigated for its selective COX-1/2 inhibitory properties. An in vitro model of inflammation using lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) was used as a model to investigate the effect of hop extract on PGE₂ production. COX-1/2 selective inhibition by the hop extract was investigated in a COX-1 whole blood assay (WBA) and a COX-2 WBA. To evaluate the in vivo activity of hop extract, it was administered orally to C57BL/6 mice in which inflammation of the right joint was induced by injecting zymosan intra-articularly. Ex vivo PGE₂ production of LPS-stimulated blood cells was determined. Also, the effect of hop extract on healthy and arthritic cartilage was investigated as well as effects on inflammatory joint swelling. Hop extract inhibited PGE₂ production by LPS-stimulated PBMC without compromising the metabolic activity of these cells. Furthermore, hop extract showed a decline in PGE₂ production in the COX-2 whole blood assay (WBA) with an IC₅₀ of 20.4 μg/mL, while in the COX-1 WBA no inhibition of PGE₂ production was observed. This indicates a COX-2 selective inhibition. The COX-1 inhibitor SC-560 inhibited PGE₂ production in the COX-1 WBA but not in the COX-2 WBA. At 2 μM, celecoxib inhibited PGE₂ production in the COX-2 WBA by 92 % and in the COX-1 WBA by 50 %. When hop extract was administered orally to C57BL/6 mice in which joint inflammation was induced with zymosan, PGE₂ production in ex vivo LPS-stimulated whole blood was significantly decreased by 24 %, suggesting that hop extract becomes bioavailable. Furthermore, oral administration of hop extract showed no negative or positive effects on healthy cartilage proteoglycan synthesis, or on zymosan-induced arthritic cartilage proteoglycan synthesis. However, no effect of oral administration of 1.25 mg hop extract daily was observed on joint swelling. In conclusion, this standardized CO₂ extract of Humulus lupulus could be a useful agent for intervention strategies targeting inflammatory disorders and/or inflammatory pain.

Introduction

Cyclooxygenase (COX) is an important enzyme in inflammation and contributes to inflammatory pain. COX is the rate-limiting enzyme in the conversion of arachidonic acid (AA) to prostaglandin E₂ (PGE₂). Different COX isoforms are known, COX-1 and COX-2 [1], [2] and more recently a third isoform, COX-3, was identified, which appeared to be an alternative splicing variant of COX-1, present in the brain and heart [3]. By inhibiting COX, non-steroidal anti-inflammatory drugs (NSAIDs) alleviate pain and suppress inflammation in a variety of conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA).

COX-1 is constitutively expressed in many cells where it produces prostanoids involved in homeostatic functions, e. g.; gastric cytoprotection and platelet activation. Nevertheless, COX-1 can also be induced in some cell types such as astrocytes [4], promonocytic cell lines [5] and endothelial cells [6], although in the latter prolonged stimulation resulted in a decrease of COX-1 and a subsequent increase of COX-2.

COX-2 is generally regarded as the inducible form and plays an important role during inflammation, where it accounts for most of the produced PGE₂. The overexpression of COX-2 protein in articular tissues may be considered as a characteristic feature for arthritic diseases [7].

Chronic use of NSAIDs can result in gastrointestinal toxicity [8] which is associated with the COX-1 inhibitory activity. This phenomenon has led to the development of selective COX-2 inhibitors [9], [10] which are currently available for the treatment of inflammatory joint pain.

Humulus lupulus L. is a plant that has a long history of use. The traditional medicinal use of H. Lupulus covers a wide range of applications, including inflammation and rheumatism [11]. Interestingly, American Indians (Delawares) used hop to relieve toothache or earache [12]. Recently, it was described that humulone, one of the constituents of H. lupulus, suppressed COX-2 gene transcription in vitro [13], [14]. This would be in support of the traditional medicinal use of H. lupulus in inflammation and pain.

This study aimed to evaluate whether a standardized CO₂ extract of H. lupulus (hop extract) has in vitro COX inhibitory properties and is active in vivo. An in vitro model of inflammation using lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) was used as a model to investigate the effect of hop extract on PGE₂ production. COX-1/2 selective inhibition by the hop extract was investigated in a COX-1 whole blood assay (WBA) and a COX-2 WBA. Based on the results from the in vitro studies, one dosage of hop extract was evaluated in a mouse model of joint inflammation. Effects were monitored of orally administered hop extract on LPS-stimulated PGE₂ production in whole blood, inflammatory joint swelling and on healthy and arthritic cartilage proteoglycan synthesis in the mouse zymosan-induced acute arthritis model.

Materials and Methods

CO₂ extract of Humulus lupulus L.

The hop extract used in this study was a powdered CO₂ extract from the strobiles of H. lupulus (Canabaceae) obtained from Yakima Chief (YC-Purified alpha, Sunnyside, WA, USA). It consists of 18.1 % alpha acids, containing 71.8 % humulone and 2.3 % beta acids and is commercially available.

LPS-stimulated PBMC and metabolic activity assay

PBMC from buffy coats (Sanquin blood bank, Nijmegen, The Netherlands) from healthy human donors were isolated and stored in liquid nitrogen and subsequently thawed using standard procedures. Cell culture medium consisted of RPMI-1640 with 25 mM HEPES and 2 mM L-glutamine (Invitrogen, Merelbeke, Belgium) and further enriched with 100 U/mL penicillin/streptomycin, 1.0 mM sodium pyruvate and 10 % heat-inactivated fetal calf serum (FCS). PBMC in cell culture medium were pipetted into a 96-well flat bottom microtiter plate (BD Falcon, Erembodegem Aalst, Belgium) in a volume of 150 μL/well containing 1.5 × 10⁵ cells. Hop extract was dissolved in dimethyl sulfoxide (DMSO) and subsequently further diluted in cell culture medium and added to the cells in an individual concentration range. Final DMSO concentration in the wells was 0.1 %. In control experiments this concentration did not show any effects on the measured parameters. Subsequently, the cells were pre-incubated for 1 hour (volume per well 170 μL). Thereafter, LPS (E. coli B55:O55, Sigma, Zwijndrecht, The Netherlands) was added, resulting in a final volume of 200 μL and final concentration of 10 ng/mL LPS. Cells were incubated for 20 hours. All incubations of cells were done at 37 °C in a humidified environment containing 5 % CO₂. At the end of the 20 hours incubation period the supernatants were harvested and stored at -80 °C until further analysis. In parallel to the plates for PGE₂ analysis, identical plates were prepared and used to determine metabolic activity of the PBMC with and without the presence of test agent using the WST-1 assay (Roche Diagnostics, Almere, The Netherlands) as described elsewhere [15]. The activity of succinate dehydrogenase reflects mitochondrial activity and may therefore be indicative for metabolic activity and/or cell viability. Control values (without test agents) were set at 100 % and all values were expressed as percentage of control values.

COX-1 and COX-2 whole blood assay (WBA)

After obtaining written informed consent, ten mL blood from healthy human volunteers were collected by venupuncture into heparin (17 IU/mL). COX-1 and COX-2 WBA were adapted from Warner et al. [16]. In short, for the COX-1 assay, 70 μL test agents dissolved in DMSO and diluted in culture medium without FCS or vehicle were added and incubated with 100 μL whole blood for 1 hour after which the calcium ionophore A23187 (Calbiochem, VWR International, Amsterdam, The Netherlands) diluted in culture medium without FCS was added and incubated for 30 minutes in a final volume of 200 μL. For the COX-2 assay, 50 μL aspirin (66.7 μM in culture medium without FCS; Sigma) were added to 100 μL whole blood and incubated for 6 hours to inactivate COX-1 after which the test agents dissolved in DMSO and diluted in culture medium without FCS or vehicle and LPS (10 μg/mL) were added, followed by another 18 hours incubation. SC-560 (Calbiochem) and celecoxib (content of Celebrex capsule containing 200 mg celecoxib was corrected for its excipient content based on weight; Pfizer, local pharmacy) were used as positive controls for selective COX-1 and selective COX-2 inhibition, respectively. Concentration of DMSO (vehicle) was 0.1 % in all wells. Plates were centrifuged and supernatants were harvested and stored at -80 °C until analysis.

Zymosan-induced acute arthritis in mice

Male C57BL/6 mice (14 weeks old, Charles River, Maastricht, The Netherlands) were fed standard rodent diet (Teklad 18 % protein rodent diet, Harlan, Horst, The Netherlands) and received water ad lib. The animals received 1.25 mg hop extract suspended in tap water (n = 6) or vehicle (tap water, n = 7) once daily for 10 days by oral gavage (200 μL). At day eight, 6 μL zymosan (30 mg/mL in PBS; Sigma) were injected intra-articularly into the right knee joint under isoflurane and N₂O/O₂. Two days later the mice were bled under isoflurane and N₂O/O₂. Blood was collected into heparin collection tubes (Becton Dickinson, 85 IU heparin), after which the animals were euthanized.

^99mTechnetium accumulation in the knee joint as a measure for joint swelling

To determine the anti-inflammatory effect of 1.25 mg hop extract daily, zymosan-induced joint swelling was measured using ^99mTc pertechnetate uptake in the knee joints, as described elsewhere [17] at days 9 and 10 (1 and 2 days after zymosan injection). At day 1 after zymosan injection the joint swelling reaches a maximum and declines at day 2. A ratio of the ^99mTc uptake in the inflamed over the contralateral knee-joint of >1.1 indicated joint swelling.

³⁵SO₄ incorporation as a measure for cartilage proteoglycan synthesis

Healthy and arthritic cartilage proteoglycan synthesis has been reported to be affected by COX inhibitory agents [18] and is therefore measured in these mice. At day 2 after zymosan injection the animals were killed since at that time-point the arthritic cartilage proteoglycan synthesis is decreased maximally [17].

Patellae with a standard amount of surrounding tissue and tendons were removed and incubated for 2 hours in RPMI-1640 containing 30 μCi/mL ³⁵SO₄ (Na₂ ³⁵SO₄, Perkin-Elmer, Boston, MA, USA). To remove the non-incorporated ³⁵SO₄, the patellae were rinsed three times with 0.9 % NaCl. After overnight fixation in 10 % formaldehyde followed by 4 hours decalcification in 5 % formic acid, the patellae were removed from their tendons and incubated overnight at 60 °C with 0.5 mL tissue solubilizer (Lumasolve®, Lumac-LSC, Groningen, The Netherlands). Subsequently, scintillation fluid (Lipoluma®, Lumac-LSC) was added and the ³⁵SO₄ content of the patellae, which is a reliable measure of cartilage proteoglycan production [19], was measured by liquid scintillation analysis.

Ex vivo whole blood assay

To evaluate whether the dosage of 1.25 mg orally administered hop extract is absorbed in the blood and capable of decreasing LPS-stimulated PGE₂ production, an ex vivo WBA was performed. Heparinized blood was pipetted into a 96-well plate in a volume of 100 μL per well. Another volume of 100 μL LPS was added to the wells and incubated for 20 hours with a final concentration LPS of 1 μg/mL. Plates were than centrifuged and supernatants were harvested and stored at -80 °C until analysis of PGE₂.

PGE₂ measurement

PGE₂ from human PBMCs and human WBAs was measured in the thawed supernatants using a commercial competitive enzyme immunoassay (Biotrak Amersham, Buckinghamshire, UK) according to the manufacturer’s protocol 2. PGE₂ from murine WBA was assayed with a commercial direct enzyme immunoassay (Oxford Biomedical Research, Oxford MI, USA).

Statistical and data analysis

Curve-fitting of the inhibition of PGE₂ production by LPS-stimulated PBMC was performed using a sigmoidal dose-response curve and IC₅₀ values were derived using Graphpad Prism 4® software (Fig. [1]).

The absolute values of PGE₂ in pg/mL in the supernatants of the COX-1 WBA and the COX-2 WBA were analyzed by linear regression and followed by one-sample T-tests to test whether the slopes are < 0. To visualize the data, cubic spline curves were prepared in Graphpad Prism 4® (Fig. [2]). The IC₅₀ for inhibition PGE₂ production by hop extract in the COX-2 WBA was derived using sigmoidal dose-response curves in Graphpad Prism 4®.

PGE₂ production in supernatants of ex vivo LPS-stimulated murine whole blood was analyzed using a one-way ANOVA (since other test groups were included, not related to hop extract) followed by a one-sided post-hoc LSD multiple comparison test. Whether PGE₂ from hop extract treated mice was decreased versus PGE₂ from vehicle treated mice was evaluated. The one-sided test was chosen because hop extract decreased the PGE₂ production in both the LPS-stimulated PBMC test as well as in the COX-2 WBA. This led to the hypothesis that hop extract might decrease the PGE₂ production by ex vivo LPS-stimulated murine whole blood when administered orally (Fig. [3]).

Cartilage proteoglycan synthesis (³⁵SO₄ incorporation) was analyzed by using a one-way ANOVA and post-hoc the LSD multiple comparison test was performed (Fig. 4). Statistical analysis were performed using SPSS 12.0.1® and considered significant when p < 0.05.

Results

Hop extract dose-dependently inhibited PGE₂ production by LPS-stimulated PBMC, without compromising metabolic activity (Fig. [1] A and [1] B). The IC₅₀ for PGE₂ production is 3.6 ± 0.4 μg/mL (average ± SEM).

To differentiate whether inhibition of PGE₂ production by hop extract results from COX-1 or from COX-2 inhibition, a WBA was used. Hop extract was not able to inhibit PGE₂ after stimulation of the blood with A23187 (COX-1 WBA), indicating that hop extract did not inhibit COX-1 at the used concentrations up to 30 μg/mL. Both celecoxib and SC-560 as positive controls inhibited the PGE₂ production in the COX-1 WBA in a dose-dependent fashion. These effects were statistically significant. SC-560 did not inhibit the PGE₂ production after stimulation of the aspirin-pretreated blood with LPS (COX-2 WBA). Celecoxib dose-dependently inhibited the PGE₂ production in the COX-2 WBA which was statistically significant. At 2 μM, celecoxib inhibited PGE₂ production by 92 % in the COX-2 WBA and by 50 % in the COX-1 WBA proving its selective inhibitory properties for COX-2 over COX-1. Hop extract dose-dependently inhibited PGE₂ production in the COX-2 WBA (p < 0.05) and not in the COX-1 WBA, which indicates that hop extract selectively inhibited PGE₂ produced by COX-2 (Fig. [2]). The calculated IC₅₀ for the inhibition of PGE₂ production of hop extract in the COX-2 WBA was 20.4 ± 3.7 μg/mL (average ± SEM).

To determine the absorption of orally administered hop extract in the blood, PGE₂ was measured in ex vivo LPS-stimulated blood cells of mice with zymosan-induced acute arthritis and compared to production of PGE₂ in LPS-stimulated blood cells of vehicle-treated mice. The mean PGE₂ production in the supernatants of LPS-stimulated whole blood of mice treated with hop extract is 24 % lower (p = 0.04) than the mean PGE₂ production of vehicle treated mice (Fig. [3]), suggesting that administration of 1.25 mg hop extract becomes bioavailable.

Zymosan-induced joint swelling was optimal at day 1 and decreased at day 2. However, no effect of 1.25 mg hop extract on joint swelling was observed when compared to vehicle-treated mice (Table [1]).

Expectedly, cartilage proteoglycan synthesis of the arthritic joints was decreased compared to control joints. Nonetheless, there was no enhanced suppression of the inhibited proteoglycan synthesis by 1.25 mg hop extract treated mice compared to vehicle-treated mice. No difference of healthy cartilage proteoglycan synthesis was observed by 1.25 mg hop extract compared to vehicle-treated mice also (Table [1]).

Discussion

This study shows that a standardized CO₂ extract from Humulus lupulus (hop extract) is a potent inhibitor of the production of the inflammatory mediator PGE₂ by selectively inhibiting COX-2 in vitro. When hop extract was administered to mice at a dose of 1.25 mg daily the bioavailability was shown by a 24 % reduction of PGE₂ production of LPS-stimulated blood cells. In addition, it was found that this dose of hop extract has no negative or positive effects on cartilage proteoglycan synthesis or on joint swelling.

Inhibition of PGE₂ production by hop extract was demonstrated in LPS-stimulated PBMC (IC₅₀ = 3.6 μg/mL). Metabolic activity of these cells was not affected by any of the doses that were tested which excludes a cytotoxic effect of hop extract. In a whole blood assay (WBA) for COX-1 activity, no inhibition of PGE₂ production by hop extract was observed, in contrast to the WBA of COX-2 activity, in which PGE₂ production was strongly inhibited by hop extract (IC₅₀ = 20.4 μg/mL). The difference in IC₅₀ values of hop extract for the PBMC experiment and the COX-2 WBA is evident and can be attributed to the fact that the WBA, consisting of 50 % blood, has a higher concentration of plasma proteins that might interfere with hop extract than does the culture medium containing 10 % FCS.

In conclusion, the results from the COX-1 and COX-2 WBA, controlled by using the COX-1 inhibitor SC-560 and the selective COX-2 inhibitor celecoxib, indicate a COX-2 selective inhibitory capacity of the hop extract. It has been reported that humulone, one of the constituents of H. lupulus, suppressed the transcription of murine COX-2 in a mouse osteoblastic cell line, but was not effective on sheep COX-1 activity [14]. The data presented in the present study demonstrate the inhibition of PGE₂ production of the human COX-2 enzyme by the hop extract which supports the COX-2 inhibitory findings of humulone.

Ex vivo, it was shown that LPS-stimulated blood cells from mice receiving 1.25 mg hop extract daily, were capable of inhibiting PGE₂ by 24 % (Fig. [3]) as compared to the vehicle-treated group. This suggests that 1.25 mg hop extract, a dose based on in vitro data, was absorbed in the blood and hence became bioavailable. A recent abstract (referring to a poster) in which a non-disclosed dose of hop extract was given to healthy human volunteers mentioned its ex vivo selective COX-2 inhibition. The data presented in the present study about ex vivo inhibition of PGE₂ production of hop extract are in agreement with that abstract [20].

The zymosan-induced arthritis model in mice is characterized by the induction of joint swelling that reaches a maximum 24 hours after zymosan injection and which declines 48 hours after zymosan injection in the joint. At these time-points after intra-articular zymosan injection, effects of anti-inflammatory agents on joint swelling can be measured in this model of acute inflammation. Furthermore, this model is known for its acute cartilage damage. Cartilage proteoglycan synthesis is decreased maximally 48 hours after intra-articular zymosan injection. This gradually returns to normal values from 72 hours after zymosan injection onwards [17].

There are several studies reporting negative [21], [22], [23], [24], [25] as well as positive [26], [37], [28], [29] effects of COX inhibitory agents on healthy and arthritic cartilage proteoglycan synthesis. In order to evaluate the effects of hop extract on arthritic (and healthy) cartilage proteoglycan synthesis, the animal experiment was ended 48 hours after zymosan injection since arthritic cartilage proteoglycan synthesis is decreased maximally at that time-point. Moreover, effects of hop extract on joint swelling can be measured at optimal swelling and at sub-optimal swelling. The present study reports that no differences on healthy cartilage proteoglycan synthesis, or on zymosan-induced arthritic cartilage proteoglycan synthesis were detected between the hop extract- and vehicle-treated groups (Table [1]), identifying that the cartilage from these hop extract-treated mice is not more vulnerable to potential side effects. Joint swelling was optimal 24 hours after zymosan injection and declined 48 hours after zymosan injection for the vehicle-treated mice (Table [1]). However, the used dose of hop extract in this study was without effects on joint swelling as compared to vehicle-treated mice, suggesting that this dose has no functional anti-inflammatory capacity in vivo. One might conclude that the dose of hop extract should be elevated in order to determine whether hop extract is capable of inhibiting joint swelling. On the other hand, there are studies reporting that NSAIDs are not effective or only modestly effective at reducing inflammatory swelling, but still remain active by preventing hyperalgesia [30], [31]. It cannot be excluded that hop extract might be active by reducing hyperalgesia. Further experimentation with higher dosages of hop extract should determine its effectiveness as an anti-inflammatory natural product. Moreover, the effectiveness of hop extract as a potential analgesic agent should be explored in models of inflammatory pain.

In conclusion, this study reports that a standardized CO₂ extract from Humulus lupulus L. potently inhibits PGE₂ production of human inflammatory cells by inhibiting COX-2. In addition, orally administered hop extract becomes bioavailable as measured by PGE₂ inhibition of LPS-stimulated blood cells. However, the used dosage of 1.25 mg hop extract was without effects on joint swelling. Future work should be aimed at evaluating both the in vivo anti-inflammatory and hyperalgesic activity of hop extract at higher dosages in order to determine the potential of hop extract as a natural agent to be used in intervention strategies targeting chronic inflammatory diseases and/or inflammatory pain.

Acknowledgements

The authors wish to thank Dr. L. R. Verdooren for assisting with the statistical data analysis and E. L. Vitters for performing the intra-articular zymosan injections.

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Sander Hougee

Numico Research

P.O. Box 7005

6700 CA Wageningen

The Netherlands

Email: Sander.Hougee@Numico-Research.nl

References

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Sander Hougee

Numico Research

P.O. Box 7005

6700 CA Wageningen

The Netherlands

Email: Sander.Hougee@Numico-Research.nl