Experimental Parasitology 126 (2010) 510–516

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Experimental Parasitology

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Leishmania major: Anti-leishmanial activity of Nuphar lutea extract mediated bythe activation of transcription factor NF-jB

Lital Ozer a,b, Joseph El-On a,b,*, Avi Golan-Goldhirsh c, Jacob Gopas a,d

a The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Israelb Laboratory of Parasitology, Soroka University Medical Center, Beer Sheva, Israelc Albert Katz Department of Dryland Biotechnologies, The Jacob Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University of the Negev,Midreshet Ben-Gurion, Israeld Department of Oncology, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 August 2009Received in revised form 26 May 2010Accepted 27 May 2010Available online 31 May 2010

Keywords:Leishmania majorNuphar luteaAmastigotesMouse peritoneal macrophagesNuclear factor kappa B (NF-jB)Inducible nitric oxide synthase (iNOS)NG-monomethyl-L-arginine (NGMMA)Respiratory burst (RB)Lysozyme (EC3.2.1.17)b-Galactosidase (EC3.2.1.23)

0014-4894/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.exppara.2010.05.025

* Corresponding author at: The Shraga Segal DepaImmunology, Faculty of Health Sciences, Ben-GurionSheva 84105, Israel. Fax: +972 8 6477 626.

E-mail addresses: jelon@bgu.ac.il, jelon@bgumail.b

Here we report the effect of a partially purified alkaloid fraction (NUP) of Nuphar lutea on nuclear factorkappa B (NF-jB) expression and studied its mechanism of toxicity against Leishmania major in C3H miceperitoneal macrophages. NUP was found to be a mixture of thermo-stable dimeric sesquiterpene thioal-kaloids containing mainly thionupharidines. The anti-leishmanial activity was shown to be mediatedthrough the activation of NF-jB and increased iNOS production. Additionally, the nitric oxide inhibitor,NG-monomethyl-L-arginine (0.5 mM) totally reverted the anti-leishmanial effect of NUP (0.25 and0.5 lg/ml). NUP was also shown to act as an anti-oxidant, almost completely inhibiting the macrophagerespiratory burst activity. However, no elevated lysozyme (EC3.2.1.17) or b-galactosidase (EC3.2.1.23)activities were demonstrated in macrophages treated with NUP. This study suggests, that the activityof NUP is mediated by NF-jB activation and the production of nitric oxide which is dependent on theL-arginine:NO pathway.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Leishmaniasis caused by the obligate intracellular protozoanparasite of the genus Leishmania is still considered a major healthproblem in the rural areas of the Middle East, Africa, Asia, Europeand Central and South America. The disease is widely distributed,affecting approximately 12 million people worldwide, causing awide spectrum of diseases. Control measures of leishmaniasis mustfocus on rapid detection, vaccination and effective treatment of thedisease. Numerous attempts have been made at developing a suc-cessful vaccine against leishmaniasis. To date, three vaccines havebeen licensed for use: one live and one killed vaccine for humans inUzbekistan and Brazil, respectively, and a second-generation vac-cine for dog prophylaxis in Brazil (Palatnik-de-Sousa, 2008). Treat-ment of leishmaniasis remains problematic in developing

ll rights reserved.

rtment of Microbiology andUniversity of the Negev, Beer

gu.ac.il (J. El-On).

countries, where it is most often found. Firstly, the cost of the avail-able drugs is high, putting it out of reach for the great majority ofinfected persons. Secondly, several Leishmania species becomedrug resistant to the first line of anti-leishmanial compounds(Amato et al., 2007; Jha, 2006; Santos et al., 2008) and the patienthas to further rely on highly toxic compounds. The number ofeffective drugs available (pentostam, pentamidine, amphotericinB, paromomycin, miltefosine) is still very limited, and the develop-ment of new, cheap, effective anti-leishmanial treatment is neces-sary for the treatment and to control the disease.

During the last decade numerous systematic studies from vari-ous parts of the world on the anti-leishmanial activity of medicinalplants have been reported. Anti-leishmanial activity against L.amazonensis and L. chagasi was demonstrated with the Brazilianplants Vernonia polyanthes (Asteraceae) and Ocimum gratissimum(Lamiaceae), respectively (Braga et al., 2007). The Paraguayan plantRollinia emagrinata DC. Ex Dunal (Annonaceae) displayed leishman-icidal effect against L. donovani and L. braziliensis (Fevrier et al.,1999) and the Cameroonian medicinal plant Harungana madagas-carensis (Hypericaceae) was found highly effective against L. dono-

L. Ozer et al. / Experimental Parasitology 126 (2010) 510–516 511

vani (Ndjakou Lenta et al., 2007). Anti L. donovani activity was alsodemonstrated with the Turkish marine macroalgae Ulva lactuca(Chlorophyta) and Posidonia oceanica (L) Delile (Posidoniaceae) (Or-han et al., 2006). However, the mechanism of their anti-leishman-ial activity has not yet been clarified. Recently, the 18b-glycyrrhetinic acid (GRA), a pentacyclic triterpene derivative, iso-lated from the root of the plant licorice, Glycyrrhizza glabra L (Fab-aceae), at a dose of 50 mg/kg/day, given three times, 5 days apart,totally eliminated L. donovani from spleen and liver of a 45-day-oldmouse model (Ukil et al., 2005). The leishmanicidal effect of 18b-glycyrrhetinic was found to be mediated by the activation of nucle-ar factor kappa B (NF-jB) and NO production.

Killing of intracellular Leishmania amastigotes in the phagolyso-some within the macrophage was reported to be mediated throughnitric oxide (NO) production, and elevated oxidative burst andlysosomal enzymes activity (El-On et al., 1980, 1990; Wanasenand Soong, 2008). NO is the major anti-leishmanial oxidant pro-duced by activated macrophages (Gantt et al., 2001). It inhibitsLeishmania cysteine proteinase activity (Salvati et al., 2001) andblocks the differentiation process from amastigote to promastigote(Lemesre et al., 1997). NO is generated by inducible nitric oxidesynthase (iNOS) through the activation of NF-jB (May and Gosh,1998; Mukbel et al., 2007). Under physiologic conditions NF-jBis predominantly inactive because of the interaction with cytoplas-mic NF-jB inhibitors (IjB). Upon activation it translocates to thenucleus in which it activates gene expression (May and Gosh,1998). Most signals induce the activity of IjB protein kinases(IKK). Activation of the IKK complex leads to IjBa phosphorylationand subsequent release of NF-jB which translocates to the nucleusand activates transcription of multiple genes, including iNOS andTh1 cytokines. Leishmania promastigotes were capable of prevent-ing phosphorylation and degradation of IjB in normal bone-mar-row derived macrophages and as a result preventing thetranslocation of NF-jB to the nucleus and NO production (Priveand Descoteaux, 2000). In addition, susceptibility of c-REL KO miceto L. major infection was reported to be associated with defectiveNO production and parasite killing (Grigoriadis et al., 1996). NF-jB activation has been also shown to be a key factor in the regula-tion and induction of IL-12 and iNOS (Griscavage et al., 1996; Mayand Gosh, 1998). Both, IL-12 and iNOS play a crucial role on thesusceptibility of the Leishmania parasite within the host cell (Gri-scavage et al., 1996; May and Gosh, 1998; Ukil et al., 2005).

In our recent study, 41 plant methanol extracts belonging to 28different families were examined, of which 9 showed leishmani-cidal effect against L. major promastigotes and amastigotesin vitro (El-On et al., 2009a). None of these plant extracts totallyeliminated the intracellular amastigotes, except the Yellow Pond-lily, Nuphar lutea (Nymphaeaceae) extract and its partially purifiedalkaloids fraction (NUP), that was almost as effective in vitro asparomomycin – a drug of choice against the disease. Nuphar luteais an aquatic perennial that grows on the water surface. The plantis native to the Eastern United States, Africa, Temperate Asia, theWest Indies, and Europe and in most temperate regions of NorthAmerica. It occurs in slow-moving streams, still ponds, and lakes.In Israel, the plant is mainly found at the Hula and the Yarkon nat-ural water reserves, in northern and Central Israel, respectively. Ithas been reported that the alkaloid fraction from N. pumilum, anadditional member of Nymphaeaceae family, partially inhibitedmetastatic activity in mice (Matsuda et al., 2001, 2006). In cancercells, however, suppression rather than activation of NF-jB is cor-related to inhibition of proliferation and tumor cell invasion. Werecently reported that NUP inhibited NF-jB activity and inducedapoptosis of tumor cells (Ozer et al., 2009). The aims of this studyare, therefore, to further characterize the anti-leishmanial activityof N. lutea and to determine the leishmanicidal mechanisms in-volved in plant toxicity.

2. Materials and methods

2.1. Parasite strains and host animals

Male C3H/HeJ, 10–12 weeks old, was used as the source of per-itoneal macrophages for in vitro studies. The parasites (L. major,WHO code: MHOM/IL/80/Freidlin) were maintained at 28 �C by bi-weekly passage in RPMI-1640 plus L-glutamine (20 mM), supple-mented with 10% heat inactivated fetal calf serum (FCS),penicillin (100 U/ml) and streptomycin (100 lg/ml) (BiologicalIndustries, Beit Haemek, Israel). Parasites were also maintainedin experimentally infected Balb/c mice. All our studies with exper-imental animals were performed in accordance with the law andthe institutional regulations regarding the use of experimentationon animals.

2.2. Development of L. major amastigotes in macrophages in vitro, at37 �C

C3H/HeJ mouse peritoneal macrophages were prepared fromthioglycollate-stimulated mice as previously described (El-Onet al., 2009a). Adherent macrophages on glass cover slips 10 mm2

(3 � 105 macrophages/ml/well) in RPMI medium, in a 24-well(16 mm in diameter) microplate (Greiner, Srikenhawen, Germany)were infected with L. major promastigotes (1:5 cells/parasites ra-tio). After 24 h, free living promastigotes were removed and themedium was replaced by fresh medium containing the corre-sponding concentration of extract. The development of the para-sites was checked at various times, by the examination of threecover slips for each treatment, after staining with Giemsa stain.Control samples included: (a) growth medium without extracts(100% viability). (b) Positive control, containing the anti-leishman-ial drug, paromomycin sulfate (PR) (Farmitalia, Italy). PR was usedas the reference drug, with IC50 value of 50 lg/ml. The percentagegrowth inhibition [100% � PS (%)] of each extract on the intracellu-lar amastigotes was evaluated. The parasite survival (PS) indicatesthe total number of intracellular amastigotes in 400 cells of thetreated sample vs. the untreated control.

2.3. Plant extracts preparation

Nuphar lutea was grown in a water pool at the Blaustein Insti-tutes for Desert Research. The active fraction was prepared fromleaves as previously described (El-On et al., 2009a). Briefly, imme-diately after harvesting, the plant material was submerged in li-quid nitrogen and stored at �70 �C until extracted. Plant sample(1 g) was extracted by methanol:water in a ratio of 1:5 (w/v) bygrinding in a pre-chilled mortar containing liquid nitrogen. Themixture obtained was kept on ice for 15 min and microcentrifuged(Eppendorf, Germany) for 5 min at room temperature. The super-natant was collected and kept at �70 �C for analysis. In certaincases, the solvent was evaporated under reduced pressure at roomtemperature and the remaining material was dried by freeze-dry-ing. The dry material content of the extract was determined gravi-metrically. The materials obtained were stored either in 50%methanol or as dry powder at �70 �C. Further purification of theactive compound was achieved by fractionation on a silica gel col-umn using chloroform:ethyl-acetate:di-ethyl-amine 20:1:1(v:v:v), as eluant (Ozer et al., 2009). All fractions were collectedand assayed for anti-leishmanial activity. The fraction with thehighest anti-leishmanial activity (NUP) was used to determinethe mechanism of plant toxicity.

512 L. Ozer et al. / Experimental Parasitology 126 (2010) 510–516

2.4. Determination of iNOS and NF-jB (p50, p65) by Western blot

Infected and uninfected macrophages (5 � 106/5 ml/plate) werecultivated in 10 cm in diameter plates (Corning Incorporated, NY,USA). Twenty-four hours after infection with 25 � 106 promastig-otes (1:5 cells/parasites ratio), free living parasites were removedand the cells were treated with NUP at 0.17 lg/ml, a concentrationnon-toxic to macrophages. After 3, 6, 12 and 72 h of exposure, thecells were washed three times with PBS and then exposed to 150 llof lysis buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% sodiumdodecyl sulfate, 50 mM Tris Base, pH-8 (RIPA), containing a cocktailof protease inhibitors (3.2 mg/ml) (Roche Molecular Biochemicals,Basel, Switzerland). The cells were then collected by scraping, andafter 30 min incubation in ice, the lysate was microcentrifuged for30 min at 4 �C. The supernatant was collected and stored at �70 �C.The supernatants from infected-untreated and uninfected-treatedcell lysates were similarly prepared and used as control. The pro-tein concentration was determined by a Bio-Rad protein assay(Bio-Rad Laboratories, Hercules, CA, USA).

NF-jB and iNOS were detected by immunoblot analysis. Theamount of 30–80 lg protein of macrophage soluble fractions ofall groups was resolved by 7.5% (iNOS) or 10% (NF-jB) sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)under reducing conditions. Resolved proteins were electroblottedto nitrocellulose membrane (Scleicher & Schuell, USA), and afteran hour blocking with TBS-T, pH 7.6 (3.8 mM HCl, 136.9 mM NaCl,20 mM Tris base, 0.1% Tween 20) containing 3% skimmed milk, themembrane was probed overnight at 4 �C with murine anti-iNOS,anti-p50 and anti-p65 monoclonal antibodies (Santa Cruz Biotech-nology, USA). After extensive washing, the membranes were fur-ther incubated for an hour with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA).The bands were detected by chemiluminescence using a detectionkit (Beit Haemek, Israel). Anti-b-actin antibody (MP Biochemicals,USA) was used as internal control to determine sample loading.

2.5. Effect of NG-monomethyl-L-arginine (NGMMA) treatment onamastigotes development

In order to determine whether the anti-leishmanial effect ofNUP is mediated through elevated NO production, the competitiveinhibitor of L-arginine, NG-monomethyl-L-arginine (NGMMA) wasused. NGMMA (0.05; 0.5 mM) (Sigma), either alone or combinedwith NUP (0.25–2.5 lg/ml) was added to the macrophages in a24-well microplate, 24 h after their infection with L. major prom-astigotes. After additional 3 days incubation the inhibitory effectagainst the intracellular amastigotes was determined as describedabove.

2.6. Measurement of respiratory burst (RB) activity

The reduction of nitro blue tetrazolium (NBT) to blue formazan,which is dependent on oxygen metabolites, is considered a usefulmethod for RB response estimation. The test was performed as de-scribed by Gueron et al. (1993). The reaction mixture for NBT testincluded 0.05% (w/v) NBT, 25% (v/v) PBS, 50% (v/v) Hank’s bufferedsalt solution (HBSS), and 25% (v/v) FCS. Phorbol 12-myristate 13-acetate (PMA) (1 lg/ml) was added for maximally activating themacrophages. Infected and uninfected macrophages (3 � 105/500 ll/well) in a 24-well microplate, were treated over 3 days withNUP as described above. On the day of experiment the RPMI med-ium in the macrophage culture was replaced by NBT reaction mix-ture. The cells were incubated for 1 h at 37 �C with gentle rocking,after which the reaction was stopped with 95% ethanol (15 min).The cells were kept in Sorensen’s buffer (0.066 M KH2PO4 +0.066 M NaHPO4, pH 6) until examined for NBT reaction (blue

staining) by light microscopy. The percentage of cells with RBactivity was evaluated. Approximately 400 cells in each samplewere scored.

2.7. Lysosomal enzymes assays

Infected and uninfected macrophages (5 � 106 macrophages/5 ml/10 cm in diameter plate) were treated over 3 days with NUPas described above. On the day of experiment the RPMI mediumin the macrophage culture was removed and the cells were washedthree times with PBS, collected by scraping, re-suspended in 300 llPBS and lysed by 6 cycles of freezing in liquid nitrogen and thenthawing. The lysate was microcentrifuged for 30 min at 4 �C andthe supernatant fluid removed and stored at �70 �C. Quantitativedetermination of b-galactosidase activity was done according tothe method of Beck et al. (1968). The reaction mixture for b-galac-tosidase assay contained 200 ll of macrophage lysate (3 � 106/ml)and 200 ll of 1 mM 4-nitrophenyl-b-D-galactopyranoside (Sigma)in 0.05 M citrate phosphate buffer at pH 3.0. After 1 h incubationat 37 �C the reaction was terminated by the addition of an equalvolume of 2 M NaOH, pH 10.7. The optical density of the nitrophe-nol product was measured at 420 nm. Lysozyme activity was eval-uated according to Vantrappen et al. (1976). The reaction mixtureof lysozyme contained 200 ll of macrophage lysate (3 � 106/ml),200 ll 0.1 M PBS (pH 7) and 200 ll of bacterial suspension(1 mg/ml PBS) of Micrococcus lysodeikticus suspension (Sigma).The mixture was incubated at 37 �C, and the bacterial lysis wasmeasured at 420 nm at 15 min intervals.

2.8. Immunohistochemical analysis

Localization of the NF-jB subunits (p50, p65) in the macro-phage was determined by immunohistochemical staining usinganti-p50 and anti-p65 antibodies. Infected and uninfected macro-phages were treated with NUP as described above. On the thirdday of treatment the cells were washed three times with PBS andfixed overnight with 10% formalin. After extensive washing, thecells were incubated overnight with rabbit anti-p50 or anti-p65antibodies (Santa Cruz Biotechnology, USA), and after an additionalwashing, they were incubated with appropriate peroxidase-conju-gated anti- mouse IgG for 30 min at room temperature. The macro-phage nuclear and cytoplasmic location of NF-jB was visualizedusing ABC-Vectastin immunoperoxidase (Vector Laboratories Bur-lingame, CA, USA), (brown staining) and hematoxylin (blue stain-ing of nuclei).

3. Results

In our previous study (El-On et al., 2009b), the partially purifiedalkaloid fraction (NUP) at 0.17 lg/ml, displayed marked selectivetoxicity (IC50 = 0.087 ± 0.003 lg/ml) and totally eliminated theintracellular parasites within 3 days of treatment. Analysis of theactive mixture showed that it is composed mainly of sesquiterpenethioalkaloids such as 6-hydroxythiobinupharidine, and 6-dihydroxythiobinupharidine B (Ozer et al., 2009).

In order to determine whether NUP affects the expression of theNF-jB subunits p50 and p65, infected and non-infected macro-phages were incubated for various times with NUP. The level ofp50 and p65 was evaluated by Western blot. Quantitation of p50and p65 expression was calculated as a relative ratio of the inten-sity of the specific NF-jB bands determined by densitometry, com-pared to b-actin. In the untreated, either uninfected or infectedcells, a substantial decrease in p50 and p65 expression was ob-served after 72 h (Fig. 1). In contrast, treatment with NUP inducedhigh expression of p50 and p65 in both infected and uninfected

Fig. 1. Effect of NUP on NF-jB (p50, p65) expression in infected and uninfected macrophages. Macrophages were infected with L. major (L.m) promastigotes at a ratio of 1:5and treated with NUP at 0.17 lg/ml (active concentration, not toxic to macrophages), for up to 3 days. (A) Western blot analysis, using anti-p50 and anti-p65 monoclonalantibodies was performed. Equal protein loading was evaluated by b-actin. (B) Quantitation of p50 and p65 expression was calculated as a relative ratio of the intensity of thespecific NF-jB bands determined by densitometry, compared to b-actin.

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cells. The effect of NUP on the expression of p50 and p65 was foundto be time-dependent. This observation was further confirmed byimmunohistochemistry, showing either negative or very low stain-ing of the untreated, infected and uninfected cells, respectively, ascompared with a very high staining in the NUP-treated infectedand uninfected macrophages, on the third day of infection(Fig. 2). The staining was demonstrated both, in the cytoplasmand in the nucleus of the cells.

NO, the major anti-leishmanial oxidant produced by macro-phages is generated by iNOS and is activated by NF-jB. In thisstudy iNOS in the macrophage soluble fractions of all groups wasdetected by immunoblot analysis, using an anti-iNOS monoclonalantibody. We found that an increase of NF-jB induced by NUP(0.17 lg/ml) was followed by elevated iNOS production in macro-phages. Up-regulation of iNOS was demonstrated in treated, in-fected and uninfected macrophages, showing 4.6 and 2.3 timeshigher activity compared to untreated macrophages, respectively(Fig. 3). In order to determine if the anti-leishmanial effect ofNUP is indeed mediated through elevated NO production, the com-

petitive inhibitor of L-arginine, NG-monomethyl-L-arginine(NGMMA), was used. NGMMA (0.05; 0.5 mM) added alone to eitheruninfected or infected macrophages did not induce any effectagainst the parasites or the host cells. However, the addition ofNGMMA to the NUP (0.25–1 lg/ml) treated, infected macrophagessignificantly reduced its inhibitory effect (Table 1). NUP adminis-trated alone at 0.25, 0.5 and 1 lg/ml inhibited the parasites devel-opment by 32.1 ± 3.2%, 71.6 ± 10.9 and 100%, respectively(Table 1). The anti-leishmanial activity of NUP at 0.25 and 0.5 lg/ml was totally abrogated by treatment with 0.5 mM NGMMA. How-ever, either a partial inhibitory effect (33%) or no effect was med-iated by NGMMA on the leishmanicidal effect of NUP at 1 lg/mland 2.5 lg/ml, respectively. The effect of NGMMA was dose depen-dent, and lower concentration of NGMMA (0.05 mM) decreased theanti-leishmanial activity of NUP (0.25; 0.5 lg/ml) by approxi-mately 40.1% and 35.6%, respectively.

An effective anti-oxidant activity was found to be induced byNUP. On the third day of infection only 4.9% of the untreated mac-rophages infected with L. major showed respiratory burst activity

Fig. 2. Immunohistochemical detection of NF-jB subunit p50. Macrophages were infected with promastigotes at a ratio of 1:5 and treated with NUP at 0.17 lg/ml for 3 days.Control uninfected (M) and infected with L. major (M + L.m). NUP treated cells: uninfected (M + NUP) and infected (M + NUP + L.m).

L.m --

+-

-+

++

β-Actin

iNOS

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

M M+L.m M+NUP M+L.m+NUP

iNO

S/β

−A

ctin

NUPA

B

Fig. 3. Effect of NUP on iNOS expression in macrophages. Macrophages wereinfected with promastigotes at a ratio of 1:5 and treated with NUP at 0.17 lg/ml for24 h at 37 �C. After which a lysate was prepared from the cells and tested for iNOSby Western immunoblot. (A) Control uninfected (M) and infected with L. major(M + L.m). NUP treated cells: uninfected (M + NUP) and infected (M + L.m + NUP).Equal protein loading was evaluated by b-actin. (B) The ratio of NF-jB to b-actin asdetermined by densitometry is presented.

Fig. 4. Effect of NUP on the respiratory burst (RB) response of macrophages.Macrophages were infected with L. major (L.m) promastigotes at a ratio of 1:5 andtreated with NUP at 0.17 lg/ml for 3 days. Control uninfected (M) and infected(M + L.m). NUP treated cells: uninfected (M + NUP) and infected (M + L.m + NUP).RB activity was examined by nitro blue tetrazolium (NBT) reaction in duplicateslides. Four hundred cells were scored in each slide and the percentage of activatedcells (blue staining) was determined by microscopic examination. Results representthe average of three experiments. The significance of the results was evaluatedusing a student’s t-test (a two-tailed t-test). Data are expressed as means ± standarddeviation of the mean (SD).

514 L. Ozer et al. / Experimental Parasitology 126 (2010) 510–516

compared to 13.5% observed in the uninfected cells. During thisperiod, NUP at 0.17 lg/ml almost completely eliminated the oxida-tive burst in both, the infected and the uninfected macrophages

(Fig. 4). Surprisingly, treatment with NUP did not induce any effecton lysozyme and b-galactosidase activities in macrophages.

4. Discussion

In the vertebrate host Leishmania is an obligatory intracellularparasite of the macrophage, in which it grows and multiplies lead-ing to destruction of the host cell. Elimination of the parasites fromthe macrophage by chemotherapeutic agents might be thereforeachieved through either a direct effect of the drug on the parasiteor/and the combined effects of the drug and the macrophage leish-manicidal activity. In our previous study, N. lutea extracts were

Table 1Effect of NGMMA on the leishmanicidal activity of NUP. Peritoneal macrophages(3 � 105 cells/ml/well) were infected with L. major promastigotes (1.5 � 106). After24 h incubation at 37 �C, the free living promastigotes were removed and the cellswere treated with NUP, either alone or combined with NGMMA. Results wereevaluated on the third day of treatment. Results represent the mean of three differentexperiments.

Sample examined Growth inhibition (%)NUP concentration (lg/ml)

0.25 0.5 1 2.5

NUP 32.1 ± 3.2 71.6 ± 10.9 100 100NUP + 0.05 mM NGMMA 12.9 ± 0.9 25.8 ± 3.7 100 100NUP + 0.5 mM NGMMA 0 0 67 ± 7.5 100

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found to have extensive and selective leishmanicidal activityagainst L. major, destroying 100% of both, free living promastigotesand intracellular amastigotes within 3 days treatment (El-On et al.,2009b). The activity against promastigotes indicates a direct leish-manicidal effect of the extract. This study was therefore initiated inorder to investigate the mechanism of action of NUP on the mod-ulation of the host immune response, and as a consequence onthe parasites killing within the host cell – the macrophage.

The induction of IL-12 and iNOS activity mediated by the activa-tion of NF-jB has been shown to play a crucial role on the suscep-tibility of the Leishmania parasite within the host cell (Griscavageet al., 1996; Ukil et al., 2005). Nitric oxide (NO) was shown to beresponsible for the cytotoxic effected against Leishmania intracellu-lar amastigotes (Green et al., 1990; Mauel et al., 1991). NO produc-tion is mediated by inducible nitric oxide synthase (iNOS) throughthe activation of NF-jB (Marletta et al., 1988; Mukbel et al., 2007).NO is generated by the oxidation of one of the nitrogens in the ami-no acid L-arginine (Hibbs et al., 1988). NGMMA, the competitiveinhibitor of L-arginine has been shown to inactivate the induciblemurine macrophage nitric oxide synthase (Griffith and Kilbourn,1996). Leishmania promastigotes were capable of preventing phos-phorylation and degradation of IjB in normal bone-marrow de-rived macrophages and as a result preventing the translocationof NF-jB to the nucleus and NO production (Prive and Descoteaux,2000). In this study, the anti-leishmanial activity of NUP wasshown to correlate with the up-regulation of NF-jB in the infectedmacrophages, leading to increased iNOS production. Treatment re-sulted initially in a decreased NF-jB expression during the first 3 h,which increased above the base level on the third day of treatment.NUP has been found to induce the NF-jB p50/p65 heterodimer innormal and L. major infected macrophages, leading to a total elim-ination of intracellular parasites, probably by iNOS/NO production.These results were further confirmed by immunohistochemicalanalysis, showing that NUP also induced NF-jB migration intothe nucleus of infected and uninfected-treated cells. A more directevidence for the role of NUP activity was demonstrated using theiNOS-selective inhibitor, NGMMA that reversed the anti-leishman-ial activity exerted by NUP.

Different signaling pathways that trigger NF-jB activation mayaffect differently disease development. Activation of NF-jB wasfound important for preventing apoptosis during rickettsial (Rikett-sia rikettsii) infection (Sporn et al., 1997), and mRNA up-regulationfor p105 and p65 was demonstrated in cultured fibroblasts in-fected by human cytomegalovirus (Yurochko et al., 1997). In addi-tion, increased expression of NF-jB in tumors is correlated withresistance to anti-tumor therapy. Conversely, suppression of NF-jB enables sensitivity to treatment, increased apoptosis anddiminished proliferation and tumor progression (Izzo et al.,2006). Similarly, we have shown in a tumor cell system that NUPinhibits NF-jB, it induces apoptosis and sensitizes the tumor cellsto chemotherapy (Ozer et al., 2009). In leishmaniasis, however, anelevation of NF-jB activity induces a lethal effect on the parasites

as reported by several studies (Ukil et al., 2005) and further con-firmed in this study. Activation of NF-jB induces the expressionof iNOS, leading to NO production and efficient killing of the intra-cellular parasites.

Hydrogen peroxide and superoxide anion produced in the respi-ratory burst, NO production and elevated lysosomal activity havebeen all implicated as major mechanisms of macrophage againstLeishmania. In this study, however, NUP was shown to act asanti-oxidant, reducing the oxidative burst activity in both infectedand uninfected macrophages. This finding was demonstrated byinhibition of the NBT reaction. We confirmed the potent anti-oxi-dant activity of NUP by determining its efficiency coefficient forhydroxyl radicals which was found to be 1.85 mM�1 (unpublisheddata). Ding et al. (1988) have shown that the pathways leading toproduction of H2O2 and NO� are independent. Therefore, a lack ofcorrelation between iNOS expression and oxidative burst activity ispossible. Nevertheless, the anti-oxidant activity of NUP and its cor-relation to elevated iNOS and NO production should be furtherinvestigated.

In this study, no elevated b-galactosidase and lysozyme activitywas induced by NUP. In our previous study, we suggested that theinhibition of b-galactosidae from mouse peritoneal macrophagesby the leishmanial lipophosphoglycan (LPG) resulted from a strongnegatively charged molecule (LPG) that electrically interacts withthe enzyme (El-On et al., 1980). NUP was found to be free of chargeat natural pH (El-On et al., 2009b). Therefore, it may not directlyinteract with neither the parasite’s negatively charged LPG northe positively charged b-galactosidase of the host cell, conse-quently having no effect in their activity. However, further studiesare required to clarify this possibility.

Plant-derived products have long been and will continue to beimportant sources of medicinal agents and models for the design,synthesis, and semi-synthesis of novel substances. The macro-phage protein kinases cascade and the NF-jB pathway were foundto play important roles in regulation of functions involved ininflammation and host defense. The anti-leishmanial activity ofNUP was shown to be mediated through the activation of NF-jBleading to elevate NO production. Therefore, activation of NF-jBmight be found as a useful target to control leishmanial infection.

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