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The hepatoprotective activity of blue green algae in Schistosoma mansoni infected mice
Accepted Manuscript The hepatoprotective activity of blue green algae in Schistosoma mansoni infected mice Azza H. Mohamed, Gamalat Y. Osman, Tarek A. Salem, Alshimaa M. Elmalawany PII: DOI: Reference:
S0014-4894(14)00164-7 http://dx.doi.org/10.1016/j.exppara.2014.06.016 YEXPR 6900
To appear in:
Experimental Parasitology
Received Date: Revised Date: Accepted Date:
11 September 2013 7 June 2014 23 June 2014
Please cite this article as: Mohamed, A.H., Osman, G.Y., Salem, T.A., Elmalawany, A.M., The hepatoprotective activity of blue green algae in Schistosoma mansoni infected mice, Experimental Parasitology (2014), doi: http:// dx.doi.org/10.1016/j.exppara.2014.06.016
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The hepatoprotective activity of blue green algae in Schistosoma mansoni infected mice Azza H. Mohamed1, Gamalat Y. Osman1, Tarek A. Salem2# and Alshimaa M. Elmalawany1* (1)
Zoology Department, Faculty of Science, Menufiya University and (2) Molecular
Biology Department, Genetic Engineering and Biotechnology Institute, Sadat City University, Egypt. (#Current address: College of medicine, Qassim University, KSA) *Corresponding author (Email: [email protected]; Tel: +201090788873; Fax: +20482235689) ABSTRACT This study aims to evaluate the immunomodulatory effects of a natural product, blue green algae (BGA) (100 mg/kg BW), alone or combined with praziquantel PZQ (250 mg/kg BW) on granulomatous inflammation, liver histopathology, some biochemical and immunological parameters in mice infected with Schistosoma mansoni. Results showed that the diameter and number of egg granuloma were significantly reduced after treatment of S. mansoni-infected mice with BGA, PZQ and their combination. The histopathological alterations observed in the liver of S. mansoni-infected mice were remarkably inhibited after BGA treatments. BGA decreased the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) as well as the level of total protein (TP) while the level of albumin was increased. Treatment of infected mice with BGA, PZQ as well as their combination led to significant elevation in the activities of hepatic antioxidant enzymes glutathione peroxidase (GPX) and glutathione-S-transferase (GST) as compared with control group. Combination of BGA and PZQ resulted in significant reduction in the level of intercellular adhesion molecules-1 (ICAM-1), vascular adhesion molecules-1 (VCAM-1) and tumor necrosis factor-alpha (TNF-α) when compared to those of the S. mansoni-infected group. Overall, BGA significantly inhibited the liver damage accompanied with schistosomiasis, exhibited a potent antioxidant and immunoprotective activities. This study suggests that BGA can be considered as promising for development a complementary and/or alternative medicine against schistosomiasis.
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Key words: Adhesion molecules, Antioxidant Enzymes, Blue green alga, Granuloma, Schistosoma mansoni, Tumor necrosis factor-Alpha.
1. INTRODUCTION Hepatic schistosomiasis is one of the most prevalent forms of chronic liver diseases in the world, often leading to the liver fibrosis (El-Lakkany et al., 2012 and Dias et al., 2013). The immunopathology of schistosomiasis is generally attributed to the granulomatous reaction around the tissue deposited eggs and is considered to be a T cell mediated immune response (Alves-Oliveria et al., 2006). Granuloma formation requires the recruitment of inflammatory cells from blood stream, via up-regulation of adhesion molecules on activated vascular endothelial cells, induced by cytokines and chemokines released at the site of inflammation (Pearce and McDonald, 2002). In addition, infection with Schistosoma mansoni alters enzymatic and non-enzymatic antioxidant status (de Oliveira et al., 2013). Schistosomiasis control represents the target of many research programs all over the world (Murray, 2005). The effect of praziquantel (PZQ) on schistosomes has been studied in many countries. Most of the studies made are on its efficacy in the treatment of S. mansoni and have reported different cure and egg excretion reduction rates (Mekonnen et al., 2013). Ismail et al. (1999) reported that PZQ does not prevent re-infection, is inactive against juvenile schistosomes and only has a limited effect on already developed liver and spleen lesions. Concern over the development of such resistance has also drawn the attention of many investigators to alternative drugs. Blue-green algae (BGA) have attracted attention as health beneficial foods and as source materials for drug development (Schaap et al., 2012). Aphanizomenon flosaquae (AFA) is a fresh water unicellular blue-green alga that spontaneously grows in Upper Klamath Lake (Germany) and that is consumed as a nutrient-dense food source and for its health-enhancing properties (Pugh et al., 2001). AFA is an important source of the blue photosynthetic pigment phycocyanin (PC), which has been described as a strong antioxidant (Bhat and Madyastha, 2001) and anti-inflammatory (Reddy et al., 2000). Also, Vadiraja et al. (1998) and Romay and Gonzalez, (2000) reported that the C-phycocyanin, a constituent of BGA, is shown to be hepatoprotective, antiarthritic, and most importantly anti-inflammatory in nature. 2
This study aims to evaluate the immunomodulatory effects of BGA alone or combined with PZQ on granulomatous inflammation, liver histopathology, some biochemical and immunological markers in mice infected with S. mansoni. Meanwhile, the role of the BGA in reducing the harmful side effects generated by PZQ treatment was assessed. 2. MATERIALS AND METHODS 2.1. Materials Praziquantel (PZQ) tablets (600 mg/tablet) were obtained from SEDICO Pharmaceutical Company (6th October City - Egypt). Blue green algae (BGA) tablet (250 mg) (Aphanizomenon flos aquae) was obtained from German Pharmaceutical Industries, (Life Blau-Green Alge, Hergestellt, Deutschland). Tablets of BGA or PZQ were ground and suspended in distilled water for oral administration by stainless steel bent feed needle (length metric 50.8 mm and gage 18) from A Harvard BioScience Company. 2. 2. Animals and study design Seventy adult male Swiss albino mice (25.00 ± 2.00g) were purchased from Schistosome Biological Supply Program (SBSP) unit at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Animals were quarantined and allowed to acclimate for a week prior to experimentation at the animal room of Zoology Department, Faculty of Science, Menufiya University. Animals were handled under standard laboratory conditions with a 12-h light/dark cycle at a temperature of 25 ± 2oC. They had free access to standard food and water; all experiments were done in compliance with the guide lines for the care and use of laboratory animals. Cercariae of Schistosoma mansoni Egyptian strain were obtained from infected Biomphalaria alexandrina snails purchased from the Schistosome Biological Supply Center at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Forty mice for infected groups were subcutaneously infected with (70.00 ± 5.00 cercariae/mouse) (Peters and Warren, 1969). Animals were randomly divided into seven groups, 10 mice each as follows: Group I (N): Non-infected control mice. Group II (BGA): Non-infected mice treated with BGA daily for 15 consecutive days. Group III (PZQ): Non-infected mice treated
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with PZQ daily for 3 consecutive days. Group IV (S. mansoni-infected): infected control mice. Group V (Infected+BGA): infected mice treated with BGA, for 15 consecutive days, 7th weeks post infection. Group VI (Infected+PZQ): infected mice treated with PZQ, for 3 consecutive days, 7th weeks post infection. Group VII (Infected+PZQ and BGA): infected mice treated with a combination of BGA for 15 consecutive days and PZQ for 3 consecutive days, 7th weeks post infection. All animals were sacrificed after the end of treatment (10th weeks post infection) by decapitation. For the groups receiving PZQ, it was administrated orally to mice in 3 doses each of 250 mg/kg/mouse for three consecutive days according to Utzinger et al. (2003). For the groups receiving BGA, it was administrated orally to mice in dose of 100 mg/kg/mouse for 15 consecutive days according to Kuriakose and Kurup (2010). 2. 3. Blood and tissue sampling At the end of experiment, all animals were sacrificed by decapitation and peripheral blood was collected and serum was separated by centrifugation at 3000 rpm for 5 minutes and kept at -20ºC until use. Livers were removed and rinsed with physiological saline. A measure of 0.5 grams of liver was weighed and mechanically homogenized by using electrical homogenizer (Potter-Elvehjem) in a 10-fold volume of ice-cold 20.00 mM tris-HCl buffer (pH 7.4). The homogenate was divided into aliquoted and kept at -70°C for biochemical studies, and small pieces of liver were separated immediately fixed in 10% formalin for histological detection. 2. 4. Liver function tests The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated according to the method of Reitman and Frankel (1957). The activity of serum alkaline phosphatase (ALP) was measured colorimetricaly according to the method of Kind and King (1954). Serum total protein (TP) content was determined by a colorimetric method using bovine serum albumin as standard as described by Jacobs et al. (1964). The level of serum albumin (Alb) was determined by using the method of Baure (1982). 2. 5. Assessment of hepatic GPX and GST activities The activity of hepatic glutathione peroxidase (GPX) was measured by spectrophotometrically assayed by using 1-chloro-2-4-dinitrobenzene (CDNB) and 4
glutathione as described by Habig et al. (1974). While, the activity of hepatic and glutathione-S-transferase (GST) was measured according to the method described by Paglia and Valentine (1967). 2. 6. Measurement of hepatic granuloma diameter, number and histopathological investigations For determination of granuloma diameter, small pieces of liver were separated and immediately fixed in 10% formalin and the tissues were impeded in paraffin wax at 60ºC (von Lichtenberg, 1962). Serial sections were cut at 5.00 µm and stained with Ehrlich´s Haematoxylin and Eosin (H&E). The slides were cleared in xylene and mounted in DPX for histological investigation. Micrographs and measurements were taken under a Carl Zeiss microscope with an automatic camera attachment. Granulomas that contained a single egg were selected for measurements. Granulomas were measured by a graduated lens and scale. The mean diameter of each granuloma in µm was calculated by measuring two diameters of the lesion at right angles to each other and the arithmetic mean of the two measurements was then calculated (Mahmoud and Warren, 1974). Liver egg granulomas were counted in five successive low-power fields (100x), and their diameters were measured using a graduated eyepiece, choosing only lobular granulomas containing central ova (Humason, 1972). 2. 7. Determination of serum ICAM-1, VCAM-1 and TNF-α The serum concentration of intracellular adhesion molecule-1(ICAM-1) was determined according to the method of Horie et al. (1997), and by using a commercial ELISA kit (Thermo-Scientific; Cat. No. 1407), the result was presented as ng/ml. The serum concentration of vascular adhesion molecule-1 (VCAM-1) was measured by using ELISA kit (Biosciences; Cat. No. 2107-2P) according to the method described by Gearing et al. (1992), the result was presented as ng/ml. Measurement of serum level of TNF-α was performed according to the method of Chan and Perlstein (1987) and by using a commercial ELISA kit (Thermo-Scientific; Cat. No. K1347), the result was presented as Pg/ml.
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2. 8. Statistical analysis The data were presented as mean ± SD. The significance of the difference between the means was compared using the Student's t- test (Sokal and Rohlf, 1981). The level of significance was accepted as P < 0.05.
3. RESULTS 3.1. Effect of BGA and PZQ on liver function Results illustrated significant increases (P < 0.05) in serum ALT, AST, ALP activities and TP level while a significant decrease (P < 0.05) in the level of serum albumin in S. mansoni-infected group when compared to those of normal group (Table 1). The activities of serum ALT, AST and ALP as well as TP level were significantly reduced (P < 0.05) after treatment of S. mansoni-infected mice with BGA, PZQ and their combination when compared with untreated S. mansoni-infected mice. In contrast, the level of serum albumin was significantly increased (P < 0.05) after treatment with BGA, PZQ and their combination when compared with the S. mansoni-infected mice. Table (1) 3. 2. Effect of BGA and PZQ on hepatic activities of GPX and GST As shown in table (2), GST and GPX activities were significantly decreased (P < 0.05) in S. mansoni-infected mice compared with those of the control group. Significant increases (P < 0.05) in the GST and GPX activities were observed after treatment of S. mansoni-infected mice with BGA, PZQ and their combination as compared with those of untreated S. mansoni-infected mice. Results also indicated that the treatment of S. mansoni-infected mice with combination of BGA and PZQ caused a significant (P < 0.05) reduction in hepatic GST and GPX activities as compared to those treated with PZQ only. In addition, treatment of normal mice with BGA led to a significant increase in the GST and GPX activities (P < 0.05) as compared with untreated normal control group.
Table (2)
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3. 3. Effect of BGA and PZQ treatment on the liver histology of Schistosoma mansoni infected mice. The effect of BGA and PZQ on the diameter of hepatic granuloma is shown in table (3). The results indicated a significant decrease in the mean diameter of hepatic granuloma (P < 0.05) of infected mice treated with BGA (324.0 ± 16.0 µm diameter), PZQ (294.0 ± 5.0 µm diameter) and their combination (240.0 ± 6.6 µm diameter) as compared to those of S. mansoni-infected mice (405.0 ± 11.0 µm diameter). The histopathological examination showed a significant reduction (P < 0.05) in the mean number of portal egg granuloma of S. mansoni-infected mice treated with BGA (40.8 ± 3.7), PZQ (37.9 ± 2.1) and their combination (23.8 ± 2.4) as compared to those of the infected control group (44.7 ± 3.7). Overall, the results indicated that the treatment of S. mansoni-infected mice with combination of BGA and PZQ caused a significant reduction (P < 0.05) in the diameter and number of hepatic granuloma as compared to those of S. mansoni-infected mice treated with PZQ alone. Table (3) As shown in figure (1), liver sections of infected mice showed a greater number of large egg granulomas. The granuloma is formed of an ovum surrounded by large number of leukocytic inflammatory cells and fibrocytic cells. The pathological alterations observed in the liver of S. mansoni-infected mice were remarkably inhibited after treatment with BGA, PZQ and their combination. There is a significant reduction in the diameter of granuloma after treatment with BGA, PZQ and their combination as compared with those of the S. mansoni-infected control group (Figure 1 c,d,e). A remarkable amelioration was observed in liver after treatment of S. mansoni-infected mice with the combination of BGA and PZQ as compared to that of S. mansoni-infected mice with PZQ alone. Figure 1
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3. 4. Effect of BGA and PZQ on serum levels of ICAM-1 and VCAM-1 in S. mansoni-infected mice Results showed that the infection of mice with S. mansoni caused significant increases in the serum levels of ICAM-1 and VCAM-1 (P < 0.05) as compared with the uninfected normal control group (Figure 2). On the other hand, the treatment of S. mansoni-infected mice with BGA, PZQ and their combination resulted in a significant reduction (P < 0.05) in the serum levels of ICAM-1 and VCAM-1 as compared with those of S. mansoni-infected mice. Meanwhile, the serum levels of ICAM-1 and VCAM-1 were significantly decreased (P < 0.05) in S. mansoni-infected mice after treatment with combination of BGA and PZQ when compared to those of S. mansoniinfected mice treated with PZQ alone.
Figure 2
3. 5. Serum level of TNF-α As shown in figure (3), the infection of mice with S. mansoni caused a significant increase (P < 0.05) in the level of TNF-α (409.0 ± 1.7 Pg/ml) as compared to that of the normal control group (324.8 ± 0.6 Pg/ml). On the other hand, treatment of S. mansoni infected mice with BGA, PZQ and their combination resulted in significant decreases (P < 0.05) in the level of TNF-α (350 ± 0.7, 364.0 ± 3.4 and 338.5 ± 1.2 Pg/ml, respectively) as compared with that of S. mansoni-infected group. Meanwhile, the level of TNF-α was significantly decreased (P < 0.05) in serum of S. mansoni-infected mice after treatment with combination of BGA and PZQ when compared to those of S. mansoni-infected mice treated with PZQ alone.
Figure 3
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4. DISCUSSION The current study indicated that S. mansoni infection resulted in alteration in the secretory and synthetic functions of liver. Whereas, the activities of AST, ALT and ALP as well as the level of TP were elevated, the level of serum albumin was significantly reduced in mice infected with S. mansoni when compared to those of the normal control group. The treatments of S. mansoni-infected mice with BGA, PZQ or a combination of BGA with PZQ lead to a significant improvement in the liver function parameters. These findings are in agreement with the results of El-Lakkany et al. (2011a, b) and Abdel-Hafeez et al. (2012) who reported that treatment of mice infected with S. mansoni with PZQ led to amelioration in liver function. Previous studies of Kuriakose and Kurup (2008 and 2010) indicated that BGA exhibited a hepatoprotective effect against experimental drug toxicity. Also, Xin et al. (2007) reported that C-phycocyanin, which is found in blue green algae, has a hepatoprotective effect in experimental hepatitis. The results also illustrated that infection of mice with S. mansoni produced a remarkable oxidative stress of hepatic GPX and GST activities were significantly reduced as compared to those of the normal mice. These findings were previously reported in mouse and human schistosomiasis by Elmalawany (2009), El-Lakkany et al. (2011a), Mantawy et al. (2011) and Abdel-Hafeez et al. (2012). The treatment of S. mansoni-infected mice with BGA, PZQ or their combination led to significant increases in the activities of hepatic GPX and GST as compared to those of the S. mansoni-infected control group. As compared to PZQ treated mice, treatment with a combination of PZQ and BGA resulted in a significant increase in the activities of GPX and GST. These findings indicate the ability of BGA in triggering the antioxidant potential activity against the generation of reactive oxygen species (ROS) accompanied with schistosomiasis. The antioxidant capacity of BGA is attributed to the presence of potent antioxidants constituents such as vitamin A, vitamin E, vitamin C and C-phycocyanin (Scoglio et al., 2009). This assumption is supported by the results of El-Lakkany et al. (2011a) reported that treatment of S. mansoni-infected mice with β-carotene and/or PZQ produced a pronounced elevation in GPX activity. Kuriakose and Kurup (2010) showed that the treatment of paracetamol-intoxicated rats with BGA resulted in a significant elevation in the GPX activity when compared with control group. 9
The results showed that the diameter and number of egg granuloma were significantly reduced (P < 0.05) after treatment of S. mansoni-infected mice with BGA, PZQ and a combination of BGA with PZQ as compared to that of S. mansoniinfected group. Massoud et al. (2004) showed a significant reduction in the number and size of granulomas after the treatment of S. mansoni-infected mice with PZQ. Similar studies reported that the treatment of S. mansoni-infected mice with either PZQ alone or combined with pentoxifylline (El-Lakkany et al., 2011b) α-lipoic acid (Abdel-Hafeez et al., 2012) Nigella sativa oil (Mahmoud et al., 2002) and N-acetyl-Lcysteine (De-Lima et al., 2012) resulted in a significant decrease in the size and number of liver granuloma. The histopathological examination of livers from S. mansoni infected mice showed many lesions of granulomatous inflammation which developed around eggs and subsequent fibrosis. These pathological alterations were remarkably modulated after treatment of mice with BGA, PZQ and their combination. Botros et al. (2010) indicated that orally administration of a combination of PZQ with artemether inhibited the histopathological manifestations in the liver of S. mansoni-infected mice. Also, Toussom et al. (2012) showed that treatment of S. mansoni-infected mice with either silymarin alone or in combination with PZQ led to a significant reduction in granuloma and decreased histopathological lesions. In addition, Kuriakose and Kurup (2010) showed that BGA improved the histopathological texture of liver in a paracetamol toxicity model. Significant increases (P < 0.05) in the serum levels of TNF-α, ICAM-1 and VCAM-1 in S. mansoni-infected mice were observed as compared to that of the normal mice. These results are in agreement with previous studies of Mantawy et al. (2011), Rathore et al. (1996), el-Ahl et al. (2000), Loebermann et al. (2009) and Azevedo et al. (2012). Treatment of S. mansoni-infected mice with BGA, PZQ and a combination of BGA with PZQ resulted in significant reductions in the serum level of TNF-α, ICAM1 and VCAM-1when compared to those of the infected mice. In contrast to PZQ treated mice, treatment of infected mice with a combination of PZQ and BGA resulted in significant reduction in the serum levels of TNF-α, ICAM-1 and VCAM-1. Previous studies reported significant reductions in the serum level of TNF-α in S. mansoni infected mice after treatment with either PZQ alone (Yu et al., 2012) or a 10
combination of PZQ and pentoxifylline (El-Lakkany et al., 2011a,b). On the other hand, Yakoot and Salem (2012) indicated that the treatment of chronic hepatitis C patient with 500mg of BGA for 3 months resulted in a significant decrease in serum level of TNF-α. It was revealed that PZQ caused a significant reduction in the serum level of ICAM-1 (Esterre et al., 1998). Murphy et al. (2005) indicated that the serum level of ICAM-1 and VCAM-1 was significantly decreased after treatment of atherosclerotic mice with vitamin E, vitamin C and β-carotene for 15 weeks. Li et al. (2006) and Yamagata et al. (2012) revealed the inhibitory action of β-carotene and Cphycocyanin on the level of ICAM-1 and VCAM-1. Taken together with our previous results (Elmalawany, 2012); we can conclude that BGA exhibited antischistosomal activity where the treatment with BGA resulted in a significant reduction in worm burden and the ova count/gm liver or intestine (data are not mentioned here). BGA also improves the antischistosomal efficacy of PZQ by scavenging the ROS associated with schistosomiasis. In addition, it exhibited a hepatoprotective activity. 5. ACKNOWLEDGMENTS To Dr., Claire J Standley (Ecology and Evolutionary Biology, Princeton University, Princeton, NJ, USA), for her critical reading, helpful discussions and suggestions during the preparation of this manuscript.
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Mantawy, M.M., Ali, H.F., Rizk, M.Z., 2011. Therapeutic effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection. Rev. Inst. Med. Trop. Sao. Paulo. 53,155-163. Massoud, A.M., El-Ebiary, F.H., Abou-Gamra, M.M., Mohamed, G.F., Shaker, S.M., 2004. Evaluation of schistosomicidal activity of myrrh extract: parasitological and histological study. J. Egy. Soc. Parasitol. 34, 1051-1076. Mekonnen,
A., Legesse,
M., Belay,
M., Tadesse,
K., Torben,
W., Zelalem,
Teklemariam , Z., Erko, B., 2013. Efficacy of praziquantel against Schistosoma haematobiumin dulshatalo village, western Ethiopia. BMC Res. Notes, 6:392. Murphy, N., Grimsditch, D.C., Vidgeon-Hart, M., Groot, P.H., Overend, P., Benson G.M., Graham, A., 2005. Dietary antioxidants decrease serum soluble adhesion molecule (sVCAM-1, sICAM-1) but not chemokine (JE/MCP-1, KC) concentrations, and reduce atherosclerosis in C57BL but not apoE Leiden mice fed an atherogenic diet. Dis. Markers, 21, 181-190 Murray, P.R., 2005. Immune responses to infectious agents: Specific Immune Response to Parasites. In: Murray, P.R.; Rosenthal, F.S. and Pfaller, M.A. (eds.). Medical Microbiology, (5th Ed). El-Seveir mosby.1600 Jhon F. Kennedy Boulevard, Suite 1800 Philadelphia, Pennsylvenia. P. 151. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158-169. Pearce, E.J., MacDonald, A.S., 2002. The immunobiology of schistosomiasis. Nat. Rev. Immunol. 2, 499-511. Peters, P.A., Warren, K.S., 1969. A rapid method of infecting mice and other laboratory animals with Schistosoma mansoni: subcutaneous injection. J. Parasitol. 55, 558. Pugh, N., Pasco, D.S., 2001. Characterization of human monocyte activation by a water soluble preparation of Aphanizomenon flos-aquae. Phytomed., 8, 445-53. Rathore, A., Sacristán, C., Ricklan, D.E., Flores Villanueva, P.O., Stadecker, M.J., 1996. In situ analysis of B7-2 costimulatory, major histocompatibility complex class II, and adhesion molecule expression in schistosomal egg granulomas. 15
Am. J. Pathol. 149, 187-194 Reddy, C.M., Bhat, V.B., Kiranmai, G., Reddy M.N., Reddanna, P., Madyastha, K.M., 2000. Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina plantesis. Biochem. Biophys. Res. Commun. 277, 599-603. Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Path. 28, 56-63. Romay, C., Gonzalez, R., 2000. Phycocyanin is an antioxidant protector of human erythrocytes against lysis by peroxyl radicals. J. Pharm. Pharmacol. 52, 367368. Schaap, A., Rohrlack, T., Bellouard, Y., 2012. Optical classification of algae species with a glass lab-on-a-chip. Lab. Chip. 12, 1527- 1532. Scoglio, S., Benedetti, S., Canino, C., Santagni, S., Rattighieri, E., Chierchia, E., Canestrari, F., Genazzani, A.D., 2009. Effect of a 2-month treatment with Klamin, a Klamath algae extract, on the general well-being, antioxidant profile and oxidative status of postmenopausal women. Gynecol. Endocrinol. 25, 235240. Sokal, R. R. and Rohlf, F. J. (1981): Biometry, 2nd Ed, W. H. Freeman & Co., San Francisco, 241- 242. Tousson, E., Beltagy, D.M., Gazia, M.A., Al-Behbehani, B., 2012. Expressions of P53 and CD68 in mouse liver with Schistosoma mansoni infection and the protective role of silymarin. Toxicol. Ind. Health, 29, 761-770. Utzinger, J., Keiser, J., Shuhua, X., Tanner, M., Singer, B.H., 2003. Combination chemotherapy of schistosomiasis in laboratory studies and clinical trials. Antimicrob. Agents Chemother. 47, 1487-1495. Vadiraja, B.B., Gaikwad, N.W., Madyastha, K.M., 1998. Hepatoprotective effect of C-phycocyanin protective for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicity in rats. Biochem. Biophys. Res. Commun. 249, 428-431. von Lichtenberg, F.C., 1962. Host response to eggs of Schistsoma mansoni. I. Granuloma formation in un-sensitized laboratory mouse. Am. J. Pathol. 41, 71116
731. Xin, Y.F., Zhou, G.L., Shen, M., Chen, Y.X., Liu, S.P., Chen, G.C., Chen, H., You, Z.Q., Xuan Y.X., 2007. Angelica sinensis: A novel adjunct to prevent doxorubicin induced chronic cardiotoxicity. Basic Clin. Pharmacol. Toxicol. 101, 421-426. Yakoot, M., Salem, A., 2012. Spirulina platensis versus silymarin in the treatment of chronic hepatitis C virus infection. A pilot randomized, comparative clinical trial. BMC Gastroenterol. 12, 32. Yamagata, K., Tanaka, N., Matsufuji, H., Chino, M., 2012. Beta-carotene reverses the IL-1β-mediated reduction in paraoxonase-1 expression via induction of the CaMKKII pathway in human endothelial cells. Microvasc. Res. 84, 297-305. Yu, L., Sun, X., Yang, F., Yang, J., Shen, J., Wu Z., 2012. Inflammatory cytokines IFN-gamma, IL-4, IL-13 and TNF-alpha alterations in schistosomiasis: A metaanalysis. Parasitol. Res. 110, 1547-1552.
17
G.
C.V.
A
B
C.V.
G.
G. G.
C
D
E
Figure 1: liver section stained with (H&E x100). (A) Showing normal hepatic architecture, central vein (CV). (B) Section of liver tissue of S. mansoni-infected mice ten weeks post infection showing Schistosoma granuloma formed of leukocytic inflammatory cells and fibrocytic cells surrounding Schistosoma-ova. G. Granuloma. (C) Liver section of BGA treated mouse (100mg/kg orally for 15 consecutive days) showing fibrocellular granuloma of intermediate size. G. Granuloma. Central vein (CV). (D) Liver section of PZQ treated mouse (250 mg/ kg o r a l l y for 3 consecutive days 7th weeks post infection) showing fibrocellular granuloma. G. Granuloma. (E) Liver section of infected mice treated with BGA+PZQ showing small size late fibrocellular granuloma, in addition, decreased cellular constituents, degenerative changes in the ova and the pigments were minimal. G. Granuloma.
200 $
180 160
*
*
*,♣
140 120
ng/ml
100 80 60
$
40
*
*
*,♣
20 0 N
BGA (100mg/kg)
PZQ (250mg/kg)
ICAM-1
S. mansoniinfected
Infected + BGA (100mg/kg)
Infected + PZQ
Infected + BGA (100mg/kg) and PZQ (250mg/kg)
VCAM-1
Figure 2: Effect of S. mansoni infection and treatment with PZQ, BGA and their combination on serum levels of ICAM-1 and VCAM-1 in mice Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
$
450 400
*
*
TNF-ALPHA(Pg\ml)
350
*,♣
300 250 200 150 100 50 0 N
BGA (100mg/kg)
PZQ (250mg/kg)
S. mansoniinfected
Infected + BGA (100mg/kg)
Infected + PZQ
Infected + BGA (100mg/kg) and PZQ (250mg/kg)
Figure 4: Effect of S. mansoni infection and treatment with PZQ, BGA and their combination on serum levels of TNF-α in mice Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
Table (1): Effect of S. mansoni infection and treatment with PZQ, BGA and in combination on liver function in mice
Groups
ALT (U/L)
AST (U/L)
ALP (U/L)
TP (g/dl)
Alb (g/dl)
Normal control
29.7 ± 3.7
72.5 ± 4.6
29.2 ± 1.9
5.3 ± 0.3
3.4 ± 0.1
Normal + BGA
29.7 ± 3.3
73.1 ± 5.4
29.9 ± 2.2
5.6 ± 0.2
Normal + PZQ
31.2 ± 1.9
73.7 ± 3.3
30.2 ± 2.7
5.8 ± 0.2
3.3 ± 0.2
Infected control
172.0 ± 9.7$
239.5 ± 12.3$
92.2 ± 2.3$
7.3 ± 0.3$
2.1 ± 0.2$
Infected + BGA
62.2 ± 4.4*
131.7 ± 5.6*
38.9 ± 2.0*
5.8 ± 0.2*
3.2 ± 0.2*
Infected + PZQ
92.3 ± 7.9*
156.2 ± 6.4*
42.7 ± 1.7*
6.1 ± 0.2*
2.9 ± 0.2*
Infected +BGA /PZQ
40.3 ± 2.8*,♣
106.7 ± 5.5*,♣
32.1 ± 1.6*,♣
5.3 ± 0.1*
3.3 ± 0.2*
3.4 ± 0.2
Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
Table (2): Effect of S. mansoni infection and treatment with PZQ, BGA and in combination on hepatic GPX and GST activities in mice GST (µmol/min/g wt tissue)
GPX (U/L)
Normal control
0.56 ± 0.03
42.9 ± 1.1
Normal + BGA
0.86 ± 0.02$
56.0 ± 0.7$
Normal + PZQ
0.43 ± 0.01
36.7 ± 1.9
Infected control
0.18 ± 0.01$
29.9 ± 1.4$
Infected + BGA
0.68 ± 0.04*
50.0 ±1.0*
Infected + PZQ
0.33 ± 0.02*
35.1 ± 1.2*
0.76 ± 0.01*,♣
53.1 ± 1.2*,♣
Groups
Infected + BGA/PZQ
Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
Table (3): Changes in mean granuloma count and diameter in liver sections from mice treated treatment with PZQ, BGA and in combination, 7 weeks postinfection. Number of granulomas in 5 successive power fields (X100)
Mean granuloma diameter in ߤm
Infected control
44.7 ± 3.7
405 ± 11
Infected + BGA
40.8 ± 3.7
324 ± 16*
Infected + PZQ
37.9± 2.1*
Infected + BGA/PZQ
23.8± 2.4*,
Groups
♣
294 ± 5* ♣
240 ± 6.6*,
Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
hepatoprotective efficiency of blue green algae (BGA) (Aphanizomenon flos-aquae) (100 mg/kg) alone or/and in combination with praziquantel (PZQ) (250 mg/kg) on mice infected with Schistosoma mansoni
BGA 100 mg/kg Seven weeks post infection be treated the groups of mice with
PZQ 250 mg/kg BGA 100 mg/kg +PZQ 250 mg/kg
Results
Diameter and number of granuloma were significantly reduced after treatment
Elevation in hepatic glutathione-S-transferase and glutathione peroxidase activities
Reduction of the elevated levels of AST, ALT and ALP as well as TP
Reduction of the elevated levels of TNF-α, ICAM-1 and VCAM-1
Highlights - The effect of blue green algae (BGA) on S. mansoni infected mice was studied. - BGA ameliorated the biochemical function of liver of S. mansoni infected mice. - BGA improved the antioxidant status of S. mansoni infected mice. - BGA reduced the elevation of TNF-α, ICAM-1 and VCAM-1. - BGA reduced the harmful side effect of antischistosomal drug Praziquantel.
S0014-4894(14)00164-7 http://dx.doi.org/10.1016/j.exppara.2014.06.016 YEXPR 6900
To appear in:
Experimental Parasitology
Received Date: Revised Date: Accepted Date:
11 September 2013 7 June 2014 23 June 2014
Please cite this article as: Mohamed, A.H., Osman, G.Y., Salem, T.A., Elmalawany, A.M., The hepatoprotective activity of blue green algae in Schistosoma mansoni infected mice, Experimental Parasitology (2014), doi: http:// dx.doi.org/10.1016/j.exppara.2014.06.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The hepatoprotective activity of blue green algae in Schistosoma mansoni infected mice Azza H. Mohamed1, Gamalat Y. Osman1, Tarek A. Salem2# and Alshimaa M. Elmalawany1* (1)
Zoology Department, Faculty of Science, Menufiya University and (2) Molecular
Biology Department, Genetic Engineering and Biotechnology Institute, Sadat City University, Egypt. (#Current address: College of medicine, Qassim University, KSA) *Corresponding author (Email: [email protected]; Tel: +201090788873; Fax: +20482235689) ABSTRACT This study aims to evaluate the immunomodulatory effects of a natural product, blue green algae (BGA) (100 mg/kg BW), alone or combined with praziquantel PZQ (250 mg/kg BW) on granulomatous inflammation, liver histopathology, some biochemical and immunological parameters in mice infected with Schistosoma mansoni. Results showed that the diameter and number of egg granuloma were significantly reduced after treatment of S. mansoni-infected mice with BGA, PZQ and their combination. The histopathological alterations observed in the liver of S. mansoni-infected mice were remarkably inhibited after BGA treatments. BGA decreased the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) as well as the level of total protein (TP) while the level of albumin was increased. Treatment of infected mice with BGA, PZQ as well as their combination led to significant elevation in the activities of hepatic antioxidant enzymes glutathione peroxidase (GPX) and glutathione-S-transferase (GST) as compared with control group. Combination of BGA and PZQ resulted in significant reduction in the level of intercellular adhesion molecules-1 (ICAM-1), vascular adhesion molecules-1 (VCAM-1) and tumor necrosis factor-alpha (TNF-α) when compared to those of the S. mansoni-infected group. Overall, BGA significantly inhibited the liver damage accompanied with schistosomiasis, exhibited a potent antioxidant and immunoprotective activities. This study suggests that BGA can be considered as promising for development a complementary and/or alternative medicine against schistosomiasis.
1
Key words: Adhesion molecules, Antioxidant Enzymes, Blue green alga, Granuloma, Schistosoma mansoni, Tumor necrosis factor-Alpha.
1. INTRODUCTION Hepatic schistosomiasis is one of the most prevalent forms of chronic liver diseases in the world, often leading to the liver fibrosis (El-Lakkany et al., 2012 and Dias et al., 2013). The immunopathology of schistosomiasis is generally attributed to the granulomatous reaction around the tissue deposited eggs and is considered to be a T cell mediated immune response (Alves-Oliveria et al., 2006). Granuloma formation requires the recruitment of inflammatory cells from blood stream, via up-regulation of adhesion molecules on activated vascular endothelial cells, induced by cytokines and chemokines released at the site of inflammation (Pearce and McDonald, 2002). In addition, infection with Schistosoma mansoni alters enzymatic and non-enzymatic antioxidant status (de Oliveira et al., 2013). Schistosomiasis control represents the target of many research programs all over the world (Murray, 2005). The effect of praziquantel (PZQ) on schistosomes has been studied in many countries. Most of the studies made are on its efficacy in the treatment of S. mansoni and have reported different cure and egg excretion reduction rates (Mekonnen et al., 2013). Ismail et al. (1999) reported that PZQ does not prevent re-infection, is inactive against juvenile schistosomes and only has a limited effect on already developed liver and spleen lesions. Concern over the development of such resistance has also drawn the attention of many investigators to alternative drugs. Blue-green algae (BGA) have attracted attention as health beneficial foods and as source materials for drug development (Schaap et al., 2012). Aphanizomenon flosaquae (AFA) is a fresh water unicellular blue-green alga that spontaneously grows in Upper Klamath Lake (Germany) and that is consumed as a nutrient-dense food source and for its health-enhancing properties (Pugh et al., 2001). AFA is an important source of the blue photosynthetic pigment phycocyanin (PC), which has been described as a strong antioxidant (Bhat and Madyastha, 2001) and anti-inflammatory (Reddy et al., 2000). Also, Vadiraja et al. (1998) and Romay and Gonzalez, (2000) reported that the C-phycocyanin, a constituent of BGA, is shown to be hepatoprotective, antiarthritic, and most importantly anti-inflammatory in nature. 2
This study aims to evaluate the immunomodulatory effects of BGA alone or combined with PZQ on granulomatous inflammation, liver histopathology, some biochemical and immunological markers in mice infected with S. mansoni. Meanwhile, the role of the BGA in reducing the harmful side effects generated by PZQ treatment was assessed. 2. MATERIALS AND METHODS 2.1. Materials Praziquantel (PZQ) tablets (600 mg/tablet) were obtained from SEDICO Pharmaceutical Company (6th October City - Egypt). Blue green algae (BGA) tablet (250 mg) (Aphanizomenon flos aquae) was obtained from German Pharmaceutical Industries, (Life Blau-Green Alge, Hergestellt, Deutschland). Tablets of BGA or PZQ were ground and suspended in distilled water for oral administration by stainless steel bent feed needle (length metric 50.8 mm and gage 18) from A Harvard BioScience Company. 2. 2. Animals and study design Seventy adult male Swiss albino mice (25.00 ± 2.00g) were purchased from Schistosome Biological Supply Program (SBSP) unit at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Animals were quarantined and allowed to acclimate for a week prior to experimentation at the animal room of Zoology Department, Faculty of Science, Menufiya University. Animals were handled under standard laboratory conditions with a 12-h light/dark cycle at a temperature of 25 ± 2oC. They had free access to standard food and water; all experiments were done in compliance with the guide lines for the care and use of laboratory animals. Cercariae of Schistosoma mansoni Egyptian strain were obtained from infected Biomphalaria alexandrina snails purchased from the Schistosome Biological Supply Center at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Forty mice for infected groups were subcutaneously infected with (70.00 ± 5.00 cercariae/mouse) (Peters and Warren, 1969). Animals were randomly divided into seven groups, 10 mice each as follows: Group I (N): Non-infected control mice. Group II (BGA): Non-infected mice treated with BGA daily for 15 consecutive days. Group III (PZQ): Non-infected mice treated
3
with PZQ daily for 3 consecutive days. Group IV (S. mansoni-infected): infected control mice. Group V (Infected+BGA): infected mice treated with BGA, for 15 consecutive days, 7th weeks post infection. Group VI (Infected+PZQ): infected mice treated with PZQ, for 3 consecutive days, 7th weeks post infection. Group VII (Infected+PZQ and BGA): infected mice treated with a combination of BGA for 15 consecutive days and PZQ for 3 consecutive days, 7th weeks post infection. All animals were sacrificed after the end of treatment (10th weeks post infection) by decapitation. For the groups receiving PZQ, it was administrated orally to mice in 3 doses each of 250 mg/kg/mouse for three consecutive days according to Utzinger et al. (2003). For the groups receiving BGA, it was administrated orally to mice in dose of 100 mg/kg/mouse for 15 consecutive days according to Kuriakose and Kurup (2010). 2. 3. Blood and tissue sampling At the end of experiment, all animals were sacrificed by decapitation and peripheral blood was collected and serum was separated by centrifugation at 3000 rpm for 5 minutes and kept at -20ºC until use. Livers were removed and rinsed with physiological saline. A measure of 0.5 grams of liver was weighed and mechanically homogenized by using electrical homogenizer (Potter-Elvehjem) in a 10-fold volume of ice-cold 20.00 mM tris-HCl buffer (pH 7.4). The homogenate was divided into aliquoted and kept at -70°C for biochemical studies, and small pieces of liver were separated immediately fixed in 10% formalin for histological detection. 2. 4. Liver function tests The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated according to the method of Reitman and Frankel (1957). The activity of serum alkaline phosphatase (ALP) was measured colorimetricaly according to the method of Kind and King (1954). Serum total protein (TP) content was determined by a colorimetric method using bovine serum albumin as standard as described by Jacobs et al. (1964). The level of serum albumin (Alb) was determined by using the method of Baure (1982). 2. 5. Assessment of hepatic GPX and GST activities The activity of hepatic glutathione peroxidase (GPX) was measured by spectrophotometrically assayed by using 1-chloro-2-4-dinitrobenzene (CDNB) and 4
glutathione as described by Habig et al. (1974). While, the activity of hepatic and glutathione-S-transferase (GST) was measured according to the method described by Paglia and Valentine (1967). 2. 6. Measurement of hepatic granuloma diameter, number and histopathological investigations For determination of granuloma diameter, small pieces of liver were separated and immediately fixed in 10% formalin and the tissues were impeded in paraffin wax at 60ºC (von Lichtenberg, 1962). Serial sections were cut at 5.00 µm and stained with Ehrlich´s Haematoxylin and Eosin (H&E). The slides were cleared in xylene and mounted in DPX for histological investigation. Micrographs and measurements were taken under a Carl Zeiss microscope with an automatic camera attachment. Granulomas that contained a single egg were selected for measurements. Granulomas were measured by a graduated lens and scale. The mean diameter of each granuloma in µm was calculated by measuring two diameters of the lesion at right angles to each other and the arithmetic mean of the two measurements was then calculated (Mahmoud and Warren, 1974). Liver egg granulomas were counted in five successive low-power fields (100x), and their diameters were measured using a graduated eyepiece, choosing only lobular granulomas containing central ova (Humason, 1972). 2. 7. Determination of serum ICAM-1, VCAM-1 and TNF-α The serum concentration of intracellular adhesion molecule-1(ICAM-1) was determined according to the method of Horie et al. (1997), and by using a commercial ELISA kit (Thermo-Scientific; Cat. No. 1407), the result was presented as ng/ml. The serum concentration of vascular adhesion molecule-1 (VCAM-1) was measured by using ELISA kit (Biosciences; Cat. No. 2107-2P) according to the method described by Gearing et al. (1992), the result was presented as ng/ml. Measurement of serum level of TNF-α was performed according to the method of Chan and Perlstein (1987) and by using a commercial ELISA kit (Thermo-Scientific; Cat. No. K1347), the result was presented as Pg/ml.
5
2. 8. Statistical analysis The data were presented as mean ± SD. The significance of the difference between the means was compared using the Student's t- test (Sokal and Rohlf, 1981). The level of significance was accepted as P < 0.05.
3. RESULTS 3.1. Effect of BGA and PZQ on liver function Results illustrated significant increases (P < 0.05) in serum ALT, AST, ALP activities and TP level while a significant decrease (P < 0.05) in the level of serum albumin in S. mansoni-infected group when compared to those of normal group (Table 1). The activities of serum ALT, AST and ALP as well as TP level were significantly reduced (P < 0.05) after treatment of S. mansoni-infected mice with BGA, PZQ and their combination when compared with untreated S. mansoni-infected mice. In contrast, the level of serum albumin was significantly increased (P < 0.05) after treatment with BGA, PZQ and their combination when compared with the S. mansoni-infected mice. Table (1) 3. 2. Effect of BGA and PZQ on hepatic activities of GPX and GST As shown in table (2), GST and GPX activities were significantly decreased (P < 0.05) in S. mansoni-infected mice compared with those of the control group. Significant increases (P < 0.05) in the GST and GPX activities were observed after treatment of S. mansoni-infected mice with BGA, PZQ and their combination as compared with those of untreated S. mansoni-infected mice. Results also indicated that the treatment of S. mansoni-infected mice with combination of BGA and PZQ caused a significant (P < 0.05) reduction in hepatic GST and GPX activities as compared to those treated with PZQ only. In addition, treatment of normal mice with BGA led to a significant increase in the GST and GPX activities (P < 0.05) as compared with untreated normal control group.
Table (2)
6
3. 3. Effect of BGA and PZQ treatment on the liver histology of Schistosoma mansoni infected mice. The effect of BGA and PZQ on the diameter of hepatic granuloma is shown in table (3). The results indicated a significant decrease in the mean diameter of hepatic granuloma (P < 0.05) of infected mice treated with BGA (324.0 ± 16.0 µm diameter), PZQ (294.0 ± 5.0 µm diameter) and their combination (240.0 ± 6.6 µm diameter) as compared to those of S. mansoni-infected mice (405.0 ± 11.0 µm diameter). The histopathological examination showed a significant reduction (P < 0.05) in the mean number of portal egg granuloma of S. mansoni-infected mice treated with BGA (40.8 ± 3.7), PZQ (37.9 ± 2.1) and their combination (23.8 ± 2.4) as compared to those of the infected control group (44.7 ± 3.7). Overall, the results indicated that the treatment of S. mansoni-infected mice with combination of BGA and PZQ caused a significant reduction (P < 0.05) in the diameter and number of hepatic granuloma as compared to those of S. mansoni-infected mice treated with PZQ alone. Table (3) As shown in figure (1), liver sections of infected mice showed a greater number of large egg granulomas. The granuloma is formed of an ovum surrounded by large number of leukocytic inflammatory cells and fibrocytic cells. The pathological alterations observed in the liver of S. mansoni-infected mice were remarkably inhibited after treatment with BGA, PZQ and their combination. There is a significant reduction in the diameter of granuloma after treatment with BGA, PZQ and their combination as compared with those of the S. mansoni-infected control group (Figure 1 c,d,e). A remarkable amelioration was observed in liver after treatment of S. mansoni-infected mice with the combination of BGA and PZQ as compared to that of S. mansoni-infected mice with PZQ alone. Figure 1
7
3. 4. Effect of BGA and PZQ on serum levels of ICAM-1 and VCAM-1 in S. mansoni-infected mice Results showed that the infection of mice with S. mansoni caused significant increases in the serum levels of ICAM-1 and VCAM-1 (P < 0.05) as compared with the uninfected normal control group (Figure 2). On the other hand, the treatment of S. mansoni-infected mice with BGA, PZQ and their combination resulted in a significant reduction (P < 0.05) in the serum levels of ICAM-1 and VCAM-1 as compared with those of S. mansoni-infected mice. Meanwhile, the serum levels of ICAM-1 and VCAM-1 were significantly decreased (P < 0.05) in S. mansoni-infected mice after treatment with combination of BGA and PZQ when compared to those of S. mansoniinfected mice treated with PZQ alone.
Figure 2
3. 5. Serum level of TNF-α As shown in figure (3), the infection of mice with S. mansoni caused a significant increase (P < 0.05) in the level of TNF-α (409.0 ± 1.7 Pg/ml) as compared to that of the normal control group (324.8 ± 0.6 Pg/ml). On the other hand, treatment of S. mansoni infected mice with BGA, PZQ and their combination resulted in significant decreases (P < 0.05) in the level of TNF-α (350 ± 0.7, 364.0 ± 3.4 and 338.5 ± 1.2 Pg/ml, respectively) as compared with that of S. mansoni-infected group. Meanwhile, the level of TNF-α was significantly decreased (P < 0.05) in serum of S. mansoni-infected mice after treatment with combination of BGA and PZQ when compared to those of S. mansoni-infected mice treated with PZQ alone.
Figure 3
8
4. DISCUSSION The current study indicated that S. mansoni infection resulted in alteration in the secretory and synthetic functions of liver. Whereas, the activities of AST, ALT and ALP as well as the level of TP were elevated, the level of serum albumin was significantly reduced in mice infected with S. mansoni when compared to those of the normal control group. The treatments of S. mansoni-infected mice with BGA, PZQ or a combination of BGA with PZQ lead to a significant improvement in the liver function parameters. These findings are in agreement with the results of El-Lakkany et al. (2011a, b) and Abdel-Hafeez et al. (2012) who reported that treatment of mice infected with S. mansoni with PZQ led to amelioration in liver function. Previous studies of Kuriakose and Kurup (2008 and 2010) indicated that BGA exhibited a hepatoprotective effect against experimental drug toxicity. Also, Xin et al. (2007) reported that C-phycocyanin, which is found in blue green algae, has a hepatoprotective effect in experimental hepatitis. The results also illustrated that infection of mice with S. mansoni produced a remarkable oxidative stress of hepatic GPX and GST activities were significantly reduced as compared to those of the normal mice. These findings were previously reported in mouse and human schistosomiasis by Elmalawany (2009), El-Lakkany et al. (2011a), Mantawy et al. (2011) and Abdel-Hafeez et al. (2012). The treatment of S. mansoni-infected mice with BGA, PZQ or their combination led to significant increases in the activities of hepatic GPX and GST as compared to those of the S. mansoni-infected control group. As compared to PZQ treated mice, treatment with a combination of PZQ and BGA resulted in a significant increase in the activities of GPX and GST. These findings indicate the ability of BGA in triggering the antioxidant potential activity against the generation of reactive oxygen species (ROS) accompanied with schistosomiasis. The antioxidant capacity of BGA is attributed to the presence of potent antioxidants constituents such as vitamin A, vitamin E, vitamin C and C-phycocyanin (Scoglio et al., 2009). This assumption is supported by the results of El-Lakkany et al. (2011a) reported that treatment of S. mansoni-infected mice with β-carotene and/or PZQ produced a pronounced elevation in GPX activity. Kuriakose and Kurup (2010) showed that the treatment of paracetamol-intoxicated rats with BGA resulted in a significant elevation in the GPX activity when compared with control group. 9
The results showed that the diameter and number of egg granuloma were significantly reduced (P < 0.05) after treatment of S. mansoni-infected mice with BGA, PZQ and a combination of BGA with PZQ as compared to that of S. mansoniinfected group. Massoud et al. (2004) showed a significant reduction in the number and size of granulomas after the treatment of S. mansoni-infected mice with PZQ. Similar studies reported that the treatment of S. mansoni-infected mice with either PZQ alone or combined with pentoxifylline (El-Lakkany et al., 2011b) α-lipoic acid (Abdel-Hafeez et al., 2012) Nigella sativa oil (Mahmoud et al., 2002) and N-acetyl-Lcysteine (De-Lima et al., 2012) resulted in a significant decrease in the size and number of liver granuloma. The histopathological examination of livers from S. mansoni infected mice showed many lesions of granulomatous inflammation which developed around eggs and subsequent fibrosis. These pathological alterations were remarkably modulated after treatment of mice with BGA, PZQ and their combination. Botros et al. (2010) indicated that orally administration of a combination of PZQ with artemether inhibited the histopathological manifestations in the liver of S. mansoni-infected mice. Also, Toussom et al. (2012) showed that treatment of S. mansoni-infected mice with either silymarin alone or in combination with PZQ led to a significant reduction in granuloma and decreased histopathological lesions. In addition, Kuriakose and Kurup (2010) showed that BGA improved the histopathological texture of liver in a paracetamol toxicity model. Significant increases (P < 0.05) in the serum levels of TNF-α, ICAM-1 and VCAM-1 in S. mansoni-infected mice were observed as compared to that of the normal mice. These results are in agreement with previous studies of Mantawy et al. (2011), Rathore et al. (1996), el-Ahl et al. (2000), Loebermann et al. (2009) and Azevedo et al. (2012). Treatment of S. mansoni-infected mice with BGA, PZQ and a combination of BGA with PZQ resulted in significant reductions in the serum level of TNF-α, ICAM1 and VCAM-1when compared to those of the infected mice. In contrast to PZQ treated mice, treatment of infected mice with a combination of PZQ and BGA resulted in significant reduction in the serum levels of TNF-α, ICAM-1 and VCAM-1. Previous studies reported significant reductions in the serum level of TNF-α in S. mansoni infected mice after treatment with either PZQ alone (Yu et al., 2012) or a 10
combination of PZQ and pentoxifylline (El-Lakkany et al., 2011a,b). On the other hand, Yakoot and Salem (2012) indicated that the treatment of chronic hepatitis C patient with 500mg of BGA for 3 months resulted in a significant decrease in serum level of TNF-α. It was revealed that PZQ caused a significant reduction in the serum level of ICAM-1 (Esterre et al., 1998). Murphy et al. (2005) indicated that the serum level of ICAM-1 and VCAM-1 was significantly decreased after treatment of atherosclerotic mice with vitamin E, vitamin C and β-carotene for 15 weeks. Li et al. (2006) and Yamagata et al. (2012) revealed the inhibitory action of β-carotene and Cphycocyanin on the level of ICAM-1 and VCAM-1. Taken together with our previous results (Elmalawany, 2012); we can conclude that BGA exhibited antischistosomal activity where the treatment with BGA resulted in a significant reduction in worm burden and the ova count/gm liver or intestine (data are not mentioned here). BGA also improves the antischistosomal efficacy of PZQ by scavenging the ROS associated with schistosomiasis. In addition, it exhibited a hepatoprotective activity. 5. ACKNOWLEDGMENTS To Dr., Claire J Standley (Ecology and Evolutionary Biology, Princeton University, Princeton, NJ, USA), for her critical reading, helpful discussions and suggestions during the preparation of this manuscript.
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Azevedo, G.M.Jr., Costa, R.A., Resende, M.A., Rodrigues C.M., Vaz N.M., Carvalho C.R., 2012. Indirect effects of oral tolerance inhibit pulmonary granulomas to Schistosoma mansoni eggs. Clin. Dev. Immunol. DOI:10.1155/2012:293625. Baure, J.D., 1982. Creatinin method using pucric acid in clinical laboratory. Clinical Lab Methods. 9th ed. Saint Louis USA: Mobsy CV Company. P. 495-956. Bhat, V.B., Madyastha, K.M., 2001. Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: Protection against oxidative damage to DNA. Biochem. Biophys. Res. Commun. 285, 262-266. Botros, S., Hammam, O., Mahmoud, M.A., Bergquist, R., 2010. Praziquantel efficacy in mice infected with PZQ non-susceptible Schistsoma mansoni isolate treated with artemether: parasitological, biochemical and immunohistochemical assessment. APMIS, 118, 692-702. Chan, D.W., Perlstein, N.T., 1987. Immunoassay: A practical guide. Ed. Acad. Press, Oxford, U.K. Vol. (II), P. 71. de Oliveira, R.B., Senger, M.R., Vasques,
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Mantawy, M.M., Ali, H.F., Rizk, M.Z., 2011. Therapeutic effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection. Rev. Inst. Med. Trop. Sao. Paulo. 53,155-163. Massoud, A.M., El-Ebiary, F.H., Abou-Gamra, M.M., Mohamed, G.F., Shaker, S.M., 2004. Evaluation of schistosomicidal activity of myrrh extract: parasitological and histological study. J. Egy. Soc. Parasitol. 34, 1051-1076. Mekonnen,
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Am. J. Pathol. 149, 187-194 Reddy, C.M., Bhat, V.B., Kiranmai, G., Reddy M.N., Reddanna, P., Madyastha, K.M., 2000. Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina plantesis. Biochem. Biophys. Res. Commun. 277, 599-603. Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Path. 28, 56-63. Romay, C., Gonzalez, R., 2000. Phycocyanin is an antioxidant protector of human erythrocytes against lysis by peroxyl radicals. J. Pharm. Pharmacol. 52, 367368. Schaap, A., Rohrlack, T., Bellouard, Y., 2012. Optical classification of algae species with a glass lab-on-a-chip. Lab. Chip. 12, 1527- 1532. Scoglio, S., Benedetti, S., Canino, C., Santagni, S., Rattighieri, E., Chierchia, E., Canestrari, F., Genazzani, A.D., 2009. Effect of a 2-month treatment with Klamin, a Klamath algae extract, on the general well-being, antioxidant profile and oxidative status of postmenopausal women. Gynecol. Endocrinol. 25, 235240. Sokal, R. R. and Rohlf, F. J. (1981): Biometry, 2nd Ed, W. H. Freeman & Co., San Francisco, 241- 242. Tousson, E., Beltagy, D.M., Gazia, M.A., Al-Behbehani, B., 2012. Expressions of P53 and CD68 in mouse liver with Schistosoma mansoni infection and the protective role of silymarin. Toxicol. Ind. Health, 29, 761-770. Utzinger, J., Keiser, J., Shuhua, X., Tanner, M., Singer, B.H., 2003. Combination chemotherapy of schistosomiasis in laboratory studies and clinical trials. Antimicrob. Agents Chemother. 47, 1487-1495. Vadiraja, B.B., Gaikwad, N.W., Madyastha, K.M., 1998. Hepatoprotective effect of C-phycocyanin protective for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicity in rats. Biochem. Biophys. Res. Commun. 249, 428-431. von Lichtenberg, F.C., 1962. Host response to eggs of Schistsoma mansoni. I. Granuloma formation in un-sensitized laboratory mouse. Am. J. Pathol. 41, 71116
731. Xin, Y.F., Zhou, G.L., Shen, M., Chen, Y.X., Liu, S.P., Chen, G.C., Chen, H., You, Z.Q., Xuan Y.X., 2007. Angelica sinensis: A novel adjunct to prevent doxorubicin induced chronic cardiotoxicity. Basic Clin. Pharmacol. Toxicol. 101, 421-426. Yakoot, M., Salem, A., 2012. Spirulina platensis versus silymarin in the treatment of chronic hepatitis C virus infection. A pilot randomized, comparative clinical trial. BMC Gastroenterol. 12, 32. Yamagata, K., Tanaka, N., Matsufuji, H., Chino, M., 2012. Beta-carotene reverses the IL-1β-mediated reduction in paraoxonase-1 expression via induction of the CaMKKII pathway in human endothelial cells. Microvasc. Res. 84, 297-305. Yu, L., Sun, X., Yang, F., Yang, J., Shen, J., Wu Z., 2012. Inflammatory cytokines IFN-gamma, IL-4, IL-13 and TNF-alpha alterations in schistosomiasis: A metaanalysis. Parasitol. Res. 110, 1547-1552.
17
G.
C.V.
A
B
C.V.
G.
G. G.
C
D
E
Figure 1: liver section stained with (H&E x100). (A) Showing normal hepatic architecture, central vein (CV). (B) Section of liver tissue of S. mansoni-infected mice ten weeks post infection showing Schistosoma granuloma formed of leukocytic inflammatory cells and fibrocytic cells surrounding Schistosoma-ova. G. Granuloma. (C) Liver section of BGA treated mouse (100mg/kg orally for 15 consecutive days) showing fibrocellular granuloma of intermediate size. G. Granuloma. Central vein (CV). (D) Liver section of PZQ treated mouse (250 mg/ kg o r a l l y for 3 consecutive days 7th weeks post infection) showing fibrocellular granuloma. G. Granuloma. (E) Liver section of infected mice treated with BGA+PZQ showing small size late fibrocellular granuloma, in addition, decreased cellular constituents, degenerative changes in the ova and the pigments were minimal. G. Granuloma.
200 $
180 160
*
*
*,♣
140 120
ng/ml
100 80 60
$
40
*
*
*,♣
20 0 N
BGA (100mg/kg)
PZQ (250mg/kg)
ICAM-1
S. mansoniinfected
Infected + BGA (100mg/kg)
Infected + PZQ
Infected + BGA (100mg/kg) and PZQ (250mg/kg)
VCAM-1
Figure 2: Effect of S. mansoni infection and treatment with PZQ, BGA and their combination on serum levels of ICAM-1 and VCAM-1 in mice Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
$
450 400
*
*
TNF-ALPHA(Pg\ml)
350
*,♣
300 250 200 150 100 50 0 N
BGA (100mg/kg)
PZQ (250mg/kg)
S. mansoniinfected
Infected + BGA (100mg/kg)
Infected + PZQ
Infected + BGA (100mg/kg) and PZQ (250mg/kg)
Figure 4: Effect of S. mansoni infection and treatment with PZQ, BGA and their combination on serum levels of TNF-α in mice Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
Table (1): Effect of S. mansoni infection and treatment with PZQ, BGA and in combination on liver function in mice
Groups
ALT (U/L)
AST (U/L)
ALP (U/L)
TP (g/dl)
Alb (g/dl)
Normal control
29.7 ± 3.7
72.5 ± 4.6
29.2 ± 1.9
5.3 ± 0.3
3.4 ± 0.1
Normal + BGA
29.7 ± 3.3
73.1 ± 5.4
29.9 ± 2.2
5.6 ± 0.2
Normal + PZQ
31.2 ± 1.9
73.7 ± 3.3
30.2 ± 2.7
5.8 ± 0.2
3.3 ± 0.2
Infected control
172.0 ± 9.7$
239.5 ± 12.3$
92.2 ± 2.3$
7.3 ± 0.3$
2.1 ± 0.2$
Infected + BGA
62.2 ± 4.4*
131.7 ± 5.6*
38.9 ± 2.0*
5.8 ± 0.2*
3.2 ± 0.2*
Infected + PZQ
92.3 ± 7.9*
156.2 ± 6.4*
42.7 ± 1.7*
6.1 ± 0.2*
2.9 ± 0.2*
Infected +BGA /PZQ
40.3 ± 2.8*,♣
106.7 ± 5.5*,♣
32.1 ± 1.6*,♣
5.3 ± 0.1*
3.3 ± 0.2*
3.4 ± 0.2
Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
Table (2): Effect of S. mansoni infection and treatment with PZQ, BGA and in combination on hepatic GPX and GST activities in mice GST (µmol/min/g wt tissue)
GPX (U/L)
Normal control
0.56 ± 0.03
42.9 ± 1.1
Normal + BGA
0.86 ± 0.02$
56.0 ± 0.7$
Normal + PZQ
0.43 ± 0.01
36.7 ± 1.9
Infected control
0.18 ± 0.01$
29.9 ± 1.4$
Infected + BGA
0.68 ± 0.04*
50.0 ±1.0*
Infected + PZQ
0.33 ± 0.02*
35.1 ± 1.2*
0.76 ± 0.01*,♣
53.1 ± 1.2*,♣
Groups
Infected + BGA/PZQ
Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) ($) Significant difference when compared to normal control group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
Table (3): Changes in mean granuloma count and diameter in liver sections from mice treated treatment with PZQ, BGA and in combination, 7 weeks postinfection. Number of granulomas in 5 successive power fields (X100)
Mean granuloma diameter in ߤm
Infected control
44.7 ± 3.7
405 ± 11
Infected + BGA
40.8 ± 3.7
324 ± 16*
Infected + PZQ
37.9± 2.1*
Infected + BGA/PZQ
23.8± 2.4*,
Groups
♣
294 ± 5* ♣
240 ± 6.6*,
Data are expressed as Mean ± SD Mice were orally administrated with 100 mg/kg of BGA for 15 consecutive days. Mice were orally administrated with 250 mg/kg for 3 days, 7th weeks post infection. (*) Significant difference when compared to control infected group at (P < 0.05) (♣) Significant difference between Infected+PZQ group and Infected+BGA/PZQ group at (P < 0.05)
hepatoprotective efficiency of blue green algae (BGA) (Aphanizomenon flos-aquae) (100 mg/kg) alone or/and in combination with praziquantel (PZQ) (250 mg/kg) on mice infected with Schistosoma mansoni
BGA 100 mg/kg Seven weeks post infection be treated the groups of mice with
PZQ 250 mg/kg BGA 100 mg/kg +PZQ 250 mg/kg
Results
Diameter and number of granuloma were significantly reduced after treatment
Elevation in hepatic glutathione-S-transferase and glutathione peroxidase activities
Reduction of the elevated levels of AST, ALT and ALP as well as TP
Reduction of the elevated levels of TNF-α, ICAM-1 and VCAM-1
Highlights - The effect of blue green algae (BGA) on S. mansoni infected mice was studied. - BGA ameliorated the biochemical function of liver of S. mansoni infected mice. - BGA improved the antioxidant status of S. mansoni infected mice. - BGA reduced the elevation of TNF-α, ICAM-1 and VCAM-1. - BGA reduced the harmful side effect of antischistosomal drug Praziquantel.