Effects of Some Natural Carotenoids on TRPA1- and TRPV1-Induced Neurogenic Inflammatory Processes In Vivo in the Mouse Skin Györgyi Horváth, Ágnes Kemény, Loránd Barthó, Péter Molnár, József Deli, Lajos Szente, Tamás Bozó, Szilárd Pál, Katalin Sándor, et al. Journal of Molecular Neuroscience ISSN 0895-8696 J Mol Neurosci DOI 10.1007/s12031-014-0472-7
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Author's personal copy J Mol Neurosci DOI 10.1007/s12031-014-0472-7
Effects of Some Natural Carotenoids on TRPA1- and TRPV1-Induced Neurogenic Inflammatory Processes In Vivo in the Mouse Skin Györgyi Horváth & Ágnes Kemény & Loránd Barthó & Péter Molnár & József Deli & Lajos Szente & Tamás Bozó & Szilárd Pál & Katalin Sándor & Éva Szőke & János Szolcsányi & Zsuzsanna Helyes
Received: 19 August 2014 / Accepted: 18 November 2014 # Springer Science+Business Media New York 2015
Abstract Mechanisms of the potent anti-inflammatory actions of carotenoids are unknown. Since carotenoids are incorporated into membranes, they might modulate transient receptor potential ankyrin 1 and vanilloid 1 (TRPA1 and TRPV1) activation predominantly on peptidergic sensory nerves. We therefore investigated the effects of three carotenoids (β-carotene, lutein and lycopene) on cutaneous neurogenic inflammation. Acute neurogenic edema and inflammatory cell recruitment were induced by smearing the TRPA1 agonist mustard oil (5 %) or the TRPV1 activator capsaicin (2.5 %) on the mouse ear. Ear thickness was then determined by micrometry, microcirculation by laser Doppler imaging and neutrophil accumulation by histopathology and spectrophotometric determination of myeloperoxidase activity. The effects of lutein on the
stimulatory action of the TRPA1 agonist mustard oil were also tested on the guinea-pig small intestine, in isolated organ experiments. Mustard oil evoked 50–55 % ear edema and granulocyte influx, as shown by histology and myeloperoxidase activity. Swelling was significantly reduced between 2 and 4 h after administration of lutein or β-carotene (100 mg/kg subcutane three times during 24 h). Lutein also decreased neutrophil accumulation induced by TRPA1 activation, but did not affect mustard oil-evoked intestinal contraction. Lycopene had no effect on any of these parameters. None of the three carotenoids altered capsaicin-evoked inflammation. It is proposed that the dihydroxycarotenoid lutein selectively inhibits TRPA1 activation and consequent neurogenic inflammation, possibly by modulating lipid rafts.
G. Horváth (*) : P. Molnár : J. Deli Department of Pharmacognosy, Medical School, University of Pécs, Rókus utca 2., Pécs 7624, Hungary e-mail:
[email protected]
Keywords Mustard oil . Transient receptor potential (TRP) vanilloid 1 ion channel (TRPV1) . TRPA1 receptor . Carotenoids . Skin inflammation . Intestinal contraction . Lipid rafts
Á. Kemény : L. Barthó : K. Sándor : É. Szőke : J. Szolcsányi : Z. Helyes Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Szigeti út 12., Pécs 7624, Hungary L. Szente CycloLab Ltd., Illatos út 7, Budapest 1097, Hungary T. Bozó Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó utca 37-47., Budapest 1094, Hungary S. Pál Institute of Pharmaceutical Technology and Biopharmacy, Medical School, University of Pécs, Rókus utca 2., Pécs 7624, Hungary Á. Kemény : É. Szőke : Z. Helyes Szentágothai Research Centre, University of Pécs, Ifjúság útja 20., Pécs 7624, Hungary
Introduction Carotenoids are important dietary nutrients with anti-oxidant potential. They are lipophilic substances derived from the same basic C40 isoprenoid skeleton (Britton et al. 1995a). Carotenoids can undergo extensive isomerization in solution, because of the large number of double bonds. The most widely occurring carotenoids are lycopene, β-carotene, lutein, violaxanthin, capsanthin and zeaxanthin. Most earlier studies examined β-carotene, but other carotenoids in the human diet have begun to receive attention recently (Molnár et al. 2005). The physico-chemical properties of carotenoids are modified by interactions with other molecules (e.g. proteins, lipids).
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These interactions may be critical in the functions and actions of carotenoids in membrane structures. Studies with model membranes enriched with polyunsaturated fatty acids indicated that the anti-oxidant/pro-oxidant activity of carotenoids was influenced by interactions with the membrane. The apolar carotenoids, lycopene and β-carotene, which distort the membrane bilayer, show a potent pro-oxidant effect, whereas the polar compound astaxanthin preserves membrane structure and exhibits significant anti-oxidant activity (McNulty et al. 2007, 2008). Systemic inflammatory response in critically ill patients is associated with increased lipid peroxidation and low carotenoid concentrations (Quasim et al. 2003). Several experimental and human studies concerning the role of carotenoids in inflammatory processes have been reported (Mayne et al. 2009; Palozza et al. 2009; Johnson and Krinsky 2009). Although their anti-inflammatory effects are well documented (Quasim et al. 2003; Yaping et al. 2003; Bhatt 2008), the mechanism of action is unknown. Neuro-immune interactions have been investigated extensively and their importance in chronic inflammatory diseases has been emphasized (Cevikbas et al. 2007; Geppetti et al. 2008). Proinflammatory neuropeptides released from capsaicinsensitive sensory nerves in response to the activation of transient receptor potential (TRP) receptors, such as ankyrin repeat 1 (TRPA1) and vanilloid 1 (TRPV1) ion channels, induce neurogenic inflammation in the innervated area (Helyes et al. 2003; Szolcsányi 2004; Helyes et al. 2009). Tachykinins (substance P: SP, neurokinin A and B) and calcitonin generelated peptide (CGRP) and the consequent neurogenic inflammation play an important role in the pathophysiological mechanisms of several chronic inflammatory diseases (ulcerative colitis, rheumatoid arthritis, allergic dermatitis) and neuropathic pain. These disorders affect a large number of people (Geppetti et al. 2006) and cannot be treated satisfactorily by the conventional anti-inflammatory and analgesic drugs. There is, therefore, a great need to understand the mechanisms precisely and identify novel targets for drug development. Lipid rafts surrounding the TRP channels/receptors regulate their function (Szőke et al. 2010). Lipid rafts, as microdomains rich in cholesterol, sphingomyelin and gangliosides, have been shown to have important functional significance in relation to plasma proteins that function as receptors and voltage-gated ion channels (Simons and Toomre 2000; Sjögren and Svenningsson 2007). Since carotenoids have the ability to incorporate into membranes and modulate membrane properties, they could influence the activation of TRP channels and, consequently, the function of the sensory nerve terminals that mediate neurogenic inflammatory processes. The aim of this study was, therefore, to investigate the effects of three major natural dietary carotenoids, with distinct structural features, on neurogenic inflammatory responses of
the mouse ear and guinea-pig small intestine induced by application of the TRPV1 agonist capsaicin (N-vanillylnonenamide) or the TRPA1 agonist mustard oil (allyl isothiocyanate). Studies with the intestine in vitro also add to our knowledge about the general neuro-pharmacology and smooth muscle pharmacology of lutein.
Materials and Methods Preparation of the Carotenoid Solutions The carotenoids investigated were the dicyclic compounds βcarotene, a hydrocarbon (Fig. 1a), lutein, a dihydroxy compound (Fig. 1b), and acyclic hydrocarbon lycopene (Fig. 1c). Solutions of β-carotene, lutein and lycopene (3, 10 and 15 mg/ml) were prepared freshly each experimental day with sterile castor oil (Oleum ricini virginale; Hungarian Pharmacopoeia VIII.) and stored in a dark bottle to avoid photo-degradation. Lycopene was isolated from tomato (Lycopersicon esculentum Mill., syn. Solanum lycopersicum L.) (Britton 1995b). As described by Horváth et al. (2010), β-carotene and lutein were isolated from flowers of Canadian goldenrod (Solidago canadensis L.) identified by Dr. Nóra Papp botanist. The purity, as determined by HPLC, was >97 % for β-carotene and lutein and >96 % for lycopene. Experimental Animals Experiments were performed on 12-week-old CD1 mice (25– 30 g, both genders) and on adult guinea pigs (350–450 g, short-haired, coloured, both genders) bred and kept in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy, University of Pécs.
Fig. 1 The chemical structure of the three natural carotenoids investigated in this study: β-carotene, lutein and lycopene. β-Carotene (a) and lutein (b) contain α- and β-ionone rings at the ends of the molecules; in the latter case, these rings are hydroxylated at the 3 and 3′ positions. Lycopene (c) has open structures on both sides
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The animals were maintained at 24–25 °C and provided with standard rat chow and water ad libitum. All experimental proced ures were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/ 1988) and complied with the recommendations of the International Association for the Study of Pain and with the Helsinki Declaration. The studies were approved by the Ethics Committee on Animal Research of University of Pécs according to the Ethical Codex of Animal Experiments (licence No.: BA 02/2000- 5-2011). Acute Neurogenic Inflammation Models Animals (n=10/group) were treated s.c. with the 100 mg/kg dose of lutein or β-carotene or lycopene (10 mg/ml, 0.1 ml/ 10 g) three times (0-, 8- and 24-h timepoints) throughout the 24-h experimental period. These doses were selected on the basis of previous exploratory experiments as well as published results obtained with the Rameb complex of lutein (Horváth et al. 2012). For lutein, two additional doses (30 or 150 mg/kg; 3 or 15 mg/ml, 0.3 or 0.5 ml/10 g) were also investigated; higher doses could not be applied due to solubility problems. In one series of experiments, ear inflammation was induced by the selective TRPV1 agonist capsaicin (N-vanillylnonenamide (Sigma); 2.5 %, in 96 % ethanol) applied topically on each surface (15–15 μl) of the ears at the beginning of the experiment and at the 3-h timepoint (Bánvölgyi et al. 2004). In another series, the TRPA1 agonist mustard oil (allylisothiocyanate (Sigma); 5 %, in paraffin oil) was administered (15–15 μl on each surface) every hour during a 6-h period (Bánvölgyi et al. 2004). Based on our previous experiments, a single administration of capsaicin or mustard oil only results in a transient, exclusively non-neurogenic edema formation without significant cellular infiltration (Bánvölgyi et al. 2004). In order to induce later, cellular inflammatory responses, we applied capsaicin twice (0- and 3-h timepoints) and mustard oil repeatedly every hour during the 6-h period (Bánvölgyi et al. 2004, 2005). Investigation of the Inflammatory Process To examine the inflamed mouse ear, ear diameter was measured with an engineer’s micrometer every hour after the induction of the inflammation throughout the 6-h experiment. Swelling was expressed as percentage of the initial control values. For histological examination, the ear sections were stained with haematoxylin-eosin (HE). AnalySIS software was used for histological scoring of mouse ear cross-sections. Ear thickness was measured at three different sites on three slides from each group and the number of accumulated
inflammatory cells was counted in three different fields of view on each sample, at ×400 magnification (n=3). Myeloperoxidase (MPO) enzyme activity, which correlates with the number of accumulated granulocytes (Bánvölgyi et al. 2004) was also measured from the ear homogenates by spectrophotometric assay (Bánvölgyi et al. 2004) 6 h after the induction of the inflammation. Measurement of Cutaneous Blood Flow in the Mouse ear Animals (n=6/group) were treated s.c. with 100 mg/kg castor oil as a solvent of carotenoids, β-carotene or lutein (10 mg/ml, 0.1 ml/10 g) three times (0-, 8- and 24-h timepoints) throughout the 24-h experimental period. Mice were then anaesthetized with ketamine and xylazine (100 and 5 mg/kg, s.c., respectively) and placed on a heating pad to maintain core body temperature. Blood flow was recorded by laser Doppler perfusion imaging (PeriScan PIM-II, Perimed, Sweden). The scanned area was set to 30×64 sampling points. Scanning of this area took 2 min. This procedure allowed simultaneous scanning of both ears. User-defined colour code was used to visualize blood flow values. Black represented areas with low blood perfusion. Colours ranging through blue, green and yellow to red represent increasing blood flow values. Baseline microcirculation values of the ears were measured for 6 min to provide three baseline images. After determination of baseline, the left ears were smeared with paraffin oil, the right ears were treated with 5 % mustard oil dissolved in paraffin oil, applied to the dorsal surface of the ears. Blood perfusion of the ears was then monitored for 30 min. Two regions of interest were chosen to represent the total area of both ears. Blood flow of the ears was calculated by comparing mean microcirculation values for the treated ears to the mean of the three baseline images (Pozsgai et al. 2012). Effects of Lutein in the Guinea-Pig Small Intestine Model In Vitro In these experiments, only lutein (6 or 10 μM concentration) was investigated. β-Carotene could not be tested because of solubility problems and lycopene was also not investigated because of its inactivity on either TRPA1 or TRPV1 ion channels. Adult guinea pigs were killed by a blow on the occiput and bled out. Segments of the pre-terminal ileum were set up as preparations along the longitudinal axis. The load on the tissue was 6 mN (constant throughout, since isotonic recording of movements was carried out). The medium was Krebs’ solution (composition [mM]: NaCl 119, NaHCO3 25, KH2PO4 1.2, CaCl2 2.5, MgSO4 1.5, KCl 2.5, and glucose 11), aerated by bubbling 5 % CO2 in O2. In some experiments, electrical field stimulation was performed through a pair of platinum electrodes, situated at the top and bottom of the organ bath,
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4 cm apart. Parameters of the stimulation were single square-wave impulses in each 20s, 80 V amplitude, 0.1 ms pulse width. The stimulant compounds, allyl isothiocyanate (mustard oil) in 200 μM concentration and N-vanillylnonenamide (capsaicin) in 0.3 μM concentration that caused approximately half-maximal contractions of the preparations (Barthó and Szolcsányi 1978; Barthó et al. 1999; 2013), were tested against lutein (6 or 10 μM). Also, single electrical pulses (see above) caused halfmaximal Btwitch^ contractions. Contact time for lutein was 20 min, and the concentration of the solution used was 2 mM (more concentrated solutions could not be prepared). Lutein’s solvent (50 % ethanol, 50 % DMSO) was also tested against the spasmogens (see Table 1 for mustard oil). Statistical Analysis Results are shown as means±S.E.M of ten or six mice per group and 5–7 guinea-pig small intestine preparation/experiments. Statistical analysis was performed by one-way or two-way (for time-dependent curves) repeated measures ANOVA followed by Bonferroni’s modified t test; in all cases, p
