http://informahealthcare.com/imt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2014.938874

RESEARCH ARTICLE

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Intravenous anesthetic propofol suppresses prostaglandin E2 and cysteinyl leukotriene production and reduces edema formation in arachidonic acid-induced ear inflammation Takefumi Inada, Kiichi Hirota, and Koh Shingu Department of Anesthesiology, Kansai Medical University, Osaka, Japan

Abstract

Keywords

Propofol is an intravenous drug widely used for anesthesia and sedation. Previously, propofol was shown to inhibit cyclo-oxygenase (COX) and 5-lipoxygenase (5-LOX) activities. Because these enzyme-inhibiting effects have only been demonstrated in vitro, this study sought to ascertain whether similar effects might also be observed in vivo. In the current studies, effects of propofol were tested in a murine model of arachidonic acid-induced ear inflammation. Specifically, propofol – as a pre-treatment – was intraperitoneally and then topical application of arachidonic acid was performed. After 1 h, tissue biopsies were collected and tested for the presence of edema and for levels of inflammatory mediators. The results indicated that the administration of propofol significantly suppressed ear edema formation, tissue myeloperoxidase activity, and tissue production of both prostaglandin E2 and cysteinyl leukotrienes. From the data, it can be concluded that propofol could exert anti-COX and anti-5-LOX activities in an in vivo model and that these activities in turn could have, at least in part, suppressed arachidonic acid-induced edema formation in the ear.

Arachidonic acid, cysteinyl leukotrienes, indomethacin, inflammation, propofol, prostaglandin E2

Introduction Propofol (2,6-di-isopropylphenol) is an intravenous general anesthetic widely used in the operating room for general anesthesia and in intensive care units for sedation (Vanlersberghe & Camu, 2008). In addition to its anesthetic effect, propofol is reported to have a variety of non-anesthetic effects, including anti-emesis, anti-epileptogenesis, and oxygen radical scavenging (Vasileiou et al., 2009). Also, due to its phenol-based structure, propofol has long been thought to have an anti-inflammatory effect (Vasileiou et al., 2009). Indeed, in vitro studies have indicated that propofol suppressed prostaglandin (PG) E2 and leukotriene (LT) production (Inada et al., 2011, 2013); this could partially explain the observed anti-inflammatory effect. These anti-cyclooxygenase (COX) and anti-5-lipoxygenase (5-LOX) properties of propofol could have far-reaching implications in clinical settings because these enzymes are involved in a variety of inflammatory states, including neuroinflammation (Teismann & Ferger, 2001; Manev et al., 2011). Nevertheless, although propofol may seem to be possibly an ideal extant anesthetic, many of its reported effects still must be demonstrated in vivo. Specifically, the enzyme-inhibitory properties of propofol have only been demonstrated in vitro and, as such, it remains to be seen if such effects can be directly extrapolated to in vivo scenarios. Therefore, the present study was undertaken to

History Received 27 March 2014 Revised 22 May 2014 Accepted 23 June 2014 Published online 21 July 2014

determine, using an arachidonic acid (AA)-induced ear inflammation mouse model, whether propofol could mitigate ear edema and whether the drug could affect tissue eicosanoid production as well.

Materials and methods Reagents Propofol (Diprivan) was purchased from Astra Zeneca (Osaka, Japan) and Intralipos was purchased from Otsuka Pharmaceuticals (Osaka, Japan). Acetone was purchased from Sigma Aldrich (St. Louis, MO) and dimethyl sulfoxide (DMSO) from Wako Pure Chemical Industries (Osaka, Japan). Arachidonic acid (AA), indomethacin, and MK 886 were obtained from Cayman Chemical (Ann Arbor, MI). Animals BALB/c mice (male, 8–9-weeks-of-age) were purchased from CLEA (Osaka, Japan) and housed in an animal facility maintained at 20–26  C with 30–70% relative humidity, and with a 12-h light/dark cycle. All animals were handled according to institutional guidelines. All mice were allowed ad libitum access to standard rodent chow and autoclaved water. The Institutional Animal Care and Use Committee of the Kansai Medical University approved this study. Arachidonic acid-induced ear inflammation

Address for correspondence: Takefumi Inada, MD, Department of Anesthesiology, Kansai Medical University, 5-1 Shin-machi 2-Cho-me, Hirakata, Osaka 573-1010, Japan. Tel: 81728040101. E-mail: [email protected]

For the arachidonic acid (AA)-induced ear inflammation model (Inoue et al., 1988; Young et al., 1984), mice were given a topical application (10 ml) of AA (1 mg/mouse) in acetone that was

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applied to the inner surface of the right ear. Acetone alone was applied to the inner surface of the left ear as an untreated reference control. At 1 h after AA application, an 8-mm diameter disc of tissue was isolated from the center of the ear using a sterile hole-punch (Kai Medical, Gifu, Japan). The period of 1 h was selected because it was previously reported that the ear reached maximum swelling 45–60 min after AA application (Inoue et al., 1988; Young et al., 1984). Edema was measured by determining the wet weight differences between the right and left ear samples.

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Test drug administration Propofol was injected intraperitoneally (IP, at 100 mg/kg) 5 min before the application of the AA. This dose was selected for use here as it was previously shown to be sufficient to sedate BALB/c mice (evidenced by loss of righting reflex for 10 s) for 30–60 min (Inada et al., 2004). Further, the timing of the administration of propofol was selected to align with the period of expected maximum AA-induced edema formation (i.e. 45–60 min) (Inoue et al., 1988; Young et al., 1984). In clinical practice, propofol is emulsified in a lipid solution (i.e. as Diprivan). This lipid vehicle is similar to 10% Intralipos which was used as the vehicle control. As a positive control, indomethacin was prepared as a 15 mg/ml (in DMSO) stock solution and dissolved further in 10% Intralipos for dosing. For this study, the indomethacin (delivered at 15 mg/kg) was injected IP 30 min before the application of AA, as suggested in Teeling et al. (2010).

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(or cysLTs, LTB4) and an acetyl-cholinesterase conjugate of PGE2 (or cysLTs, LTB4) for binding to a limited amount of anti-PGE2 (or -cysLTs, -LTB4) antibody. The detection limits for the PGE2, cysLTs, and LTB4 assays were, respectively, 36, 20, and 45 pg/ml. Eicosanoid concentrations were each presented as pg/mg protein in the starting supernatant sample. Statistical analysis All data were expressed as mean ± SEM. For statistical analysis, an unpaired Student’s t-test and a 1-way analysis of variance (ANOVA) with a Bonferroni correction were performed using Prism 4 software (GraphPad, San Diego, CA). Statistical significance was assigned at a p-value50.05.

Results AA-induced ear edema Topical application of AA to the ear of the murine hosts provoked an increase in weight relative to that of the counterpart ear that was treated with vehicle only (Figures 1A). The degree of edema induced was reflected by the net increase in weight of each ear-punch sample (Figure 1B). In the present study, the propofol treatment significantly suppressed AA-induced edema by 55.7 (± 4.3)%. As a positive control, this study confirmed that intraperitoneal treatment with 15 mg indomethacin/kg (injected 30 min before AA application) caused significant reduction in edema [by 52.0 (± 5.0)%], mimicking the effect of propofol.

Tissue sample preparation After weighing, each ear tissue punch sample was placed in 200 ml cold phosphate buffered saline (PBS) that contained an eicosanoid inhibitor cocktail of indomethacin (10 mM final concentration) and MK 886 (2 mM final concentration) and then homogenized on ice with a Plus-One Sample Grinding Kit (GE Healthcare, Buckinghamshire, UK). Thereafter, each sample was centrifuged at 15 000 rpm (10 min, 4  C) and resulting supernatants were collected and frozen (80  C) until used for subsequent measurements. Protein concentrations in the supernatants were measured via a Bradford assay (Bio-Rad, Hercules, CA).

MPO activity Topical application of AA to the ear caused a small but significant increase in MPO activity (vs AA [] value). MPO activity increased from 47.03 (± 2.86) to 60.78 (± 2.14) mU/ml (Figure 2). This change in activity was blocked by propofol pretreatment, with the value now significantly decreased to 49.55 (± 1.33) mU/ml. Eicosanoid production in tissue after AA application

Myeloperoxidase (MPO) activity in each supernatant was measured with a commercially available kit (BioVision, Milpitas, CA) according to manufacturer instructions. Briefly, in the assay (kit), MPO was incubated with H2O2 and Cl for 1 h at 25  C to produce hypochlorous acid (HClO) that, in turn, reacted with taurine to generate taurine chloramine. A 5-thio-2-nitrobenzoic acid (TNB) color probe was then added, and the absorbance was measured (l ¼ 412 nm) in an EnSpire plate reader (PerkinElmer, Waltham, MA). The MPO enzyme activity in a given sample was directly proportional to the extent of TNB bleaching. All data were reported in terms of MPO activity (mU/ml) in the starting supernatant from the sample of homogenate (200 ml). One unit of MPO was defined as the amount of MPO that generated enough taurine chloramine to consume 1.0 mmol TNB/min at 25  C.

Application of AA to the ear significantly increased PGE2 concentrations in the ear tissue. This increase was suppressed by pre-treatment of the host with propofol (Figure 3A). Similarly, pre-treatment with indomethacin also significantly reduced tissue PGE2 concentrations. Compared to levels in the vehicle-treated hosts, PGE2 decreased from 54.06 (± 7.19) pg/mg to 28.47 (± 2.29) and 9.33 (± 1.18) pg/mg, respectively, in the tissues from the propofol and indomethacin mice. Application of AA also caused a significant increase in tissue cysLT concentrations, albeit these concentrations were generally low (Figure 3B). As with PGE2, this increase was inhibited with propofol pre-treatment. Tissue LTB4 concentrations were below the detection limit, both before and after AA. Pre-treatment with indomethacin before topical application of AA significantly increased cysLT concentrations in the tissues (Figure 3C); the same treatment also raised LTB4 levels to detectable levels. The concentrations in five indomethacin-treated ear samples were 0.52, 0.51, 0.66, 0.17*, and 0.37 pg/mg (*below the detection limit).

Measurement of PGE2 and LT concentrations

Discussion

Eicosanoids in the supernatant samples were purified via elution through solid phase extraction (SPE) cartridges (C-18) (Cayman Chemical) according to manufacturer instructions. The concentrations of PGE2, cysteinyl leukotrienes (cysLTs), and LTB4 were then determined using an enzyme immunoassay (Cayman Chemical). The assay was based on competition between PGE2

In the present study, an arachidonic acid (AA)-induced ear inflammation model was used to test the in vivo anti-inflammatory properties of propofol. Overall, the results showed that propofol ameliorated ear edema formation and decreased production of prostaglandin (PG)-E2 and cysteinyl leukotrienes (cysLTs) in AA-treated ear tissues. To the best of our knowledge,

Myeloperoxidase assay

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DOI: 10.3109/1547691X.2014.938874

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Figure 2. Myeloperoxidase (MPO) activity in arachidonic acid (AA)-induced ear inflammation. AA (1 mg/mouse) was topically applied to the right ear; 1 h later, ear biopsies were taken, and MPO activity was subsequently measured. Propofol (100 mg/kg) was given IP 5 min before AA application. One unit of MPO was defined as the amount of MPO that generated taurine chloramine to consume 1.0 mmol TNB/min at 25  C. n ¼ 7–8/group. *p50.005 vs AA (). #p50.01 vs 0.

Figure 1. Arachidonic acid (AA)-induced ear inflammation. AA (1 mg/mouse) was topically applied to the right ear and vehicle alone to the left ear. One hour later, discs of ear tissue were excised and edema determined by the difference in weight between the right (AA-treated) and left (AA-untreated) ear samples. To test drug effects, propofol (100 mg/kg) or indomethacin (15 mg/kg) was given IP at 5 and 30 min, respectively, before the AA application. Weights of the right (AA-treated) and left (AA-untreated) ears of the mice (A), and the differences (B) between these weights (reflecting level of edema) are shown. n ¼ 5/group. *p50.001 vs 0 (that of right ear in (A)).

this is the first report to show that propofol imparts COX/5-LOX inhibiting effects in vivo. In vitro studies had previously shown that propofol had COX/ 5-LOX-inhibiting effects in a variety of cells (Inada et al., 2010, 2011, 2013; Kambara et al., 2009; Kubo et al., 2011). Propofol itself does not change the availability of AA (the substrate of COX/5-LOX enzymes) or the expression levels of the enzymes; rather, it appears that propofol directly suppresses the activity of the COX/5-LOX. Suppression in a cell-free system with COX/5LOX recombinant enzymes provided the evidence that propofol impacts directly on COX/5-LOX activity (Inada et al., 2013; Kubo et al., 2011). The AA-induced ear inflammation model is a classic, simple, readily reproducible model. It is based on the principle that the metabolism of AA into eicosanoids leads to inflammation, which results in edema formation (Inoue et al., 1988; Young et al., 1984). The model was reported to be useful for testing the

efficacy of anti-inflammatory drugs, such as non-steroidal antiinflammatory drugs (NSAID), including indomethacin. The most important mediator in that inflammatory model may be PGE2, although the exact mechanism of inflammation remains to be defined. A recent study demonstrated that a crucial role of PGE2 is to bind to the EP3 receptor on mast cells in ear tissues. This binding leads to release of pro-inflammatory mediators including histamine and interleukin (IL)-6 (Morimoto et al., 2014). A contribution of LTs has also been suggested (Haribabu et al., 2000; Rao et al., 2007). In the present study, propofol significantly reduced AA-induced increases in PGE2 and cysLT production. It is conceivable that the lesser tissue production of PGE2 (and presumably to a lesser extent cysLTs) could have alleviated the ear edema in the model. Myeloperoxidase (MPO) is abundant in neutrophils (PMN); thus, MPO activity may generally correspond to the extent of PMN recruitment to tissues (Bradley et al. 1982). However, MPO is also present, at much lower concentrations, in monocytes and macrophages. Thus, the considerable MPO activity in ear samples without AA stimulation (Figure 2) may be ascribed to the MPO in resident macrophages and neutrophils/monocytes in blood vessels in the ear tissues. Upon AA stimulation, a small significant increase in MPO activity was detected. One could assume the small increase in MPO activity after the AA treatment was primarily due to hyperemia and a corresponding increase in numbers of PMN in the ear vessels, although extravasated PMN may also have contributed to MPO activity (see below). Propofol treatment reversed the AA-induced increased MPO activity to levels similar to those seen without AA treatment. Presumably, the propofol-induced decrease in MPO activity arose mainly due to a reduction of hyperemia (which should decrease PMN numbers in the ear vessels). The present study could not detect LTB4 in the AA-treated tissues; this absence suggested that LTB4 (a PMN chemoattractant; Henderson, 1994) may only have made a minor contribution to the influx of PMN into these tissues. The latter finding was inconsistent with previous studies that implied LTB4 played an important role due to significant PMN tissue recruitment in the same mouse model (Haribabu et al., 2000; Rao et al., 2007). This discrepancy may be due to the different mouse strains used here vs elsewhere; inflammatory reactions may be predicted to be

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Figure 3. Tissue prostaglandin E2 (PGE2) and cysteinyl leukotriene (cysLTs) concentrations in AA-induced ear inflammation. (A) Tissue PGE2. (B) cysLTs. AA (1 mg/mouse) was topically applied to the right ear; 1 h later, ear biopsies were taken for measurement. Propofol (100 mg/kg) and indomethacin (15 mg/kg) were given IP 5 and 30 min, respectively, before the AA application. (C) Production of cysLTs after AA alone or AA combined with indomethacin pre-treatment. From a statistical point of view, indomethacin results are presented separately due to the high cysLT concentrations. n ¼ 5/group. *p50.001 vs AA (); #p50.01, ##p50.001 vs 0.

considerably different between C57BL6 mice (Haribabu et al., 2000) and BALB/c mice (present study). Alternatively, the study design here may have allowed insufficient time for PMN to enter the tissues after AA application; i.e. 1 h (here) vs 3 h (Rao et al., 2007). In addition to the main findings, the study here showed that indomethacin increased tissue cysLT and LTB4 concentrations. This intriguing finding may explain a curious phenomenon that occurs with NSAID suppression of COX enzyme activity; i.e. that suppression results in a diversion of AA (which fuels LT synthesis) and leads to a paradoxical enhancement of inflammation. Thus, it is possible, in inflammatory situations where a considerable LT contribution is also expected in addition to PG, the dual COX/5-LOX inhibiting activity of propofol could be more effective at alleviating inflammation than the COX inhibition alone by an NSAID.

Conclusions The present study showed that the intravenous anesthetic propofol suppressed tissue PGE2 and cysLT production in a murine AA-induced ear inflammation model. Edema of the ear was also mitigated by the propofol treatment. Therefore, it seems likely that propofol may serve as a dual COX/5-LOX inhibitor in vivo and, thus, this drug could be associated with anti-inflammatory effects beneficial in the context of surgery. Clearly, further work needs to be done before any clinical benefits/implications can be reported concerning the inhibition of these enzyme activities by propofol.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) (C)-25462419 from the Japan Society for the Promotion of Science, and by the Ministry of Education, Culture, Sports, Science, and Technology in Japan (MEXT)-supported Program for the Strategic Research Foundation at Private Universities 2011–2015.

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Inada, T., Ueshima, H., and Shingu, K. 2013. Intravenous anesthetic propofol suppresses leukotriene production in murine dendritic cells. J. Immunotoxicol. 10:162–169. Inoue, H., Mori, T., and Koshihara, Y. 1988. Sulfidopeptide-leukotrienes are major mediators of arachidonic acid-induced mouse ear edema. Prostaglandins. 36:731–739. Kambara, T., Inada, T., Kubo, K., and Shingu, K. 2009. Propofol suppresses prostaglandin E2 production in human peripheral monocytes. Immunopharmacol. Immunotoxicol. 31:117–126. Kubo, K., Inada, T., and Shingu, K. 2011. Possible role of propofol’s cyclooxygenase-inhibiting property in alleviating dopaminergic neuronal loss in the substantia nigra in an MPTP-induced murine model of Parkinson’s disease. Brain. Res. 1387:125–133. Manev, H., Chen, H., Dzitoyeva, S., and Manev, R. 2011. Cyclooxygenases and 5-lipoxygenase in Alzheimer s disease. Prog. Neuropsychopharmacol. Biol. Psychiatry 35:315–319. Morimoto, K., Shirata, N., Taketomi, Y., et al. 2014. Prostaglandin E2-EP3 signaling induces inflammatory swelling by mast cell activation. J. Immunol. 192:1130–1137.

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Rao, N. L., Dunford, P. J., Xue, X., et al. 2007. Anti-inflammatory activity of a potent, selective leukotriene A4 hydrolase inhibitor in comparison with the 5-lipoxygenase inhibitor zileuton. J. Pharmacol. Exp. Ther. 21:1154–1160. Teeling, J. L., Cunningham, C., Newman, T. A., and Perry, V. H. 2010. The effect of non-steroidal anti-inflammatory agents on behavioural changes and cytokine production following systemic inflammation: Implications for a role of COX-1. Brain Behav. Immun. 24:409–419. Teismann P, and Ferger, B. 2001. Inhibition of the cyclooxygenase isoenzymes COX-1 and COX-2 provide neuroprotection in the MPTPmouse model of Parkinson’s disease. Synapse 39:167–174. Vanlersberghe, C., and Camu, F. 2008. Propofol. Handbook Exp. Pharmacol. 182:227–252. Vasileiou, I., Xanthos, T., Koudouna, E., et al. 2009. Propofol: A review of its non-anesthetic effects. Eur. J. Pharmacol. 605:1–8. Young, J. M., Spires, D. A., Bedord, C. J., et al. 1984. The mouse ear inflammatory response to topical arachidonic acid. J. Invest. Dermatol. 82:367–371.

Intravenous anesthetic propofol suppresses prostaglandin E2 and cysteinyl leukotriene production and reduces edema formation in arachidonic acid-induced ear inflammation.

Propofol is an intravenous drug widely used for anesthesia and sedation. Previously, propofol was shown to inhibit cyclo-oxygenase (COX) and 5-lipoxyg...
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