Fitoterapia 95 (2014) 160–174
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Antiinflammatory activity of glucomoringin isothiocyanate in a mouse model of experimental autoimmune encephalomyelitis Maria Galuppo a,1, Sabrina Giacoppo a,1, Gina Rosalinda De Nicola b, Renato Iori b, Michele Navarra c, Giovanni Enrico Lombardo c, Placido Bramanti a, Emanuela Mazzon a,⁎ a b c
IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, contrada Casazza, 98124 Messina, Italy Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali (CRA-CIN), Via di Corticella 133, 40128 Bologna, Italy Università degli Studi di Messina, Facoltà di Farmacia, Dipartimento di Scienze del farmaco e dei Prodotti per la Salute, Viale Annunziata, 98168 Messina, Italy
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Article history: Received 6 February 2014 Accepted in revised form 12 March 2014 Available online 29 March 2014 Keywords: Glucomoringin Isothiocyanate Inflammation Multiple sclerosis Cytokines
a b s t r a c t Glucomoringin (4(α-L-rhamnosyloxy)-benzyl glucosinolate) (GMG) is an uncommon member of glucosinolate group belonging to the Moringaceae family, of which Moringa oleifera Lam. is the most widely distributed. Bioactivation of GMG with the enzyme myrosinase forms the corresponding isothiocyanate (4(α-L-rhamnosyloxy)-benzyl isothiocyanate) (GMG-ITC), which can play a key role in antitumoral activity and counteract the inflammatory response. The aim of this study was to assess the effect of GMG-ITC treatment in an experimental mouse model of multiple sclerosis (MS), an inflammatory demyelinating disease with neurodegeneration characterized by demyelinating plaques, neuronal, and axonal loss. For this reason, C57Bl/6 male mice were injected with myelin oligodendrocyte glycoprotein35–55 which is able to evoke an autoimmune response against myelin fibers miming human multiple sclerosis physiopatogenesis. Results clearly showed that the treatment was able to counteract the inflammatory cascade that underlies the processes leading to severe MS. In particular, GMG-ITC was effective against proinflammatory cytokine TNF-α. Oxidative species generation including the influence of iNOS, nitrotyrosine tissue expression and cell apoptotic death pathway was also evaluated resulting in a lower Bax/Bcl-2 unbalance. Taken together, this work adds new interesting properties and applicability of GMG-ITC and this compound can be suggested as a useful drug for the treatment or prevention of MS, at least in association with current conventional therapy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: GMG, glucomoringin; GMG-ITC, glucomoringin-isothiocyanate; MS, multiple sclerosis; GLs, glucosinolates; ITCs, isothiocyanates; EAE, experimental autoimmune encephalomyelitis; TNF-α, tumor necrosis factor alpha; IL-10, interleukin-10; CFA, complete Freund's adjuvant; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; (LFB), luxol fast blue; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling; MAPKs, mitogenactivated protein kinases; NSAIDs, nonsteroidal anti-inflammatory drugs. ⁎ Corresponding author. Tel.: +39 09060128708; fax: +39 09060128850. E-mail address: [email protected] (E. Mazzon). 1 These authors share the first authorship.
http://dx.doi.org/10.1016/j.fitote.2014.03.018 0367-326X/© 2014 Elsevier B.V. All rights reserved.
Phytomedicines have been playing a significant role in the prevention and treatment of various human aliments and it is a long time that the folk medicine relies to the medical value of leaves and seeds of Moringa oleifera Lam. [1]. M. oleifera, which belong to the Moringaceae family, is a plant widely cultivated in many countries of the tropics and it is known as a medicinal plant. More in detail, M. oleifera is a multipurpose tree which grows in many tropical or equatorial regions, around dry tropical areas in Northwestern India at the Southwestern foot of the Himalaya that is used for human and animal nutrition, and medicinal
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purposes. Hypoglycaemic, hypotensive, liver-protective, immunomodulatory, antioxidant and antitumor activities of M. oleifera have been assessed and demonstrated by several pharmacological studies [2–4]. Moreover, leaves are useful for preventing malnutrition and seeds are used safely for water purification [5]. Glucosinolates (GLs) are well known sulfur-containing secondary metabolites (ca. 130 molecules identified to date), which display a structural homogeneity based on a β-D-glucopyranosyl unit and an O-sulfated anomeric (Z)-thiohydroximate function connected to a variable side chain depending on the amino acid metabolism of the plant species [6]. GLs are bioactivated by enzymatic hydrolysis involving myrosinase (E.C. 3.2.1.147), releasing at neutral pH values health-promoting isothiocyanates (ITCs) [7,8]. The numerous effects exhibited by these natural compounds through which they exerted a protective action against cancer progression [9] and neurodegenerative disease were recently reported [10–12]. Glucomoringin (GMG; 4(α-L-rhamnosyloxy)-benzyl GL), a typical secondary metabolite present in large amount in seeds of M. oleifera [13], is an uncommon member of the GLs family and presents a unique characteristic consisting in a second glycosidic residue in its side chain. The glycosylated isothiocyanate 4(alpha-L-rhamnosyloxy)-benzyl isothiocyanate (GMG-ITC), resulting from myrosinase-hydrolysis (N99%) of GMG at neutral condition (Fig. 1), has been shown to exhibit a broad biological activity and it was also shown to exert an effective antitumor promoting activity [14,15].
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Gram-scale production of natural GMG has been recently set up starting from M. oleifera seeds according to a procedure developed at CRA-CIN of Bologna, Italy [2]. The aim of this work was to test GMG bioactivated with myrosinase that provides the therapeutic natural agent GMG-ITC, in an animal model of experimental autoimmune encephalomyelitis (EAE). The EAE mimics the physiopathology of the human multiple sclerosis (MS), with weight loss and progressive paralysis [16]. Basically, MS is a chronic inflammatory disease, attacking central nervous system and resulting in demyelination as well as oligodendrocyte loss, glial scar formation and subsequent degeneration of axonal and neuronal damage [17,18]. The pathogenesis of MS has been related to the unbalance invasive (Th1 and Th17) and defensive (Th2 and Treg) T cell responses. This is believed crucial for the development and progression of MS both in humans and EAE in rodents. Also, since an involvement of vascular endothelial is supposed, being alterations in blood vessel properties of central importance in the initiation and/or maintenance of the disease [19,20], our research started from the activation of a systemic mediators in response to EAE-induction, such as tumor necrosis factor alpha (TNF-α). Moreover, we tested the correlated molecular pathways involved in serious inflammatory disease, such as the possible activation of oxidative cascade (NOS2 and nitrotyrosine formation) as well as the state of neuronal cell life (Tunel assay) following injection of myelin oligodendrocyte glycoprotein (MOG)35–55, that leads to alterations of Bax/Bcl-2 stability.
Fig. 1. A) Production of 4-(α-L-rhamnosyloxy)benzyl isothiocyanate (GMG-ITC). Reaction of myrosinase catalyzed hydrolysis of glucomoringin (GMG), purified from Moringa oleifera seeds, in phosphate buffered saline (PBS) solution (pH 7.2) to produce GMG-ITC. B) Body weight. C57Bl/6 mice have been daily weighed. After EAE induction, a significative reduced increasing in body weight was observed in EAE mice compared to the naive group. Difference in body weigh between the naive group and the “EAE + GMG-ITC group” or the “GMG-ITC control group” didn't result statistically significative. * p b 0.05 vs naive.
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The relevance and innovation of the present study lie in the possible use of a safe new formulation of GMG bioactivated with myrosinase that provides the therapeutic natural agent GMG-ITC, which can be considered as a useful drug for the treatment or prevention of MS.
5 = complete hind limb paralysis; 6 = moribund or dead animal. Mice with high score of disease were sacrificed to avoid animal suffering.
2. Materials and methods
GMG was isolated from M. oleifera L. (fam. Moringaceae) seeds according to a method previously described [2,21]. In brief, GMG was purified by two sequential chromatographic steps: isolation through anion exchange chromatography, followed by gel filtration to achieve purification to homogeneity. GMG was unambiguously characterized by 1H and 13C NMR spectrometry and the purity was assessed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method accepted by European Economic Community, Commission Regulation, EEC No. 1864/90, yielding GMG with a purity of 99% based on peak area value, and about 90–92% on a weight basis due to its high hygroscopic properties. The enzyme myrosinase was isolated from seeds of Sinapis alba L. as described by Pessina et al. [22] with some modification. The specific activity of the stock solution used in the present study was 60 U/mg of soluble protein. The enzymatic activity was 32 U/ml and the solution was stored at 4 °C in sterile saline solution at neutral pH until use. One myrosinase unit was defined as the amount of enzyme able to hydrolyze 1 μmol/min of sinigrin at pH 6.5 and 37 °C [8].
2.1. Animals Male adult C57Bl/6 mice (20-25 g, Harlan Nossan, Milan, Italy) were used for all studies. Mice were housed in stainless steel cages and maintained under 12 h/12 h light/dark cycle at 21 ± 1 °C and 50 ± 5% humidity. Animals were acclimated to their environment for 1 week and food and water were given ad libitum. 2.2. Ethics statement This study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the National Institutes of Health. The protocol was approved by the Ministry of Health “General Direction of animal health and veterinary drug”. In particular, animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116/92) as well as with the EEC regulations (O.J. of E.C.L 358/1 12/18/1986). Experimental procedures did not cause any significant animal suffering. 2.3. Reagents MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) was synthesized and purified by Cambridge Research Biochemicals (Billingham, UK). Complete Freund's adjuvant (CFA) containing Mycobacterium tubercolosis H37Ra strain was purchased from Difco Laboratories (Sparks, MD, USA), while Bordetella pertussis toxin was from Sigma–Aldrich Company Ltd. (Milan, Italy). All other chemicals were of the highest commercial grade available. 2.4. EAE induction After anaesthesia, mice were immunized subcutaneously with 300 μl/flank of the emulsion consisting of 300 μg of MOG35–55 in phosphate-buffered saline (PBS) combined with an equal volume of CFA containing 300 μg heat-killed M. tubercolosis H37Ra. After emulsion–injection, animals were immunized with an injection of 100 μl of B. pertussis toxin (500 ng/100 μl, i.p), repeated 48 h later. The disease follows a course of progressive degeneration, with visible signs of pathology consisting of flaccidity of the tail and loss of motion of the hind legs. 2.5. Body weight and clinical score Mice were daily weighed and observed for signs of EAE. Clinical score was evaluated using a standardized scoring system [30]. Briefly, clinical signs were scored as follows: 0 = no signs; 1 = partial flaccid tail; 2 = complete flaccid tail; 3 = hind limb hypotonia; 4 = partial hind limb paralysis;
2.6. GMG and myrosinase purification.
2.7. Enzyme bioactivation of GMG and animal treatment GMG (10 mg/kg) was dissolved in PBS solution pH 7.2 and mouse treatment required the enzyme bioactivation of the phytochemical. The action of myrosinase enzyme (5 μl/mouse) for 15 min allowed to have GMG-ITC quickly, before the i.p treatment (Fig. 1). The total conversion of pure GMG into GMG-ITC was confirmed by HPLC analysis of the GMG desulfo-derivative, which allowed us to monitor the reduction of GMG until its complete disappearance in the reaction mixture [21]. 2.8. Experimental design Mice were randomly allocated into the following groups (n = 30 total animals): 1. EAE group (n = 10): mice subjected to EAE that did not receive GMG-ITC; 2. EAE + GMG-ITC group (n = 10): mice subjected to EAE were treated with bioactive GMG-ITC (10 mg/kg GMG + 5 μl/mouse myrosinase). GMG-ITC was daily i.p. administrated 1 week before the induction of EAE and, after immunization the treatment was daily protracted until the sacrifice; 3. Naive group (n = 5): mice that received neither vehicle (saline) nor MOG35–55; 4. GMG-ITC control group (n = 5): mice daily i.p. treated with bioactive GMG-ITC (10 mg/kg GMG + 5 μl/mouse myrosinase) for all the duration of the experiment. GMG-ITC was given as pretreatment once a day for 7 days via i.p. injection. The disease was induced according to the experimental procedure reported above and “EAE + GMG-ITC”
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group received a post-treatment prolonged until twenty-first day. At the end of the experiment, animals were sacrificed and spinal cord tissues were harvested and processed in order to evaluate parameters of disease. 2.9. Western blot analysis All the extraction procedures were performed on ice using ice-cold reagents. In brief, spinal cord tissues were suspended in extraction buffer containing 0.32 M sucrose, 10 mM Tris– HCl, pH 7.4, 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM 2-mercaptoethanol, 50 mM NaF, protease inhibitor tablets (Roche), and they were homogenized at the highest setting for 2 min. The homogenates were chilled on ice for 15 min and then centrifuged at 1000 g for 10 min at 4 °C, and the supernatant (cytosol + membrane extract from spinal cord tissue) was collected. The pellets were suspended in the supplied complete lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA protease inhibitors tablets (Roche), and then they were centrifuged for 30 min at 15,000 g at 4 °C, and the supernatant (nuclear extract) was collected. Supernatants were stored at –80 °C until use. Protein concentration in homogenate was estimated by the Bio-Rad Protein Assay (Bio-Rad) using BSA as standard, and 50 μg both of cytosol and nuclear extract from each sample was analyzed. Proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Westran S PVDF Membranes, Whatman™, GE Healthcare Companies, Dassel, Germany), blocked with PBS containing 5% nonfat dried milk for 45 min at room temperature, and subsequently probed at 4 °C overnight with specific antibodies for Bax (1:500 Santa Cruz Biotechnology, Inc), phospho-ERK p42/44 (1:500 Cell Signaling), ERK-2 (1:1000; Cell Signaling), in 1 × PBS, 5% (w/v) non fat dried milk, 0.1% Tween-20 (PMT). Membranes were incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG secondary antibodies (1:2000, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of proteic lysates, they were also incubated with GAPDH-HRP conjugated primary antibody (1:1000 Cell Signaling). Relative protein band expression for Bax (23 kDa) and phospho-ERK p42/44 (42 and 44 kDa double band), was visualized using an enhanced chemiluminescence system (Luminata™ Forte, Western HRP substrate, Millipore) and proteic bands were acquired and quantified with ChemiDoc ™ MP System (Bio-Rad) and a computer program (ImageJ), respectively. 2.10. Histological evaluation After sample fixation at room temperature in 10% (w/v) PBS-buffered formaldehyde, spinal cord tissue was dehydrated in graded ethanol and embedded in Paraplast (Sherwood Medical). Thereafter, 7-μm sections were deparaffinized with xylene, stained with hematoxylin and observed in a microscope (LEICA DM 2000 combined with LEICA ICC50 HD camera).
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2.11. Luxol Fast Blue (LFB) To show myelin and phospholipids in histological sections, LFB staining was performed according to the manufacturer's protocol (http://www.bio-optica.it/pdf3/200812.pdf, Bio-Optica, Milano S.P.A). LFB affinity for central nervous system is usually ascribed to the bonds it forms with phospholipidic structures such as lecithin and sphingomyelin. The staining provides: myelin in turquoise blue, neurons and glial nuclei in pink/violet and Nissl substance in pale pink. 2.12. Silver impregnation for reticulum Silver impregnation was performed as the recommended method to show argyrophilic reticular fibers in connective tissue and especially to differentiate collagen fibers from connective tissue. Silver impregnation was performed according to the manufacturer's protocol (http://www.biooptica.it/pdf3/040801.pdf, Bio-Optica, Milano S.P.A). Reticular and nervous fibers will appear in black, connective tissue in tobacco brown and collagen in gold yellow. 2.13. IHC localization After deparaffinization with xylene, sections of hemisphere samples were hydrated in graded ethanol. Detection of TNF-alpha, IL-10, NOS2, nitrotyrosine, Bax, and Bcl-2 was carried out after boiling in citrate buffer 0.01 M pH 6 for 4 min. Endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Sections were incubated overnight with: • rabbit polyclonal anti-TNF-α antibody (Novus biological, 1:100 in PBS); • mouse monoclonal anti-Foxp3 antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS); • rat monoclonal anti-CD44 antibody (Abcam, 1:100 in PBS); • rabbit polyclonal anti-NOS2 antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS); • rabbit polyclonal anti-nitrotyrosine antibody (Millipore, Milan, Italy, 1:1000 in PBS); • rabbit polyclonal anti-Bax antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS); • rabbit polyclonal anti-Bcl2 antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS). Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were washed with PBS and incubated with secondary antibody. Specific labelling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vectastain ABC kit, VECTOR). The counterstain was developed with diaminobenzidine (brown colour) and hematoxylin (blue background). To verify the binding specificity, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections,
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indicating that the immunoreaction was positive in all the experiments carried out. All sections were obtained using light microscopy (LEICA DM 2000 combined with LEICA ICC50 HD camera) and studied via an Imaging computer program (Leica Application Suite V4.1). 2.14. Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay To test whether spinal cord damage was associated with cell death by apoptosis, we measured TUNEL-like staining in the perilesional spinal cord tissue. TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instruction (Apotag, HRP kit DBA, Milan, Italy). Sections were incubated with 15 mg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated dUTP, incubated in a humid atmosphere at 37 °C for 1 h, and then washed with PBS. Sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine and controstained with methyl green. 2.15. Statistical evaluation GraphPad Prism 6 (registered trademark of GraphPad Software, Inc.) was used to run all statistical tests. Comparison among two groups was performed with T-student test. A p value of b0.05 was considered to be statistically significant. Results are expressed as the mean ± S.E.M. of n experiments. 3. Results and discussion MS is recognized as a worldwide health problem. Today, statistical data show that about more than 2 million people have MS and the disease is among the most common causes of neurological disability in young adults, which typically presents in the third or fourth decade of life [23], with an increased incidence over time and a prevalence especially in women [24,25] and a high variability in the distribution between regions and populations [23]. About the etiology of the disease, both genetic predispositions and environmental risk factors given possibly contribute to developing MS [26]. EAE is a well characterized, validated and recognized model of multiple sclerosis in mouse [25]. The first sign of disease is a reduced increasing in body weight in EAE mice when compared with all other groups (Fig. 1B). Moreover, mice belonging to the EAE group showed the highest score of disease (4/5 points in the grading scale of disease, data not shown) and a percentage of disease's incidence at the onset (14th day) of 60% against the 37.5% of EAE + GMG-ITC mice. These data confirm both the disability in mice affected by EAE and the belief that chronic inflammation and autoimmune conditions in animals are associated with substantial feeding alterations [27].
Histological evaluation of spinal cord sections from the “EAE group” (Fig. 2C, D and E represents histological damage in the absence of pharmacological treatment) showed significant differences comparing to the “EAE + GMG-ITC group” (Fig. 2 F), the “naive group” (Fig. 2A) and the “GMG-ITC control group” (Fig. 2B). More in details, H&E staining for EAE group displayed a wide area of infiltrating lymphocyte cells, confirming the important role of the cellular components in spinal cord tissue that undergoes degenerative conditions. GMG-ITC treatment attenuated the histological EAE score, suggesting a protective effect on CNS tissue. As EAE is a demyelinating disease, GMG-ITC was also evaluated for the protective action on myelin sheath integrity by LFB staining. Compared to naive and GMG-ITC control mice (Fig. 3A and B, respectively), EAE untreated animals exhibited remarkably reduced myelin and axonal structures in the spinal cord (Fig. 3C). Consistently, corroborating these evidences, other authors have reported myelin and axonal loss along different white matter tracts in EAE mice [28,29], displaying a significant loss in LFB staining. Similarly, our evidences showed that treatment with GMG-ITC reduced demyelination and axonal loss in EAE mice with an intense LFB positive staining (Fig. 3D). Moreover, silver impregnation highlighted a severe condition of connective tissue breaking up in spinal cord sections cut from EAE mice (Fig. 4C). Otherwise, spinal cord tissues harvested from the EAE + GMG-ITC group” (Fig. 4D), the “naive group” (Fig. 4A) and the “GMG-ITC control group” (Fig. 4B) display a normal trend and architecture of vascular collagen fibers. The current literature shows that the EAE model has an unambiguous immune system involvement [30,31]. Gonçalves Zorzella-Pezavento et al. in a recent paper [32] analyzed the typical profile of mediators released during acute and chronic stages of EAE. Our results corroborate these data confirming a clear engagement of T cells with increasing Foxp3 in both phases. It has been demonstrated that myelin-specific Treg cells (CD4 + CD25 + Foxp3+ T cells) are able to migrate and to accumulate in the CNS in animals with EAE (Fig. 5C, see densitometric analysis Fig. 14A). Probably due to the protective role of GMG-ITC we didn't find Foxp3+ cells in the spinal cord tissue of mice MOG35–55-injected but pharmacologically treated (Fig. 5D, see densitometric analysis Fig. 14A), as well as reported in the “naive group” (Fig. 5A, see densitometric analysis Fig. 14A) and the “GMG-ITC control group” (Fig. 5B, see densitometric analysis Fig. 14A). This is of particular interest and it is also understandable if we look at CD44 expression, a key ligand at the focal inflammatory demyelinating lesion site in the spinal cord. Therefore, this marker of lymphocyte adhesion reveals as EAE-affected mice (Fig. 6C, see densitometric analysis Fig. 14B) express higher tissue levels of CD44 with respect to all other groups (Fig. 6A, B and D, see densitometric analysis Fig. 14B). This data lead to believe that the spinal cord tissue, in severe disease, attracts infiltrating cells at the site of damage. The MAP kinase family includes ERK 1/2 (p42/44) member, a protein kinase that, when phosphorylated, is widely used as a measurable endpoint from many multiple cellular cascades. Its activation is instrumental for many signaling pathway, kinase targets, and receptor systems [33]. For example, activation of MAPKs, such as ERK1/2, JNK and p38 subfamilies, causes IκBα phosphorylation that, in turn, induces NF-κB nuclear translocation and gene transcription of inflammatory cytokines and
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Fig. 2. Histological evaluation. Spinal cord sections from the “EAE group” (C, D and E) showed significant differences compared to the “EAE + GMG-ITC group” (F), the “naive group” (A) and the “GMG-ITC control group” (B). H&E staining highlights the presence of infiltrating cells confirming the important role of the cellular components in the spinal cord tissue that undergoes degenerative conditions.
mediators [34]. Also, MAPK activation is triggered following nerve injury in spinal glial cells and MAPK inhibitors appear to decrease injury-induced pain hypersensitivity [35]. In particular, phospho-ERK p42/44 spinal cord triggering is associated to models of neuropathic pain, where ERK phosphorylation results were upregulated [36]. Being this one a common secondary complication in human MS [37], interestingly, GMG-ITC treatment, as anti-inflammatory, showed a typical feature of nonsteroidal anti-inflammatory drugs (NSAIDs) traceable to phospho-ERK p42/44 inhibition (Fig. 7). Modulation of inflammatory mediators in the mouse spinal cord, particularly with regard to two important cytokines altered in the MS patient profile [38], was investigated to understand and assess the effects of GMG-ITC treatment on the molecular mechanisms of inflammation. For this reason, TNF-α (proinflammatory regulator) tissue expression was detected. We have clearly confirmed an increase in TNF-α release and,
consequently, in tissue localization over the course of EAE (Fig. 8C, see densitometric analysis Fig. 14C). On the contrary, reduced expression of TNF-α was observed in mice which received GMG-ITC treatment (Fig. 8D, see densitometric analysis Fig. 14C), so we can speculate that probably, GMG-ITC works through a strict control of the cytokine production. As regards to the “naive group” and the “GMG-ITC control group”, the immuno-sections resulted negative for TNF-α (Fig. 8A and B, respectively, see densitometric analysis Fig. 14C). EAE onset and progression have been related to the generation of oxidative stress, that seems to play a main role [20], confirmed by several experimental studies that showed the beneficial treatment with antioxidants against the progression of the disease [39]. In both MS and EAE, demyelination and axonal damage can be related to reactive oxygen species (ROS) action [40,41]. Moreover, some authors reported that iNOS knockout mice
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Fig. 3. LFB staining. Compared to naive (A) and GMG-ITC control mice (B), EAE untreated animals exhibited remarkably reduced myelin and axonal structures in the spinal cord (C). Moreover, treatment with GMG-ITC reduces demyelination and axonal loss in EAE mice with an intense LFB positive staining (D).
show low signs of disease [42]. Our results demonstrated that GMG-ITC reduced the generation of reactive species through the immunolocalization of NOS2 (Fig. 9D, see densitometric
analysis Fig. 14D) and nitrotyrosine, taken as an indirect marker of peroxynitrite activity [43] (Fig. 10D, see densitometric analysis Fig. 14E). This is evident when the activity of the
Fig. 4. Silver impregnation. A severe condition of vascular tissue breaking up in spinal cord sections cut from EAE mice (C). Otherwise, spinal cord harvested from the EAE + GMG-ITC group” (D), the “naive group” (A) and the “GMG-ITC control group” (B) displays a normal trend and architecture.
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Fig. 5. Foxp3 immunohistochemical localization. A high Foxp3 tissue expression was detected in the spinal cord of mice subjected to EAE (C). Probably due to the protective role of GMG-ITC we didn't find Foxp3+ cells in the spinal cord tissue of mice MOG35–55-injected but pharmacologically treated (D). Naive animals (A) and GMG-ITC control mice (B) resulted negative too.
Fig. 6. CD44 immunohistochemical localization. CD44 expression reveals express higher tissue levels of this markers in EAE-affected mice (C) with respect to the EAE + GMG-ITC group” (D), the “naive group” (A) and the “GMG-ITC control group” (B) This data leads to believe that the spinal cord tissue, in severe disease, attracts infiltrating cells at the site of damage.
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Fig. 7. MAP kinase signal pathway. GMG-ITC treatment following EAE induction caused phospho-ERK p42/44 inhibition reducing the citoplasmatic level of the protein when compared with untreated EAE mice. This could be associated with the typical feature of nonsteroidal anti-inflammatory drugs (NSAIDs).
treatment is compared with immunohistochemical image of EAE mice not pharmacologically treated (Fig. 9C, see densitometric analysis Figs. 14D and 10C, see densitometric analysis Fig. 14E, respectively). Moreover, it is well known that reactive radicals and apoptosis are strictly linked and protective mechanisms involved in the treatment of MS/EAE go through oxidative stress downregulation that seem in turn related to apoptosis pathway shutdown [44]. Also, authors have correlated the EAE model with apoptosis, mostly of oligodendrocytes, neurons and astrocytes in percentage of 45, 20 and 13% respectively [45]. The unbalance between Bax, a proapoptotic protein, and Bcl-2, an anti-apoptotic protein, plays an important role in the triggering of cellular apoptotic cascade during EAE [46], since, basically, their ratio provides the fate for cell survival [47,48]. A negative Bax expression level in EAE group treated with GMG-ITC (Fig. 11D, see densitometric analysis Fig. 14 F) leads to believe that the ITC exerted a beneficial protective effect against the mechanisms of cellular apoptotic death. Conversely, untreated EAE mice displayed high immune-Bax localization (Fig. 11C, see densitometric analysis Fig. 14 F). These results correlate with protective indices given by Bcl-2 highly expressed localization in a section taken from the “naive group” (Fig. 12A, see densitometric analysis Fig. 14G), the “GMG-ITC group” (Fig. 12B, see densitometric analysis Fig. 14G) as well as the “EAE + GMG-ITC group” (Fig. 12D, see densitometric analysis Fig. 14G). A negative staining in slide obtained from EAE not pharmacologically treated mice put on view a severe degree of cell death in the immunohistochemical section incubated with Bcl-2 antibody (Fig. 12C, see densitometric analysis Fig. 14G).
Fig. 8. Proinflammatory cytokine release: TNF-α. Increased TNF-α tissue localization in EAE mice was reported (C). On the contrary, reduced expression of TNF-α was observed in mice which received GMG-ITC (D). Naive mice (A) and GMG-ITC control mice (B) resulted negative at TNF-α tissue staining.
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Fig. 9. iNOS immunohistochemical localization. Increased iNOS tissue localization in EAE mice was found (C). On the contrary, reduced expression of iNOS was observed in mice which received GMG-ITC (D). Naive mice (A) and GMG-ITC control mice (B) were negative at iNOS tissue staining.
To corroborate the above results, TUNEL staining was performed providing the presence of DNA fragmentation. The severity and importance of this mechanism can be evaluated
considering some of most symptomatic events for this type of cell death, including: 1) nuclear DNA double-strand cleavage at level of the linker region between nucleosomes, 2) nuclear
Fig. 10. Nitrotyrosine tissue expression. GMG-ITC treatment resulted in negative staining for anti-nitrotyrosine antibody (D) as well as in naive mice (A) and in GMG-ITC control mice (B). On the contrary, the immunohistochemical image of EAE not pharmacologically treated mice showed a clear positivity (C).
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Fig. 11. Proapoptotic factor expression: Bax. Naive mice (A) and GMG-ITC control mice (B) sections did not stain for anti-Bax antibody. Negative Bax expression levels were found in the EAE group treated with GMG-ITC (D) leading to believe that the ITC exerted a beneficial protective effect against the mechanisms of cellular apoptotic death. Conversely, untreated EAE mice displayed high immune-Bax localization (C).
chromatin condensation at earlier stages, 3) coarse fragmentation of the nucleus at later stages, and 4) membrane-bound cellular particle production [49]. Images showed in EAE untreated mice evident tissue presence of nuclei with an intense positivity for TUNEL staining and characterized by an intensely and completely labelled nucleus broken up into smaller TUNEL-labeled brown fragments (Fig. 13C, see arrow). Differently, an absence of apobodies has been detected in tissue sections from GMG-ITC treated mice, naive mice and GMG-ITC control mice (Fig. 13D, A and B, respectively).
3.1. Conclusions Following achieved data, we can conclude that GMG-ITC has protective effects on spinal cord tissue, with a protective action on myelin loss and axonal damage. Moreover, a representative panel of all markers, distinctive of the disease, evaluated in the present work is summarized in Fig. 12 in a prospective overview that shows all densitometric analysis. To sum up, this work has shown new evidences about the bioactivity of GMG-ITC in addition to the well known anticancer [2] effects and antibiotic [21] properties. Thus,
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Fig. 12. Anti-apoptotic factor expression: Bcl-2. A Bcl-2 highly expressed localization in a section taken from the “naive group” (A), the “GMG-ITC group” (B) as well as the “EAE + GMG-ITC group” (D) was found. A negative staining in a slide obtained from EAE not pharmacologically treated mice put on view a severe degree of cell death in the immunohistochemical section incubated with Bcl-2 antibody (C).
Fig. 13. Tunel staining. EAE untreated mice (C) showed evident tissue presence of nuclei with an intense positivity for TUNEL staining and characterized by an intensely and completely labelled nucleus broken up into smaller TUNEL-labeled brown fragments (see arrow). Differently, an absence of apobodies was detected in tissue sections from GMG-ITC treated mice (D), naive mice (A) and GMG-ITC control mice (B).
172 M. Galuppo et al. / Fitoterapia 95 (2014) 160–174 Fig. 14. Densitometric quantification. For IHC images, densitometric analysis was carried out to quantify and highlight significant differences among experimental groups. Summarizing: (A) FoxP3; (B) CD44; (C) TNF-α; (D) iNOS; (E) nitrotyrosine; (F) Bax; (G) Bcl-2. A p value b0.05 was considered statistically significant.
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our results have highlighted that this compound is able to exert antinflammatory and antioxidant effects with consequences on cell apoptotic death processes. These results related to MS, a disease with neurodegenerative course, provide a new and interesting possible application of GMG-ITC, produced by GMG bioactivation with myrosinase, in the clinical treatment of this pathology.
[18]
[19]
[20]
Conflict of interest Authors have not any conflict of interest to declare.
[21]
Acknowledgments [22]
The authors would like to thank the Dr. Giuseppe Galletta and Dr. Massimo Messina belonging to the secretary office of IRCCS Centro Neurolesi “Bonino-Pulejo”- Messina, for their excellent technical assistance.
[23]
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Antiinflammatory activity of glucomoringin isothiocyanate in a mouse model of experimental autoimmune encephalomyelitis Maria Galuppo a,1, Sabrina Giacoppo a,1, Gina Rosalinda De Nicola b, Renato Iori b, Michele Navarra c, Giovanni Enrico Lombardo c, Placido Bramanti a, Emanuela Mazzon a,⁎ a b c
IRCCS Centro Neurolesi “Bonino-Pulejo”, Via Provinciale Palermo, contrada Casazza, 98124 Messina, Italy Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali (CRA-CIN), Via di Corticella 133, 40128 Bologna, Italy Università degli Studi di Messina, Facoltà di Farmacia, Dipartimento di Scienze del farmaco e dei Prodotti per la Salute, Viale Annunziata, 98168 Messina, Italy
a r t i c l e
i n f o
Article history: Received 6 February 2014 Accepted in revised form 12 March 2014 Available online 29 March 2014 Keywords: Glucomoringin Isothiocyanate Inflammation Multiple sclerosis Cytokines
a b s t r a c t Glucomoringin (4(α-L-rhamnosyloxy)-benzyl glucosinolate) (GMG) is an uncommon member of glucosinolate group belonging to the Moringaceae family, of which Moringa oleifera Lam. is the most widely distributed. Bioactivation of GMG with the enzyme myrosinase forms the corresponding isothiocyanate (4(α-L-rhamnosyloxy)-benzyl isothiocyanate) (GMG-ITC), which can play a key role in antitumoral activity and counteract the inflammatory response. The aim of this study was to assess the effect of GMG-ITC treatment in an experimental mouse model of multiple sclerosis (MS), an inflammatory demyelinating disease with neurodegeneration characterized by demyelinating plaques, neuronal, and axonal loss. For this reason, C57Bl/6 male mice were injected with myelin oligodendrocyte glycoprotein35–55 which is able to evoke an autoimmune response against myelin fibers miming human multiple sclerosis physiopatogenesis. Results clearly showed that the treatment was able to counteract the inflammatory cascade that underlies the processes leading to severe MS. In particular, GMG-ITC was effective against proinflammatory cytokine TNF-α. Oxidative species generation including the influence of iNOS, nitrotyrosine tissue expression and cell apoptotic death pathway was also evaluated resulting in a lower Bax/Bcl-2 unbalance. Taken together, this work adds new interesting properties and applicability of GMG-ITC and this compound can be suggested as a useful drug for the treatment or prevention of MS, at least in association with current conventional therapy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: GMG, glucomoringin; GMG-ITC, glucomoringin-isothiocyanate; MS, multiple sclerosis; GLs, glucosinolates; ITCs, isothiocyanates; EAE, experimental autoimmune encephalomyelitis; TNF-α, tumor necrosis factor alpha; IL-10, interleukin-10; CFA, complete Freund's adjuvant; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; (LFB), luxol fast blue; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling; MAPKs, mitogenactivated protein kinases; NSAIDs, nonsteroidal anti-inflammatory drugs. ⁎ Corresponding author. Tel.: +39 09060128708; fax: +39 09060128850. E-mail address: [email protected] (E. Mazzon). 1 These authors share the first authorship.
http://dx.doi.org/10.1016/j.fitote.2014.03.018 0367-326X/© 2014 Elsevier B.V. All rights reserved.
Phytomedicines have been playing a significant role in the prevention and treatment of various human aliments and it is a long time that the folk medicine relies to the medical value of leaves and seeds of Moringa oleifera Lam. [1]. M. oleifera, which belong to the Moringaceae family, is a plant widely cultivated in many countries of the tropics and it is known as a medicinal plant. More in detail, M. oleifera is a multipurpose tree which grows in many tropical or equatorial regions, around dry tropical areas in Northwestern India at the Southwestern foot of the Himalaya that is used for human and animal nutrition, and medicinal
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purposes. Hypoglycaemic, hypotensive, liver-protective, immunomodulatory, antioxidant and antitumor activities of M. oleifera have been assessed and demonstrated by several pharmacological studies [2–4]. Moreover, leaves are useful for preventing malnutrition and seeds are used safely for water purification [5]. Glucosinolates (GLs) are well known sulfur-containing secondary metabolites (ca. 130 molecules identified to date), which display a structural homogeneity based on a β-D-glucopyranosyl unit and an O-sulfated anomeric (Z)-thiohydroximate function connected to a variable side chain depending on the amino acid metabolism of the plant species [6]. GLs are bioactivated by enzymatic hydrolysis involving myrosinase (E.C. 3.2.1.147), releasing at neutral pH values health-promoting isothiocyanates (ITCs) [7,8]. The numerous effects exhibited by these natural compounds through which they exerted a protective action against cancer progression [9] and neurodegenerative disease were recently reported [10–12]. Glucomoringin (GMG; 4(α-L-rhamnosyloxy)-benzyl GL), a typical secondary metabolite present in large amount in seeds of M. oleifera [13], is an uncommon member of the GLs family and presents a unique characteristic consisting in a second glycosidic residue in its side chain. The glycosylated isothiocyanate 4(alpha-L-rhamnosyloxy)-benzyl isothiocyanate (GMG-ITC), resulting from myrosinase-hydrolysis (N99%) of GMG at neutral condition (Fig. 1), has been shown to exhibit a broad biological activity and it was also shown to exert an effective antitumor promoting activity [14,15].
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Gram-scale production of natural GMG has been recently set up starting from M. oleifera seeds according to a procedure developed at CRA-CIN of Bologna, Italy [2]. The aim of this work was to test GMG bioactivated with myrosinase that provides the therapeutic natural agent GMG-ITC, in an animal model of experimental autoimmune encephalomyelitis (EAE). The EAE mimics the physiopathology of the human multiple sclerosis (MS), with weight loss and progressive paralysis [16]. Basically, MS is a chronic inflammatory disease, attacking central nervous system and resulting in demyelination as well as oligodendrocyte loss, glial scar formation and subsequent degeneration of axonal and neuronal damage [17,18]. The pathogenesis of MS has been related to the unbalance invasive (Th1 and Th17) and defensive (Th2 and Treg) T cell responses. This is believed crucial for the development and progression of MS both in humans and EAE in rodents. Also, since an involvement of vascular endothelial is supposed, being alterations in blood vessel properties of central importance in the initiation and/or maintenance of the disease [19,20], our research started from the activation of a systemic mediators in response to EAE-induction, such as tumor necrosis factor alpha (TNF-α). Moreover, we tested the correlated molecular pathways involved in serious inflammatory disease, such as the possible activation of oxidative cascade (NOS2 and nitrotyrosine formation) as well as the state of neuronal cell life (Tunel assay) following injection of myelin oligodendrocyte glycoprotein (MOG)35–55, that leads to alterations of Bax/Bcl-2 stability.
Fig. 1. A) Production of 4-(α-L-rhamnosyloxy)benzyl isothiocyanate (GMG-ITC). Reaction of myrosinase catalyzed hydrolysis of glucomoringin (GMG), purified from Moringa oleifera seeds, in phosphate buffered saline (PBS) solution (pH 7.2) to produce GMG-ITC. B) Body weight. C57Bl/6 mice have been daily weighed. After EAE induction, a significative reduced increasing in body weight was observed in EAE mice compared to the naive group. Difference in body weigh between the naive group and the “EAE + GMG-ITC group” or the “GMG-ITC control group” didn't result statistically significative. * p b 0.05 vs naive.
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The relevance and innovation of the present study lie in the possible use of a safe new formulation of GMG bioactivated with myrosinase that provides the therapeutic natural agent GMG-ITC, which can be considered as a useful drug for the treatment or prevention of MS.
5 = complete hind limb paralysis; 6 = moribund or dead animal. Mice with high score of disease were sacrificed to avoid animal suffering.
2. Materials and methods
GMG was isolated from M. oleifera L. (fam. Moringaceae) seeds according to a method previously described [2,21]. In brief, GMG was purified by two sequential chromatographic steps: isolation through anion exchange chromatography, followed by gel filtration to achieve purification to homogeneity. GMG was unambiguously characterized by 1H and 13C NMR spectrometry and the purity was assessed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method accepted by European Economic Community, Commission Regulation, EEC No. 1864/90, yielding GMG with a purity of 99% based on peak area value, and about 90–92% on a weight basis due to its high hygroscopic properties. The enzyme myrosinase was isolated from seeds of Sinapis alba L. as described by Pessina et al. [22] with some modification. The specific activity of the stock solution used in the present study was 60 U/mg of soluble protein. The enzymatic activity was 32 U/ml and the solution was stored at 4 °C in sterile saline solution at neutral pH until use. One myrosinase unit was defined as the amount of enzyme able to hydrolyze 1 μmol/min of sinigrin at pH 6.5 and 37 °C [8].
2.1. Animals Male adult C57Bl/6 mice (20-25 g, Harlan Nossan, Milan, Italy) were used for all studies. Mice were housed in stainless steel cages and maintained under 12 h/12 h light/dark cycle at 21 ± 1 °C and 50 ± 5% humidity. Animals were acclimated to their environment for 1 week and food and water were given ad libitum. 2.2. Ethics statement This study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the National Institutes of Health. The protocol was approved by the Ministry of Health “General Direction of animal health and veterinary drug”. In particular, animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116/92) as well as with the EEC regulations (O.J. of E.C.L 358/1 12/18/1986). Experimental procedures did not cause any significant animal suffering. 2.3. Reagents MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) was synthesized and purified by Cambridge Research Biochemicals (Billingham, UK). Complete Freund's adjuvant (CFA) containing Mycobacterium tubercolosis H37Ra strain was purchased from Difco Laboratories (Sparks, MD, USA), while Bordetella pertussis toxin was from Sigma–Aldrich Company Ltd. (Milan, Italy). All other chemicals were of the highest commercial grade available. 2.4. EAE induction After anaesthesia, mice were immunized subcutaneously with 300 μl/flank of the emulsion consisting of 300 μg of MOG35–55 in phosphate-buffered saline (PBS) combined with an equal volume of CFA containing 300 μg heat-killed M. tubercolosis H37Ra. After emulsion–injection, animals were immunized with an injection of 100 μl of B. pertussis toxin (500 ng/100 μl, i.p), repeated 48 h later. The disease follows a course of progressive degeneration, with visible signs of pathology consisting of flaccidity of the tail and loss of motion of the hind legs. 2.5. Body weight and clinical score Mice were daily weighed and observed for signs of EAE. Clinical score was evaluated using a standardized scoring system [30]. Briefly, clinical signs were scored as follows: 0 = no signs; 1 = partial flaccid tail; 2 = complete flaccid tail; 3 = hind limb hypotonia; 4 = partial hind limb paralysis;
2.6. GMG and myrosinase purification.
2.7. Enzyme bioactivation of GMG and animal treatment GMG (10 mg/kg) was dissolved in PBS solution pH 7.2 and mouse treatment required the enzyme bioactivation of the phytochemical. The action of myrosinase enzyme (5 μl/mouse) for 15 min allowed to have GMG-ITC quickly, before the i.p treatment (Fig. 1). The total conversion of pure GMG into GMG-ITC was confirmed by HPLC analysis of the GMG desulfo-derivative, which allowed us to monitor the reduction of GMG until its complete disappearance in the reaction mixture [21]. 2.8. Experimental design Mice were randomly allocated into the following groups (n = 30 total animals): 1. EAE group (n = 10): mice subjected to EAE that did not receive GMG-ITC; 2. EAE + GMG-ITC group (n = 10): mice subjected to EAE were treated with bioactive GMG-ITC (10 mg/kg GMG + 5 μl/mouse myrosinase). GMG-ITC was daily i.p. administrated 1 week before the induction of EAE and, after immunization the treatment was daily protracted until the sacrifice; 3. Naive group (n = 5): mice that received neither vehicle (saline) nor MOG35–55; 4. GMG-ITC control group (n = 5): mice daily i.p. treated with bioactive GMG-ITC (10 mg/kg GMG + 5 μl/mouse myrosinase) for all the duration of the experiment. GMG-ITC was given as pretreatment once a day for 7 days via i.p. injection. The disease was induced according to the experimental procedure reported above and “EAE + GMG-ITC”
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group received a post-treatment prolonged until twenty-first day. At the end of the experiment, animals were sacrificed and spinal cord tissues were harvested and processed in order to evaluate parameters of disease. 2.9. Western blot analysis All the extraction procedures were performed on ice using ice-cold reagents. In brief, spinal cord tissues were suspended in extraction buffer containing 0.32 M sucrose, 10 mM Tris– HCl, pH 7.4, 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM 2-mercaptoethanol, 50 mM NaF, protease inhibitor tablets (Roche), and they were homogenized at the highest setting for 2 min. The homogenates were chilled on ice for 15 min and then centrifuged at 1000 g for 10 min at 4 °C, and the supernatant (cytosol + membrane extract from spinal cord tissue) was collected. The pellets were suspended in the supplied complete lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA protease inhibitors tablets (Roche), and then they were centrifuged for 30 min at 15,000 g at 4 °C, and the supernatant (nuclear extract) was collected. Supernatants were stored at –80 °C until use. Protein concentration in homogenate was estimated by the Bio-Rad Protein Assay (Bio-Rad) using BSA as standard, and 50 μg both of cytosol and nuclear extract from each sample was analyzed. Proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Westran S PVDF Membranes, Whatman™, GE Healthcare Companies, Dassel, Germany), blocked with PBS containing 5% nonfat dried milk for 45 min at room temperature, and subsequently probed at 4 °C overnight with specific antibodies for Bax (1:500 Santa Cruz Biotechnology, Inc), phospho-ERK p42/44 (1:500 Cell Signaling), ERK-2 (1:1000; Cell Signaling), in 1 × PBS, 5% (w/v) non fat dried milk, 0.1% Tween-20 (PMT). Membranes were incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG secondary antibodies (1:2000, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of proteic lysates, they were also incubated with GAPDH-HRP conjugated primary antibody (1:1000 Cell Signaling). Relative protein band expression for Bax (23 kDa) and phospho-ERK p42/44 (42 and 44 kDa double band), was visualized using an enhanced chemiluminescence system (Luminata™ Forte, Western HRP substrate, Millipore) and proteic bands were acquired and quantified with ChemiDoc ™ MP System (Bio-Rad) and a computer program (ImageJ), respectively. 2.10. Histological evaluation After sample fixation at room temperature in 10% (w/v) PBS-buffered formaldehyde, spinal cord tissue was dehydrated in graded ethanol and embedded in Paraplast (Sherwood Medical). Thereafter, 7-μm sections were deparaffinized with xylene, stained with hematoxylin and observed in a microscope (LEICA DM 2000 combined with LEICA ICC50 HD camera).
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2.11. Luxol Fast Blue (LFB) To show myelin and phospholipids in histological sections, LFB staining was performed according to the manufacturer's protocol (http://www.bio-optica.it/pdf3/200812.pdf, Bio-Optica, Milano S.P.A). LFB affinity for central nervous system is usually ascribed to the bonds it forms with phospholipidic structures such as lecithin and sphingomyelin. The staining provides: myelin in turquoise blue, neurons and glial nuclei in pink/violet and Nissl substance in pale pink. 2.12. Silver impregnation for reticulum Silver impregnation was performed as the recommended method to show argyrophilic reticular fibers in connective tissue and especially to differentiate collagen fibers from connective tissue. Silver impregnation was performed according to the manufacturer's protocol (http://www.biooptica.it/pdf3/040801.pdf, Bio-Optica, Milano S.P.A). Reticular and nervous fibers will appear in black, connective tissue in tobacco brown and collagen in gold yellow. 2.13. IHC localization After deparaffinization with xylene, sections of hemisphere samples were hydrated in graded ethanol. Detection of TNF-alpha, IL-10, NOS2, nitrotyrosine, Bax, and Bcl-2 was carried out after boiling in citrate buffer 0.01 M pH 6 for 4 min. Endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Sections were incubated overnight with: • rabbit polyclonal anti-TNF-α antibody (Novus biological, 1:100 in PBS); • mouse monoclonal anti-Foxp3 antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS); • rat monoclonal anti-CD44 antibody (Abcam, 1:100 in PBS); • rabbit polyclonal anti-NOS2 antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS); • rabbit polyclonal anti-nitrotyrosine antibody (Millipore, Milan, Italy, 1:1000 in PBS); • rabbit polyclonal anti-Bax antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS); • rabbit polyclonal anti-Bcl2 antibody (Santa Cruz Biotechnology, Inc., 1:100 in PBS). Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were washed with PBS and incubated with secondary antibody. Specific labelling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vectastain ABC kit, VECTOR). The counterstain was developed with diaminobenzidine (brown colour) and hematoxylin (blue background). To verify the binding specificity, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections,
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indicating that the immunoreaction was positive in all the experiments carried out. All sections were obtained using light microscopy (LEICA DM 2000 combined with LEICA ICC50 HD camera) and studied via an Imaging computer program (Leica Application Suite V4.1). 2.14. Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay To test whether spinal cord damage was associated with cell death by apoptosis, we measured TUNEL-like staining in the perilesional spinal cord tissue. TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instruction (Apotag, HRP kit DBA, Milan, Italy). Sections were incubated with 15 mg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated dUTP, incubated in a humid atmosphere at 37 °C for 1 h, and then washed with PBS. Sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine and controstained with methyl green. 2.15. Statistical evaluation GraphPad Prism 6 (registered trademark of GraphPad Software, Inc.) was used to run all statistical tests. Comparison among two groups was performed with T-student test. A p value of b0.05 was considered to be statistically significant. Results are expressed as the mean ± S.E.M. of n experiments. 3. Results and discussion MS is recognized as a worldwide health problem. Today, statistical data show that about more than 2 million people have MS and the disease is among the most common causes of neurological disability in young adults, which typically presents in the third or fourth decade of life [23], with an increased incidence over time and a prevalence especially in women [24,25] and a high variability in the distribution between regions and populations [23]. About the etiology of the disease, both genetic predispositions and environmental risk factors given possibly contribute to developing MS [26]. EAE is a well characterized, validated and recognized model of multiple sclerosis in mouse [25]. The first sign of disease is a reduced increasing in body weight in EAE mice when compared with all other groups (Fig. 1B). Moreover, mice belonging to the EAE group showed the highest score of disease (4/5 points in the grading scale of disease, data not shown) and a percentage of disease's incidence at the onset (14th day) of 60% against the 37.5% of EAE + GMG-ITC mice. These data confirm both the disability in mice affected by EAE and the belief that chronic inflammation and autoimmune conditions in animals are associated with substantial feeding alterations [27].
Histological evaluation of spinal cord sections from the “EAE group” (Fig. 2C, D and E represents histological damage in the absence of pharmacological treatment) showed significant differences comparing to the “EAE + GMG-ITC group” (Fig. 2 F), the “naive group” (Fig. 2A) and the “GMG-ITC control group” (Fig. 2B). More in details, H&E staining for EAE group displayed a wide area of infiltrating lymphocyte cells, confirming the important role of the cellular components in spinal cord tissue that undergoes degenerative conditions. GMG-ITC treatment attenuated the histological EAE score, suggesting a protective effect on CNS tissue. As EAE is a demyelinating disease, GMG-ITC was also evaluated for the protective action on myelin sheath integrity by LFB staining. Compared to naive and GMG-ITC control mice (Fig. 3A and B, respectively), EAE untreated animals exhibited remarkably reduced myelin and axonal structures in the spinal cord (Fig. 3C). Consistently, corroborating these evidences, other authors have reported myelin and axonal loss along different white matter tracts in EAE mice [28,29], displaying a significant loss in LFB staining. Similarly, our evidences showed that treatment with GMG-ITC reduced demyelination and axonal loss in EAE mice with an intense LFB positive staining (Fig. 3D). Moreover, silver impregnation highlighted a severe condition of connective tissue breaking up in spinal cord sections cut from EAE mice (Fig. 4C). Otherwise, spinal cord tissues harvested from the EAE + GMG-ITC group” (Fig. 4D), the “naive group” (Fig. 4A) and the “GMG-ITC control group” (Fig. 4B) display a normal trend and architecture of vascular collagen fibers. The current literature shows that the EAE model has an unambiguous immune system involvement [30,31]. Gonçalves Zorzella-Pezavento et al. in a recent paper [32] analyzed the typical profile of mediators released during acute and chronic stages of EAE. Our results corroborate these data confirming a clear engagement of T cells with increasing Foxp3 in both phases. It has been demonstrated that myelin-specific Treg cells (CD4 + CD25 + Foxp3+ T cells) are able to migrate and to accumulate in the CNS in animals with EAE (Fig. 5C, see densitometric analysis Fig. 14A). Probably due to the protective role of GMG-ITC we didn't find Foxp3+ cells in the spinal cord tissue of mice MOG35–55-injected but pharmacologically treated (Fig. 5D, see densitometric analysis Fig. 14A), as well as reported in the “naive group” (Fig. 5A, see densitometric analysis Fig. 14A) and the “GMG-ITC control group” (Fig. 5B, see densitometric analysis Fig. 14A). This is of particular interest and it is also understandable if we look at CD44 expression, a key ligand at the focal inflammatory demyelinating lesion site in the spinal cord. Therefore, this marker of lymphocyte adhesion reveals as EAE-affected mice (Fig. 6C, see densitometric analysis Fig. 14B) express higher tissue levels of CD44 with respect to all other groups (Fig. 6A, B and D, see densitometric analysis Fig. 14B). This data lead to believe that the spinal cord tissue, in severe disease, attracts infiltrating cells at the site of damage. The MAP kinase family includes ERK 1/2 (p42/44) member, a protein kinase that, when phosphorylated, is widely used as a measurable endpoint from many multiple cellular cascades. Its activation is instrumental for many signaling pathway, kinase targets, and receptor systems [33]. For example, activation of MAPKs, such as ERK1/2, JNK and p38 subfamilies, causes IκBα phosphorylation that, in turn, induces NF-κB nuclear translocation and gene transcription of inflammatory cytokines and
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Fig. 2. Histological evaluation. Spinal cord sections from the “EAE group” (C, D and E) showed significant differences compared to the “EAE + GMG-ITC group” (F), the “naive group” (A) and the “GMG-ITC control group” (B). H&E staining highlights the presence of infiltrating cells confirming the important role of the cellular components in the spinal cord tissue that undergoes degenerative conditions.
mediators [34]. Also, MAPK activation is triggered following nerve injury in spinal glial cells and MAPK inhibitors appear to decrease injury-induced pain hypersensitivity [35]. In particular, phospho-ERK p42/44 spinal cord triggering is associated to models of neuropathic pain, where ERK phosphorylation results were upregulated [36]. Being this one a common secondary complication in human MS [37], interestingly, GMG-ITC treatment, as anti-inflammatory, showed a typical feature of nonsteroidal anti-inflammatory drugs (NSAIDs) traceable to phospho-ERK p42/44 inhibition (Fig. 7). Modulation of inflammatory mediators in the mouse spinal cord, particularly with regard to two important cytokines altered in the MS patient profile [38], was investigated to understand and assess the effects of GMG-ITC treatment on the molecular mechanisms of inflammation. For this reason, TNF-α (proinflammatory regulator) tissue expression was detected. We have clearly confirmed an increase in TNF-α release and,
consequently, in tissue localization over the course of EAE (Fig. 8C, see densitometric analysis Fig. 14C). On the contrary, reduced expression of TNF-α was observed in mice which received GMG-ITC treatment (Fig. 8D, see densitometric analysis Fig. 14C), so we can speculate that probably, GMG-ITC works through a strict control of the cytokine production. As regards to the “naive group” and the “GMG-ITC control group”, the immuno-sections resulted negative for TNF-α (Fig. 8A and B, respectively, see densitometric analysis Fig. 14C). EAE onset and progression have been related to the generation of oxidative stress, that seems to play a main role [20], confirmed by several experimental studies that showed the beneficial treatment with antioxidants against the progression of the disease [39]. In both MS and EAE, demyelination and axonal damage can be related to reactive oxygen species (ROS) action [40,41]. Moreover, some authors reported that iNOS knockout mice
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Fig. 3. LFB staining. Compared to naive (A) and GMG-ITC control mice (B), EAE untreated animals exhibited remarkably reduced myelin and axonal structures in the spinal cord (C). Moreover, treatment with GMG-ITC reduces demyelination and axonal loss in EAE mice with an intense LFB positive staining (D).
show low signs of disease [42]. Our results demonstrated that GMG-ITC reduced the generation of reactive species through the immunolocalization of NOS2 (Fig. 9D, see densitometric
analysis Fig. 14D) and nitrotyrosine, taken as an indirect marker of peroxynitrite activity [43] (Fig. 10D, see densitometric analysis Fig. 14E). This is evident when the activity of the
Fig. 4. Silver impregnation. A severe condition of vascular tissue breaking up in spinal cord sections cut from EAE mice (C). Otherwise, spinal cord harvested from the EAE + GMG-ITC group” (D), the “naive group” (A) and the “GMG-ITC control group” (B) displays a normal trend and architecture.
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Fig. 5. Foxp3 immunohistochemical localization. A high Foxp3 tissue expression was detected in the spinal cord of mice subjected to EAE (C). Probably due to the protective role of GMG-ITC we didn't find Foxp3+ cells in the spinal cord tissue of mice MOG35–55-injected but pharmacologically treated (D). Naive animals (A) and GMG-ITC control mice (B) resulted negative too.
Fig. 6. CD44 immunohistochemical localization. CD44 expression reveals express higher tissue levels of this markers in EAE-affected mice (C) with respect to the EAE + GMG-ITC group” (D), the “naive group” (A) and the “GMG-ITC control group” (B) This data leads to believe that the spinal cord tissue, in severe disease, attracts infiltrating cells at the site of damage.
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Fig. 7. MAP kinase signal pathway. GMG-ITC treatment following EAE induction caused phospho-ERK p42/44 inhibition reducing the citoplasmatic level of the protein when compared with untreated EAE mice. This could be associated with the typical feature of nonsteroidal anti-inflammatory drugs (NSAIDs).
treatment is compared with immunohistochemical image of EAE mice not pharmacologically treated (Fig. 9C, see densitometric analysis Figs. 14D and 10C, see densitometric analysis Fig. 14E, respectively). Moreover, it is well known that reactive radicals and apoptosis are strictly linked and protective mechanisms involved in the treatment of MS/EAE go through oxidative stress downregulation that seem in turn related to apoptosis pathway shutdown [44]. Also, authors have correlated the EAE model with apoptosis, mostly of oligodendrocytes, neurons and astrocytes in percentage of 45, 20 and 13% respectively [45]. The unbalance between Bax, a proapoptotic protein, and Bcl-2, an anti-apoptotic protein, plays an important role in the triggering of cellular apoptotic cascade during EAE [46], since, basically, their ratio provides the fate for cell survival [47,48]. A negative Bax expression level in EAE group treated with GMG-ITC (Fig. 11D, see densitometric analysis Fig. 14 F) leads to believe that the ITC exerted a beneficial protective effect against the mechanisms of cellular apoptotic death. Conversely, untreated EAE mice displayed high immune-Bax localization (Fig. 11C, see densitometric analysis Fig. 14 F). These results correlate with protective indices given by Bcl-2 highly expressed localization in a section taken from the “naive group” (Fig. 12A, see densitometric analysis Fig. 14G), the “GMG-ITC group” (Fig. 12B, see densitometric analysis Fig. 14G) as well as the “EAE + GMG-ITC group” (Fig. 12D, see densitometric analysis Fig. 14G). A negative staining in slide obtained from EAE not pharmacologically treated mice put on view a severe degree of cell death in the immunohistochemical section incubated with Bcl-2 antibody (Fig. 12C, see densitometric analysis Fig. 14G).
Fig. 8. Proinflammatory cytokine release: TNF-α. Increased TNF-α tissue localization in EAE mice was reported (C). On the contrary, reduced expression of TNF-α was observed in mice which received GMG-ITC (D). Naive mice (A) and GMG-ITC control mice (B) resulted negative at TNF-α tissue staining.
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Fig. 9. iNOS immunohistochemical localization. Increased iNOS tissue localization in EAE mice was found (C). On the contrary, reduced expression of iNOS was observed in mice which received GMG-ITC (D). Naive mice (A) and GMG-ITC control mice (B) were negative at iNOS tissue staining.
To corroborate the above results, TUNEL staining was performed providing the presence of DNA fragmentation. The severity and importance of this mechanism can be evaluated
considering some of most symptomatic events for this type of cell death, including: 1) nuclear DNA double-strand cleavage at level of the linker region between nucleosomes, 2) nuclear
Fig. 10. Nitrotyrosine tissue expression. GMG-ITC treatment resulted in negative staining for anti-nitrotyrosine antibody (D) as well as in naive mice (A) and in GMG-ITC control mice (B). On the contrary, the immunohistochemical image of EAE not pharmacologically treated mice showed a clear positivity (C).
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Fig. 11. Proapoptotic factor expression: Bax. Naive mice (A) and GMG-ITC control mice (B) sections did not stain for anti-Bax antibody. Negative Bax expression levels were found in the EAE group treated with GMG-ITC (D) leading to believe that the ITC exerted a beneficial protective effect against the mechanisms of cellular apoptotic death. Conversely, untreated EAE mice displayed high immune-Bax localization (C).
chromatin condensation at earlier stages, 3) coarse fragmentation of the nucleus at later stages, and 4) membrane-bound cellular particle production [49]. Images showed in EAE untreated mice evident tissue presence of nuclei with an intense positivity for TUNEL staining and characterized by an intensely and completely labelled nucleus broken up into smaller TUNEL-labeled brown fragments (Fig. 13C, see arrow). Differently, an absence of apobodies has been detected in tissue sections from GMG-ITC treated mice, naive mice and GMG-ITC control mice (Fig. 13D, A and B, respectively).
3.1. Conclusions Following achieved data, we can conclude that GMG-ITC has protective effects on spinal cord tissue, with a protective action on myelin loss and axonal damage. Moreover, a representative panel of all markers, distinctive of the disease, evaluated in the present work is summarized in Fig. 12 in a prospective overview that shows all densitometric analysis. To sum up, this work has shown new evidences about the bioactivity of GMG-ITC in addition to the well known anticancer [2] effects and antibiotic [21] properties. Thus,
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Fig. 12. Anti-apoptotic factor expression: Bcl-2. A Bcl-2 highly expressed localization in a section taken from the “naive group” (A), the “GMG-ITC group” (B) as well as the “EAE + GMG-ITC group” (D) was found. A negative staining in a slide obtained from EAE not pharmacologically treated mice put on view a severe degree of cell death in the immunohistochemical section incubated with Bcl-2 antibody (C).
Fig. 13. Tunel staining. EAE untreated mice (C) showed evident tissue presence of nuclei with an intense positivity for TUNEL staining and characterized by an intensely and completely labelled nucleus broken up into smaller TUNEL-labeled brown fragments (see arrow). Differently, an absence of apobodies was detected in tissue sections from GMG-ITC treated mice (D), naive mice (A) and GMG-ITC control mice (B).
172 M. Galuppo et al. / Fitoterapia 95 (2014) 160–174 Fig. 14. Densitometric quantification. For IHC images, densitometric analysis was carried out to quantify and highlight significant differences among experimental groups. Summarizing: (A) FoxP3; (B) CD44; (C) TNF-α; (D) iNOS; (E) nitrotyrosine; (F) Bax; (G) Bcl-2. A p value b0.05 was considered statistically significant.
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our results have highlighted that this compound is able to exert antinflammatory and antioxidant effects with consequences on cell apoptotic death processes. These results related to MS, a disease with neurodegenerative course, provide a new and interesting possible application of GMG-ITC, produced by GMG bioactivation with myrosinase, in the clinical treatment of this pathology.
[18]
[19]
[20]
Conflict of interest Authors have not any conflict of interest to declare.
[21]
Acknowledgments [22]
The authors would like to thank the Dr. Giuseppe Galletta and Dr. Massimo Messina belonging to the secretary office of IRCCS Centro Neurolesi “Bonino-Pulejo”- Messina, for their excellent technical assistance.
[23]
[24]
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