ORIGINAL PAPER

Propolis enhances the effectiveness of praziquantelin experimental schistosomiasis: biochemicaland histopathological study

Tamer Y. Mahmoud & Sherine M. Rizk &

Amany S. Maghraby & Amira A. Shaheen

Received: 17 September 2014 /Accepted: 23 September 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Despite the wide current use of praziquantel (PZQ)in treatment of schistosomiasis, low cure rates have beenrecorded in many studies. The aim of this study was directedto evaluate the curative effect of propolis (Pps) alone or incombination with PZQ on biochemical, immunological, par-asitological, and histological changes associated with experi-mental schistosomiasis in mice. Schistosoma mansoni-infect-ed mice were divided into two experimental sets, each withfour subgroups: (i) untreated, (ii) treated with Pps/day p.o for4 weeks, (iii) treated with PZQ p.o 2×500 mg/kg bd wt, and(iv) treated with Pps+PZQ as in group ii and iii; all treatmentsstarted on the 8th week postinfection, in addition to uninfectedgroup as control for the previous groups. Treatment of infect-ed mice with Pps, although failed to eradicate the worm,significantly reduced the hepatic granuloma number, theirlymphocytic infiltration and aggregation, hepatic and splenicmyeloperoxidase (MPO) activity and plasma, and liver andthymus nitric oxide (NOx) levels together with normalizationof plasma proteins and alleviation of oxidative stress in theexamined t issues as evidenced by reduct ion ofmalondialdehyde (MDA) and normalization of glutathione(GSH). Promising results were obtained when Pps was givenin combination with PZQ, where the anti-schistosomal activ-ity of PZQ was markedly potentiated with complete allevia-tion and amelioration of the histological and biochemicalalteration associated with schistosomiasis. This study high-lights the potential usefulness of Pps as an adjunct to PZQ inschistosomiasis.

Keywords Schistosomiasis . Praziquantel . Propolis .

Biochemical . Immunological . Parasitological .

Histopathological changes

Introduction

Schistosoma mansoni (S. mansoni) causes pathologicalchanges in many of the host organs resulting from an interac-tion between the helminthous parasite and the mammalianhost. The principal pathogenic factor in this disease is theparasitic ova lodged not only in the microvasculature of theliver but also in other host tissues such as intestine, skeletalmuscle, lung, spleen, brain, heart, etc. (Warren et al. 1974).The deposited ova may interfere with the physiological andbiochemical aspects of these tissues, depending on the degreeof the host’s inflammatory and fibrotic reactions to them.Many of the arise tissue complications are continued inspiteof the treatment with schistosomicidal drugs.

PZQ is schistosomicidal drug which has been successfullyused for treatment of S. mansoni infection in Egypt. Althoughthe effectiveness of PZQ against schistosomes and other hel-minths is well documented (Fallon et al. 1996), and more than100 million people are currently being treated for schistoso-miasis with PZQ, some of them are rapidly reinfected andmust be retreated on an annual or semiannual basis (Hotezet al. 2006). Low cure rates have been recorded in manystudies in Africa including Egypt where patients have yieldedisolates that are tolerant to higher dosages of PZQ (Doenhoffet al. 2002, 2008). Drug resistance has been frequently report-ed in many endemic foci of S.mansoni, notably in Africancountries, such as Egypt and Senegal (Ismail et al. 1996;Wang et al. 2012), and after more than 20 years of large-scale use of PZQ, the spectra of drug-resistant parasites arelooming and it is time for introducing a new therapeuticapproach (Xiaonong et al. 2002). This over drug resistance

T. Y. Mahmoud : S. M. Rizk (*) :A. A. ShaheenBiochemistry Department, Faculty of Pharmacy, Cairo University,Cairo, Egypte-mail: sherine.abdelaziz@pharma.cu.edu.eg

A. S. MaghrabyTherapeutical Chemistry Department, Immunology and InfectiousDiseases Group, National Research Center, Cairo, Egypt

Parasitol ResDOI 10.1007/s00436-014-4141-0

and possible reoccurrence of infection encouraged the searchfor new therapy approach, possibly from natural resources(Abdualla et al. 2007; Abebe 2008).

Propolis (Pps) is a resinous hive product collected byhoney bees from exudates and buds of plants and mixed withwax and bee enzymes (Burdock 1998). Pps has several bio-logical and pharmacological properties, as antimicrobial (AbdEl Hady and Hegazi 2002), anti-inflammatory (Park andKahng 1999), and antioxidant (Marquele et al. 2005; Kanburet al. 2009), in addition to immune modularity and immunestimulant effects (Nassar et al. 2012). Pps has been demon-strated to increase the percentage of protected animals againstbovine herpesvirus type 5 (Fischer et al. 2007) suggesting itsuse in vaccines as an adjuvant. Due to such wide spectrum ofactivity, our interest was stimulated for researching the bene-ficial action of domestic Pps in infected schistosomiasis.

To the best of our knowledge, the effect of Pps on schisto-somiasis has not been studied yet. Therefore, the present workwas adopted to evaluate the curative effect of an ethanolicextract of Egyptian Pps when administered orally alone or incombination with PZQ against experimental challenge ofmice with S. mansoni strain. The study was based on bio-chemical, immunological, parasitological, and histopatholog-ical investigations.

Materials and methods

Animal

Adult male swiss albino mice weighing 20–25 g at 6–8 weeksold were purchased from the animal house of NationalResearch Center (Dokki, Egypt). The animals were housedin steel cages with raised mesh bottom at 24±2 °C under a12-h light-dark cycle, and they were fed with a standardpelleted chow diet and allowed to receive water ad libitum.This investigation complies with the Guide for the Care andUse of Laboratory Animals published by the US NationalInstitutes of Health (NIH Publication No. 85-23, revised1996) and was approved by the Ethics Committee forAnimal Experimentation at Faculty of Pharmacy, CairoUniversity.

Drug and chemicals

Drug PZQ was obtained from Egyptian InternationalPharmaceutical Industries Company (E.I.P.I.Co.).

Chemicals Metaphosphoric acid; 5,5 ′-dithiobis-2-ni t robenzoic ac id; th iobarb i tur ic ac id ; 1 ,1 ,3 ,3-tetraethoxypropane; vanadium trichloride; sulfanilamide;N-(1-naphthyl) ethylenediamine dihydrochloride; and o-dianisidine hydrochloride were obtained from Sigma-

Aldrich Chemical Co. (St. Louis, MO, USA). Horseradishperoxidase enzyme was obtained from Fluka. Other chemicalswere from Analar grade or from the purest grade available.

Extraction of Pps

One hundred grams of the resinous material of Egyptianpropolis (obtained from Dakahlia Governorate, Egypt) wascut into small pieces and extracted at room temperature with50 ml of 70 % ethanol. Extraction was performed twice with a24-h interval. The alcoholic extract was evaporated undervacuum at 50 °C until dryness. Total phenols and total flavo-noids were estimated in the dried ethanolic extract by theFaculty of Agriculture Research Park, Faculty ofAgriculture, Cairo University, where 1 g of dried ethanolicextract was found to contain 83.50 mg total phenols and88.67 mg total flavonoids. The obtained dried ethanolic ex-tract of propolis (28 g) was suspended in phosphate bufferedsaline (PBS) (pH 7.2).

Experimental groups

Cercariae, the infective stage of S. mansoni, were obtainedfrom Theodor Bilharz Institute (Giza, Egypt). The viability ofthe larva was verified under optical microscope. The cercarialsuspension was diluted with water so that each 0.2 ml contains80±10 cercariae. Eachmouse was injected (s.c.) with cercarialsuspension (80±10 cercariae) in the scruff of the anterior partof the neck. Seven weeks after infection, only mice passingschistosomal ova were regarded as the infected animal stock.Two sets of animals were used: the first set of animals (60mice) was employed for parasitological, immunological, andhistopathological studies. The second set of animals (100mice) was employed for biochemical studies. Each set ofanimals was equally divided and randomly assigned in thefollowing groups: group 1: healthy mice that received vehicle(bidistilled water) and served as normal control group; group2: infected mice that was left without treatment and served asinfected control; group 3: infected mice that received an oraldose of PZQ (1000 mg/kg) and divided into two successivedays (Piper et al. 1990; Shaheen et al. 1994) starting 49 daysafter infection; group 4: infected mice that received an oraldose of Pps (300 mg/kg) daily for 4 weeks starting 49 daysafter infection; group 5: infected mice that received a combi-nation of PZQ and Pps by the same route and duration asmentioned in groups 3 and 4.

Assessment of worm burden

A relatively simple and rapid method for recovery of adultschistosome worms from the portal circulation of the infectedand infected treated animals was adapted in the present workafter modification of Duvall and Dewitt (1967). A citrated

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saline solution (0.85 % NaCl+0.75 % Na-citrate w/v) wasused as perfusion fluid. The perfusion was carried out bymaking an incision in the portal vein and injecting the fluidinto the aorta under intermittent pressure. This process offlushing out continued until the brown color of the liverbecome pale and the perfused worms were counted using aninverted microscope, each paired (couple) worms counted astwo. The percentage reduction of the worm burden was cal-culated using the formula: (C−T/C)×100, where C equalsmean recovery from infected control mice and T equals themean recovery from treated mice.

Immunological study

The animals , employed for immunological andhistopatholigical studies, were sacrificed by decapitation atthe end of treatment period. The blood of each animal wascollected from the neck on a plain centrifuge tube, left for15 min at room temperature then centrifuged at 3000×g. Thesera collected were kept at −80 °C until used. Sandwichenzyme-linked immunosorbent assay (ELISA) was carriedout according to Maghraby et al. (2007) for detection ofimmunoglobulins G & M titers using (peroxidase-labeledanti-mice IgG & IgM (Sigma, Deisenhofen, Germany)). Forvisualization of the immune reaction, the soluble peroxidase-specific substrate o-Phenylenediamine (OPD; Sigma,Deisenhofen, Germany) was applied.

Biochemical analysis

At the end of treatment, animals that employed for biochem-ical analysis were anesthetized with chloroform. Each anes-thetized mouse was weighed and blood was collected in cleanheparinized tubes from brachial artery. A part of the collectedwhole blood was used for determination of reduced glutathi-one (GSH) according to the method of Beutler et al. (1963)and the remaining blood centrifuged at 3000×g for 10 min at4 °C, the separated plasma was kept at −20 °C until used fordetermination of malondialdehyde (MDA) (Mihara andUchiyama 1987), nitric oxide (NOx) (Miranda et al. 2001)levels, and ceruloplasmin (CP) activity (Schosinsky et al.1974). Plasma total protein and albumin were alsodetermined using commercially available kits ofBiodiagnostic Company as described by Gornal et al. (1949)and Doumas et al. (1971), respectively.

Liver, spleen, and thymus were excised and washed withice cold saline, and accurately weighed pieces of the usedtissues were homogenized in ice-cold saline using Potter-Elvejham glass homogenizer to make 10 % homogenate.The homogenates were then centrifuged at 3000×g for15 min at 4 °C. The supernatants were separated and storedat −20 °C until used for determination of MDA, GSH, NOx,as previously mentioned, and myeloperoxidase (MPO)

activity (Bradley et al. 1982). MPO activity was expressedas units per milligram protein, in which protein was estimatedaccording to Lowery et al. (1951).

Histopathological examination

Livers of different animal groups were processed for paraffinembedding, and 5-μm sections were prepared. The first sec-tions were stained with hematoxylin and eosin (HE), while thesecond sections were stained with Masson’s trichrome stainand examined microscopically. The macroscopic and histo-logical experiments were performed by investigators whowere blinded to the group and treatment of animals. Thehistological sections were coded to eliminate the observerbias.

Statistical analysis The results were expressed as the mean±SEM, and statistical comparisons were carried out using one-way analysis of variance (ANOVA), followed by Tukey-Kramer's multiple comparisons test. The minimal level ofsignificance was identified at P<0.05.

Results

Worm burden

The perfusion of hepatic portal system in PZQ-treatedmice showed marked reduction in the worm burden toabout 9 % of infected group with a percentage protec-tion amounting to 91 %, which demonstrated antipara-sitic influence of PZQ resulted in parasitologic cure. Onthe other hand, Pps has no anti-schistosomal activitywhen administered alone; however, it produced a slightnon-significant increase in worm burden. Combinedtreatments with PZQ resulted in a significant reductionin worm burden to about 2 % of the infected groupgiving an effective protection exceeding 98 % (Fig. 1).

Lymphocyte infiltration and lymphocyte aggregation as wellas granuloma number and diameter

Data compiled in Table 1 showed that separate admin-istration of Pps and PZQ caused a significant reductionin the degree of lymphocytic infiltration in the liver ofinfected mice to reach to about half the infected values.On combined administration of both therapies, a markedreduction in the degree of lymphocytic infiltration wasproduced reaching one eighth of the infected values ascompared to that produced by PZQ alone. On the otherhand, these therapies alone or in combination causedsignificant reductions in the degree of lymphocytic

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aggregation in infected mice livers reaching to 44, 8,and 26 %, respectively, with respect to infected controlgroup. The granuloma number, the main schistosomiasislesion, was significantly reduced in mice treated witheither Pps or PZQ to reach 60 and 55 % of the infectedcontrol, respectively. However, combined treatmentsproduced an effective reduction in number and size ofgranulomas reaching 14 and 54 % of the infected con-trol, respectively. Such reductions were significant ascompared to that produced by PZQ.

IgG and IgM levels

It is obvious from the results shown in Fig. 2 that 11 weeks ofinfection with S. mansoni were sufficient to elevate signifi-cantly the levels of IgG and IgM. However, such elevated IgGand IgM levels were not significantly lowered by administra-tion of either propolis or PZQ treatments or even theircombination.

Total proteins, albumin, and albumin/globulin (A/G) ratio

Infected mice exhibited a significant reduction in plasmaalbumin and globulins without any significant change inA/G ratio, a matter which reflected on plasma total proteinlevel which, in turn, was significantly lowered as compared tothe normal group. On administration of Pps either alone orcombinedwith PZQ, significant elevations in plasma albumin,globulins, and hence total protein were observed, coupledwith a significant reduction in A/G ratio compared to theinfected control values. Such modifications were sufficientto normalize most of these parameters as shown in Table 2.

CP activity

Infected mice with schistosomiasis exhibited a marked eleva-tion of CP activity amounting 237 % of the normal values.Therapeutic administration of Pps or PZQ either separately orin combination resulted in a further increase in its activity(Table 2).

MDA and GSH

Results in Table 3 showed that infected mice had sufferedfrom marked elevations in plasma, liver, spleen, and thymusMDA levels, together with a significant reduction in blood,liver, and thymus GSH levels in comparison with normalmice. On administration of Pps alone to infected mice, there

Fig. 1 Effect of Pps and PZQ treatments separately or in combination onworm burden in S. mansoni-infected mice. Values are expressed as % ofinfected mice (N=12). Letter b with asterisk denotes a significant differ-ence from infected group at P<0.01. Letter c with asterisk denotes asignificant difference from praziquantel-treated group at P<0.01

Table 1 Effect of Pps and PZQ treatments separately or in combination on certain histopathological parameters in liver from S. mansoni-infected mice(mean±SE, N=12)

Infected Infected treated with

Pps PZQ Pps+PZQ

Lymphocyte infiltration 4.93±0.85 2.80±0.52b 2.60±0.42b 0.60±0.24b*c

Lymphocyte aggregation 3.93±0.75 1.73±0.47b* 0.33±0.19b* 1.00±0.32b*

Granuloma number 14.23±1.03 8.60±0.79b* 7.80±0.47b* 2.00±0.54b*c*

Granuloma diameter (μm) 143.33±11.38 156.00±14.04 120.67±8.62 78.66±7.42 b*c

b Significant difference from infected group at P<0.05b* Significant difference from infected group at P<0.01c Significant difference from PZQ-treated group at P<0.05c* Significant difference from PZQ-treated group at P<0.01

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was a significant reduction in MDA level in plasma andinvestigated tissues. However, such reductions were notenough to normalize plasma, liver, and thymus MDA butwere enough for spleen MDA level to approach the normalvalue. PZQ alone had failed to produce any significant reduc-tion in the elevated plasma and liver MDA levels in infectedmice but produced a significant reduction in its level in spleenand thymus of infected animals. Such reduction was sufficientto normalize only spleen MDA level. Combined treatments toinfected mice resulted in normalization of plasma and thymusMDAwith a significant reduction in liver MDA together withreduction in spleen MDA below its normal level.

Both Pps and PZQ when given alone failed to produce anysignificant amelioration in the lowered blood GSH of infected

mice. However, their combination significantly increased thelevel of this parameter in blood to approach normality.Administration of Pps alone or in combination with PZQ notonly normalized the lowered liver GSH but also significantlyincreased its values above the normal levels, but not higherthan that produced by PZQ alone. It is worthy noticed that Ppsalone failed to modify spleen GSH level in infected mice.However, combination with PZQ resulted in marked increaseof its value to about 2.5-fold the normal value, which wassignificantly higher than that produced by PZQ alone. Dataalso showed that infected mice treated with Pps or PZQseparately or in combination exhibited marked increases inthe lowered GSH levels in thymus to reach 186, 154 and211 %, respectively, of the infected value.

Fig. 2 Effect of Pps and PZQtreatments separately or incombination on immunoglobulinsG & M (IgG & IgM) levels inplasma of S. mansoni-infectedmice. Values are expressed as %of normal ± % SE (N=6). Letter awith asterisk denotes a significantdifference from normal group atP<0.01

Table 2 Effect of Pps and PZQ treatments separately or in combination on plasma proteins and CP in S. mansoni-infected mice (Mean±SE, N=7)

Normal Infected Infected treated with

Pps PZQ Pps+PZQ

Total protein 6.75±0.23 5.14±0.21a* 6.92±0.19b*c* 5.67±0.08a* 6.85±0.29b*c

Albumin 3.46±0.27 2.86±0.04a* 3.32±0.11b*c 2.99±0.07a* 3.18±0.05b

Globulins 3.34±0.27 2.41±0.18a 3.60±0.14b*c* 2.68±0.06 3.67±0.26b*c*

A/G ratio 1.08±0.10 1.20±0.09 0.93±0.05b 1.12±0.04 0.90±0.05b*

CP 129.71±5.63 307.71±15.31a* 362.00±15.72 a* 371.00±11.14 a*b 415.71±25.42 a*b*

Plasma proteins are expressed as g/dl; CP is expressed as U/la Significant difference from normal groups at P<0.05a* Significant difference from normal groups at P<0.01b Significant difference from infected groups at P<0.05b* Significant difference from infected groups at P<0.01c Significant difference from PZQ-treated groups at P<0.05c* Significant difference from PZQ-treated groups at P<0.01

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MPO and NOx

The data included in Table 4 showed that MPO activity wasnot detected in thymus of different experimental groups. Theyalso showed that MPO activity was significantly induced inliver and spleen of infected mice compared to normal group.Administration of Pps either alone or combined with PZQresulted in a significant suppression of the enzyme activity inliver and spleen reaching to about half the infected values,which were significantly lower than that produced by PZQalone. Eleven weeks of infection with S. mansoni caused asignificant rise in the levels of NOx in plasma, liver, andthymus compared to the normal control group. Spleen on theother side did not show any significant change in NOx levelby infection or any of the treatments. However, therapeuticadministration of Pps or PZQ alone resulted in significantdecreases in NOx in plasma and liver to levels approachingthose of normal control group. However, only Pps was capa-ble of reducing the elevated NOx level in thymus approachingthe normal values; on the contrary, PZQ failed to correct suchincrease. However, in both agents, when given together, theyield was better where the values were slightly below normalin plasma and thymus or even reduced to one half the normalvalues in liver.

Histopathological findings

Liver sections from normal control mice showed normalhepatic architecture (Fig. 3A). Liver of mice challenged with

S. mansoni showed numerous large fibrocellular granulomaswith central intact ova (Fig. 3(Bi)) and dense lobular lympho-cytic aggregation (Fig. 3(Bii)). Pps-treated mice showed alarge granuloma with central intact ova (Fig. 3(Ci)) with amild lobular lymphocytic infiltration (arrow) (Fig. 3(Cii)).Liver sections from PZQ-treated mice showed numerousfibrocellular moderate-sized granulomas with almostdegenerated ova (Fig. 3(Di)) with mild lymphocytic infiltra-tion around terminal hepatic venule (arrow) (Fig. 3(Dii)). Onthe other side, Pps+PZQ-treated mice showed fibrocellularsmall granuloma with degenerated egg (Fig. 3(Ei)) togetherwith mild lobular infiltration (arrow) (Fig. 3(Eii)).

Liver fibrosis

Liver sections stained with Masson’s trichrome stain revealednormal liver architecture in control mice (Fig. 4A), whileschistosome-infected mice showed fibrocellular large granu-lomas (arrows) formed by central intact miracidium withconcentric fibrous bundles entangling lymphocytes andepitheloid cells (Fig. 4B). Moreover, liver section from Pps-treated mice showed fibrocellular large granulomas (arrows)formed by central degenerated miracidium with concentricfibrous bundles entangling lymphocytes and epitheloid cells(Fig. 4C). On the other hand, liver section from PZQ-treatedmice showed a fibrocellular moderate-sized granulomaformed by central intact miracidium with concentric fibrousbundles entangling lymphocytes and epitheloid cells(Fig. 4D). Combined treatment with Pps and PZQ showed

Table 3 Effect of Pps and PZQ treatments separately or in combination onMDA and GSH in plasma and certain tissues from S. mansoni-infected mice(mean±SE, N=10)

Normal Infected Infected treated with

Pps PZQ Pps+PZQ

Plasma MDA 3.67±0.24 10.52±0.29a* 5.14±0.51ab* 9.55±0.15a* 2.84±0.51b*c*

Liver MDA 0.72±0.03 1.88±0.06a* 1.22±0.05ab* 1.71±0.08a* 1.03±0.04a*b*

Spleen MDA 2.10±0.09 2.50±0.05a* 2.05±0.07b* 2.23±0.06b 1.65±0.07a*b*c*

Thymus MDA 0.85±0.05 1.92±0.08a* 1.14±0.03ab* 1.24±0.07 a*b* 0.91±0.08 b*c*

Blood GSH 38.29±2.53 24.50±1.35a* 30.18±1.78a 29.85±1.54a 38.95±1.96b*c*

Liver GSH 2.99±0.17 2.04±0.17a 4.27±0.34a*b* 3.29±0.21b* 3.94±0.29ab*

Spleen GSH 0.58±0.02 0.47±0.03 0.61±0.02c* 0.90±0.05a*b* 1.51±0.13a*b*c*

Thymus GSH 0.61±0.04 0.37±0.03a* 0.69±0.04b*c 0.57±0.03b* 0.78±0.03 a*b*c*

PlasmaMDA is expressed as nmol/ml; tissueMDA is expressed as nmol/g tissue and blood GSH are expressed as mg/dl; tissue GSH is expressed as mg/g tissuea Significant difference from normal groups at P<0.05a* Significant difference from normal groups at P<0.01b Significant difference from infected groups at P<0.05b* Significant difference from infected groups at P<0.01c Significant difference from PZQ-treated groups at P<0.05c* Significant difference from PZQ-treated groups at P<0.01

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fibrocellular small granuloma formed by central degeneratedmiracidium with concentric fibrous bundles entangling lym-phocytes and epitheloid cells (Fig. 4E).

Discussion

Despite the wide current use of PZQ in treatment of schisto-somiasis and other helminthal infections, low cure rates havebeen recorded in many studies in Africa including Egyptwhere patients are frequently reported to exhibit a toleranceto higher dosage of PZQ (Doenhoff et al. 2002, 2008). Thisover drug resistance and possible relapse of infection encour-aged the search for new therapeutic approach possibly fromnatural resources. Therefore, the goal of this study is to eval-uate the curative effect of Egyptian propolis when adminis-tered orally alone or in combination with PZQ against exper-imental challenge of mice with S.mansoni strain based onhistological and biochemical examination.

In this study, treatment of S. mansoni-infected mice withPZQ resulted in a marked reduction in worm burdens togetherwith a marked decline in the liver lymphocytic infiltration andaggregation. On treatment with Pps, it failed to affect wormburden but insignificantly increased it. The anti-schistosomalactivity of PZQ could be related to the efficacy of the drug incausing worm tegument damage with eventual death of theparasite that consequently limit significant immune responseof patients and generate a reversion of the tissue fibrosis(Botros et al. 2006). This clearly supports the observed histo-logical reduction of hepatic lymphocyte infiltration and ag-gregation together with lessening the fibrosis. Treatment of

S. mansoni-infected mice with Pps alone remarkably reducedthe lymphocyte infiltration and aggregation. The reduction inthese aspects together with concomitant inhibition of MPOactivity in this study lends significant anti-inflammatory ef-fects to Pps in schistosomiasis. The anti-inflammatory actionsof propolis had been frequently demonstrated by Martin et al.(2013) who showed that propolis had anti-inflammatory ac-tion in corneal inflammation in rats following alkali burns.Recently, it was found that Pps extract decreases the release ofthe inflammatory cytokines like tumor necrosis factor-alpha(TNF-α) with a simultaneous increased production of anti-inflammatory cytokines like interleukin- 4 (IL-4) in testis ofrats subjected to doxorubicin toxicity (Rizk et al. 2014). WhenPps was given in combination with PZQ, a marked eradicationof wormswas obtained resulting in parasitologic cure exceeding98%. This was associatedwith a greater reduction of the hepaticlymphocyte infiltration and aggregation together with effectivehealing of hepatic granulomatous lesions supporting the role ofPps in potentiating the anti-schistosomal activity of PZQ.

In this study, all S. mansoni-infected mice were sufferingfrom hepatic granulomas around deposited schistosome eggs.Hepatic granuloma, the principal schistosomal lesion, is mainlyarisen from mature S. mansoni worm pairs live in the portalvasculature and producing eggs. Since blood flows mainlytowards the liver in the portal circulation, many eggs fail toengage the intestine and instead are carried to the liver wherethey become trapped in the sinusoids. Egg antigens elicit strong-ly Th2-polarized cellular responses which orchestrate the devel-opment of granulomatous lesions around tissue-trapped eggs(Pearce and MacDonald 2002). Separate treatment with Pps orPZQ was effective in healing of hepatic granulomatous lesionsas evidenced histopathologically by reduction of granuloma

Table 4 Effect of Pps and PZQ treatments separately or in combination onMPO and NOx in plasma and certain tissues from S. mansoni-infected mice(mean±SE, N=10)

Normal Infected Infected treated with

Pps PZQ Pps+PZQ

Liver MPO 1.90±0.02 2.95±0.05a* 1.72±0.02a*b*c* 1.87±0.02b* 1.55±0.02a*b*c*

Spleen MPO 9.17±0.14 12.86±0.17a* 7.92±0.02 a*b*c* 9.99±0.26ab* 7.08±0.14a*b*c*

Thymus MPO ND ND ND ND ND

Plasma NOx 48.49±1.60 56.31±2.05a* 44.31±0.74b* 48.31±1.38b* 43.03±1.42b*

Liver NOx 17.92±1.09 23.80±1.19a* 18.19±0.64b* 15.05±0.80b* 8.31±0.91a*b*c*

Spleen NOx 7.42±0.99 10.01±0.86 9.05±0.58 8.51±0.57 8.61±0.76

Thymus NOx 10.51±0.76 15.56±1.35a* 9.80±0.71b*c* 14.50±0.86a 8.38±0.66b*c*

Tissue MPO is expressed as U/mg protein; plasma NOx is expressed as μmol/l; tissue NOx is expressed as μmol/g tissue

ND not detecteda Significant difference from normal groups at P<0.05a* Significant difference from normal groups at P<0.01b* Significant difference from infected groups at P<0.01c* Significant difference from PZQ-treated groups at P<0.01

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number while the combined therapy was highly effective inreduction of granuloma diameter as well.

As a result of hepatic granuloma formation, hepatocellularfunction was affected which is documented by hypoalbumin-emia and hypoglobulinemia with subsequent decrease in plas-ma total protein. Hypoalbuminemia in chronic infection in thisstudy occurs simultaneously with the increase in fibrocellularlarge granulomas with concentric fibrous bundles.Hypoalbuminemia might be also arisen from malabsorptiondue to damaged intestinal mucosa resulting from the extrusionof large numbers of eggs or due to decreased hepatic synthesis

which may result from parasitic injury (Oliveira et al. 2009).In the current study, treatment with Pps alone significantlybrought the reduced plasma albumin, globulin, and their ratioand consequently total protein concentration towards the con-trol values indicating an improvement in the hepatic metabolicprocesses. The efficacy of Pps in correcting the plasma pro-teins was found to be analogous to that produced by sylimarintreatment against CCl4 toxicity in rats (Bhadauria 2012).However, the failure of PZQ alone to restore the fall in plasmaproteins was effectively corrected by the combined Ppsadminstration.

Fig. 3 Histopathological changes in the liver of mice from different experimental groups (H & E ×200)

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Regarding plasma CP, its level was markedly elevated byinfection and progressively increased with all therapies.Generally, CP is one of the acute proteins which is widelyused for assessment of the inflammatory process. The state ofhosts inflammatory reactions develops in response to hepaticgranuloma triggered an acceleration of hepatic CP synthesiswith subsequent release to blood stream. Thus, elevated serumCP during chronic infection was an adaptive response todownregulate the inflammatory process by inactivating vari-ous mediators and inhibiting lipid peroxide formation throughoxidation of ferrous required for free radical generation(Kanner et al. 1988). Moreover, the further elevation of plas-ma CP in infected mice by Pps alone or in combination withPZQ might be beneficial to guard against the states of oxida-tive stress and inflammation observed in infected mice, sinceCP activity was considered as a protective mechanism againstoxygen radical and several inflammatory mediators.

In this study, the present data revealed a significant eleva-tion in immunoglobulins, IgG and IgM, after infection withS. mansoni. This result is in agreement with several studieswhich demonstrated a very intense humoral response with amassive production of anaphylactic antibodies including bothIgG and IgM which are increased significantly in the host inresponse to a challenge infection (Grzych et al. 1982; Capronet al. 1983). In addition, Correia et al. (2009) found an in-crease of serum IgG in patients with S. mansoni, such anincrease in immunoglobulins could be attributed to solubleS. mansoni egg-antigens or antigenic egg secretions whichinduced a marked production of IL-4, IL-5, and IL-13 accom-panied by an upregulation of immunoglobulins and

circulating eosinophils (Hams et al. 2013). Unfortunately,the elevated IgG and IgM levels were not significantlylowered by administration of either propolis or PZQ treat-ments or even their combination.

The results of the present study clearly indicate the devel-opment of a state of oxidative stress in plasma and examinedtissues of infected mice. This was demonstrated by a signifi-cant elevation in MDA and a significant reduction in reducedGSH. The highest increase of MDAwas observed in plasma,liver and, thymus confirming earlier work by Mainess andSenft (1981) and Shaheen et al. (1994). Lipid peroxidationincluding the formation of MDA requires the participation ofhighly reactive O•-

2 and other reactive oxygen metabolite inchain of biochemical reactions. In schistosomiasis, granulomamacrophages isolated from hepatic, intestinal, and other tissuelesions were found to release significant amounts of O•-

2 andHO• radicals (Feldman et al. 1990; Pascal et al. 2000).Another mechanism responsible for flooding the host tissueswith these reactive oxygen metabolites is the marked increasein host heme degradation with subsequent elevation of totalserum iron (Mainess and Senft 1981). Excessively releasediron has been recognized to be potentially toxic, since it iscapable of taking part in free radical mediated reactions(Halliwell 1987). Alternatively, the decreased GSH contentsin blood, liver, and thymus of infected mice observed here aswell as those reported about the decreased activities of blood,kidney, and spleen SOD (Shaheen et al. 1994), blood catalaseand glutathione peroxidase, and hepatic glutathione S-transferase (El-Mouelhi and Mansour 1990) in schistosomia-sis could result in increased exposure of the host tissues to

Fig. 4 Representative images of Masson’s trichrome staining by light microscope (×200)

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toxic reactive species. Excessive consumption of GSH inresponse to increased oxidative condition and/or the inhibitionof GSH-reductase (Gharib et al. 1999) could account for thesignificant reduction in blood and tissue GSH.

Treatment of infected mice with Pps succeeded in restoringtissue GSH and increasing its blood contents significantlytogether with a reversal of schistosome-induced elevation inplasma and tissues MDA levels. These beneficial effects ofPps might be attributed to its well-known antioxidant actions(Capucho et al. 2012) and its ability in suppressing the over-production of MDA and restoring the respiratory control inmitochondria (Alyane et al. 2008). On the other side, treat-ment with PZQ succeeded in restoring tissue GSH with con-comitant reduction of their MDA levels. These results mightbe attributed to the anti-schistosomal activity of PZQ and asubsequent reduction of ova deposition and granuloma num-ber. The greater improvement in redox status of the examinedtissues in group of combined therapy might be attributed to apowerful antioxidant action with an effective eradication ofparasite and greater reduction of size and number of hepaticgranuloma.

Tissue MPO activity, which is directly related to the num-ber and activity of myeloid cell infiltrate in inflamed tissue,was assayed to monitor the degree of inflammation (Bradleyet al. 1982). The present study revealed a significant increasein MPO activity in liver and spleen of infected mice withS. mansoni. Eosinophils form a prominent constituent of thegranuloma, and their marked infiltration is a cardinal featureof the Schistosoma granuloma (Hams et al. 2013).Administration of Pps or PZQ either alone or in combinationresulted in marked reduction in liver and thymus MPO. Theseresults are matched with the decreased lymphocyte infiltrationand aggregation observed in this study. In concomitant withenhanced state of oxidative stress and increased activity ofMPO, increased production of NOx was observed in infectedmice in the current study. Brunet et al. (1999) examined therole of NOx during S. mansoni infection, and they found anelevation of systemic NOx levels in IL-4−/−mice throughoutthe course of acute disease together with up-regulation ofiNOS mRNA in hepatic tissues of WT- and IL-4−/−-infectedmice at the onset of egg deposition. Therapeutic administra-tion of Pps was capable of reducing NOx in plasma, liver, andthymus approaching normal values. However, its combinationwith PZQ was highly effective in reducing NOx in differenttissues to a level even below the normal values. Inhibition ofiNOS activity and expression by Pps in carrageenin-inducedmouse paw edema was previously reported by Tan-No et al.(2006) which may account for this effect.

In conclusion, this study demonstrated an effective benefi-cial role for Pps on the liver schistosomal lesion as evidencedby reduction of hepatic granuloma number and their lympho-cytic infiltration and aggregation accompanied by restoringthe synthesis of plasma proteins and alleviating the state of

oxidative stress in most of examined tissues. Promising resultswere obtained on concurrent use of Pps with PZQ, where anti-schistosomal and curative actions of PZQ are markedly en-hanced with complete alleviation and/or amelioration of mostbiochemical and histological derangement associated with thedisease. Accordingly, these results highlight the potential use-fulness of Pps as an adjunct to PZQ therapy in schistosomia-sis. However, additional studies must be undertaken to eval-uate for the possible translation of these findings to the clinicalsetting.

Acknowledgments Authors are grateful to Prof. Dr. Gamal Sayed ElBaroty, Laboratory director of Faculty of Agriculture Research Park,Faculty of Agriculture, Cairo University, for the total phenol and flavo-noids estimation in the dried ethanolic extract of Pps in the present study.

Conflict of interest The authors had no conflicts of interest to declarein relation to this article.

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