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Influence of sativa seeds against liver fibrosis and consequence complications in murine Schistosomiasis.

Introduction

Schistosomiasis mansoni is a tropical helminthic disease characterized by parasite egg-induced granulomatous inflammation and cumulative fibrosis. Schistosoma mansoni produces several hundred eggs per day, and proportion of these eggs is trapped in hepatic tissues and in presinusoidal venules. There, they induce a granulomatous inflammation that leads to the accumulation of scar tissue in the portal spaces. Periportal fibrosis causes venous obstruction and portal blood hypertension and contributes to the development of splenomegaly and renopathy, severely affected patients develop esophageal varices, ascites, cachexia and renal failure, resulting in death in the absence of treatment (Weinstock, 1992; Johansen et al. 1994; van Velthuysen and Florquin, 2000). Fibrosis, which involves stellate cells derived from fibroblasts, is an excessive deposition of extracellular matrix proteins (ECMPs), like collagen (types I, III, which are the most abundant proteins, and IV), fibronectin, proteoglycans, laminin and elastin. It results from an imbalance between the positive and negative regulatory mechanisms controlling the production and degradation of ECMPs (Bataller and Brenner, 2005). Non-immunologic and immunologic mechanisms play a central role in the production of ECMPs and collagen deposition around schistosome eggs in liver. Complex interactions that are mediated by CD4+ T helper, type1 and 2 ([Th.sub.1] and [Th.sub.2)] cells (Wynn et al. 2004) and more recent studies have furnished numerous insights into the inflammatory mediators including glycated proteins (Shek and Benyon, 2004), enzymes, oxidant (as reactive oxygen species, ROS) (El-Sokkary et al. 2002; Eboumbou et al. 2005), cytokines (Henri et al. 2002; Davies et al. 2004) and antibodies have been described and vary according to the duration of the infection and the severity of the disease (Davies et al. 2004; Wynn et al. 2004; Torben and Hailu, 2007).

Currently, schistosomiasis control strategy is mainly based on the treatment of infected individuals with the drug of choice being praziquantel (WHO, 1999). Unfortunately, the long term world wide application of the drug coupled with the discovery of praziquantel-tolerant schistosome has generated concern over the development of drug-resistant schistosoma strains (Shengliang et al. 2001; Appiah and DeVlas, 2002). Beside, it was reported that praziquantel induce hemorrhage in the lung tissue of the host (Flisser and Mclaren, 1989). So for controlling schistosomiasis, there is an urgent need to develop new effective drug. Traditional medicinal plants were applied by some authors for the treatment of schistosomiasis (Sparg et al. 2000; Molgaard et al. 2001; Metwally, 2006). Among the promising medicinal plants, Nigella sativa L (Family, Ranunculacea), an amazing herb, is found wild in southern Europe, northern Africa and Asia Minor and it is one of the native plants that are widely distributed in Egypt (Schleicher and Saleh 1998). The seeds of N sativa are the source of the active ingredients and commonly known as black seed, black cumin or habatul Barakah. The seeds have long been used in folk medicine for a wide range of illnesses, including bronchial asthma, desentry, hypertension and diabetes (Schleicher and Saleh 1998; Al-Rowais, 2002). Pharmacological investigations revealed that the black seed oil exhibited hepato-and reno-protective effects against toxicity (Turkdogan et al. 2001; Ali, 2004). The seed oil was found to have antioxidant (Ramadan et al. 2003), anti-inflammatory (Mansour and Tornhamre, 2004), antitumor (Badary and Gamal El-Din, 2001) and immunomodulatory properties (El-Dakhakhny et al. 2000; El-Gazzar et al. 2006). Beside, the essential oil was shown to have anthelmintic activity (Aboul-Ela, 2002).

In view of the above information, the objective of this study was undertaken to evaluate the schistosomacidal capacity of N. sativa crushed seeds against different developmental stages of S mansoni infection, at the time of infection (immature schistosomula < 14 days post infection), during schistosomula maturation (14-21 days Pi), at the time of schistosomula maturation (>21days PI) and after the beginning of egg laying (>35 days PI) (Davies et al. 2004, Lescano et al. 2004) in infected mice. This can be achieved by evaluating worm recovery, egg count, number and size of granuloma, total area of infection and liver histopathology. Also the present study was extended to investigate the potential role of the black seeds in ameliorating schistosomal liver fibrosis and subsequent complications induced in both spleen and kidney. This can be achieved by measuring hydroxyproline in liver and alpha-fetoprotein in serum (markers of liver fibrosis), and some risk factors which have important role in liver fibrosis and consequence complications, including fructosamine in serum, xanthine oxidase (XO) and nitric oxide (NO) in tissues (liver, spleen and kidney) as well as tumor necrosis factor-alpha (TNF-[alpha]) in both serum and tissues of different organs and total serum immunoglobin E (IgE). Liver and kidney function markers were also measured in serum as documents of the capability of the used drug in modulating schistosomal induced pathology.

Materials and Methods

Chemicals

All chemicals used were of high analytical grade, product of Sigma (US), Merk (Germany) and BDH (England). N sativa seeds were commercially purchased and crushed in a grinder, weighed and made into suspension with 1%carboxymethylcellulose (CMC) to be used in the treatment of infected animals.

Animals

Swiss albino mice CDI strain weighing 18-22 g were raised and maintained throughout the expriment in the Schistosome Biological Materials Supply Program, Theodor Bilharz Research Institute (SBSP/TBRI), Giza, Eygpt. Mice were provided with balanced commercial pellet diet and water ad libitum during the duration of the expriment.

Parasites and Infection

Cercariae of an Eygptian strain of S mansoni were obtained from SBSP/TBRI and used for infection immediately after shedding from Biomphalaria alexandrina snails. Infection was carried out percutaneously with 70 [+ or -] 5 S mansoni cercariae/ mouse by the tail exposure method (Smithers and Terry, 1965).

Experimental Design

The animals were divided into six groups, each of ten mice. Group 1: Uninfected native control, Groups 2-6: S mansoni infected mice and classified as follow: Group 2: S mansoni-infected animals (positive control), Group 3: infected-N sativa treated group immediately at the time of infection (immature schistosomula < 14 days post infection). Group 4: infected- N sativa treated group ten days after infection (during schistosomula maturation, 14-21 days Pi). Group 5: infected- N sativa treated group twinty days after infection (at the time of schistosomula maturation > 21 Pi). Group 6: infected-N sativa treated group forty days after infection (after the beginning of egg lying >35 days PI).

The crushed seeds were given orally for ten days in a dosage of 50 mg/Kg/day (El-Daly, 1998) suspended in 1% CMC solution. Each animal in both control and infected groups (G1 and G2 respectively) was received 1.0 ml of 1% CMC solution/kg body weight daily for ten days. At the end of the experiment, 8 weeks after infection, animals in each group were anesthesized with ether and blood was collected by cardiac puncture in heparinized vials. Serum was separated by centrifugation at 3000x g for 10 minutes and used for some biochemical analysis. After liver perfusion, the organs (liver, spleen and kidney) from different animal groups were immediately removed weighed and washed using chilled saline solution. The tissues of these organs were minced and homogenized in either 10% trichloroacetic acid (for NO determination) or in ice cold bidistilled water (for hydroxyproline, XO and TNF-[alpha] determination) to yield 10% homogenates using a glass homogenizer. The homogenates were centrifuged for 15 minutes at 10000g at 4 [degrees]C and the supernatants were used for the biochemical analyses.

Liver Perfusion

Worms were recovered from the hepatic portal system and liver by a perfusion technique previously described by Smithers and Terry (1965). The worms from each mouse were left to sediment for about 20 min. in a small Petri dish to allow sex identification, examination and count. The degree of protection or the % reduction in challenge was calculated from P= C-V/Cx100, where P is the % protection, C is the mean number of the parasites recovered from infected mice and V is the mean number of the parasites recovered from treated mice.

Oogram count

The number of eggs/g liver tissue was assessed following digestion with 4%KOH according to the method of Cheever and Anderson (1971).

Histopathology

Representative slices from liver tissue were taken from the eviscerated animals and fixed in buffer formalin (10%). Paraffin embedded sections (4 [micro]m thick) were taken after fixation and slides were stained using haematoxlin and eosin (H& E) by the method of Hirsch et al. (1997).

Granuloma count

Granuloma count was carried out in five successive fields (10x10) of serial tissue section of more than 25 [micro]m apart. Granuloma dimensions were measured using an ocular micrometer for the lobular granuloma with central ova.

Biochemical analysis

Liver collagen concentration was determined by measuring hydroxyproline content using the method of Jamall et al. (1981). Nitrite concentration (an indirect measurement of NO synthesis) was assayed using Griess reagent (sulfanilamide and N-1-naphthyl-ethylenediamine dihydrochloride) in acidic medium (Moshage et al, 1995). XO activity was determined by the reduction of nitro-blue tetrazolium (NBT) in the presence of xanthine, forming formazan. The enzyme activity was calculated using the extinction coefficient of reduced NBT (7.5 [cm.sup.2/[micro]mol] at 540 nm) (Fried and Fried, 1974). TNF-[alpha] was quantified using a commercial ELISA kit (Endogen, Woburn, MA). Fructosamine (glycated serum protein) was determined using reagents, calibrators and controls from Sigma Diagnostics (St. Louis, MO) and application parameters for the Cobas Mira automated chemistry analyzer. The assay is a modification of the original method of Johnson aand colleagues (Parlin et al. 1997) where fructosamine reduces NBT under alkaline conditions and forms a purple-colored formazan with an absorption maximum at 530 nm. AFP was assayed by ELISA using commercial kit. The level of total IgE was measured by ELISA and compared with known mouse IgE standard (BD PharMingen). ALT and AST activities were determined according to the method described by Bergmeyer et al. (1986). GGT was assayed using the method of Schmidt and Schmidt (1981). ALP was measured using 4-nitrophenyl phosphate as substrate (Demetriou et al. 1974). albumin was determined using the method of Doumas et al. (1971). uric acid (Fossati et al. 1980) and creatinine (Henry, 1974) were also measured.

Statistical analyses

Data were analyzed by comparing values for different treatment groups with the values for individual controls. Results are expressed as mean [+ or -] S.D. The significant differences among values were analyzed using analysis of variance (one-way ANOVA) coupled with post-hoc (LSD).

Results

Table 1 shows the worm burden and ova count in infected and N sativa seeds treated groups. The result revealed that oral administration of N sativa seeds to S mansoni infected mice (Gs 3, 4, 5 & 6) showed significant reduction of all forms of differential worm burden, the solitary male and female and coupled which is coincided with a decrease in the total egg burden (from liver) compared to untreated infected animals (G2). Granuloma count in sativa- treated groups also revealed a marked reduction accompanied by an obvious reduction in its diameter and total area of infection in relation to infected mice as seen under the microscope in low powered field (Table 2). Histopathological studied proved the parasitological results, where the schistosomal liver showed multiple granulomatous lesions and focal areas of necrosis. The granulomatous reactions resulted from periportal cellular infiltration around mature eggs and extend towards similar lesion neighboring portal tract. It reached its maximum size surrounding the egg at the end period of infection. The brownish black schistosomal pigmentation was also observed in Kupffer cells of the infected liver section (fig. 1 B) compared to normal one (fig1 A). The liver from infected-sativa treated different groups showed improvement represented by a decrease in granuloma size with a minimal degenerative change in liver tissue (fig.1 C, D, E & F). The best result was obtained in groups 3 and 4 in wwhich sativa seeds are eitheradministered at the time of infection, G3 (in the presence of immature larvae, schistosomula) or ten days after infection, G4 (during schistosomula maturation). The results also revealed that infection with S mansoni (G2) led to the elevation of liver hydroxyproline and serum AFP (markers of liver fibrosis) versus normal uninfected animals, indicating establishment of liver fibrosis (Tables 3 & 6 respectively). Also, under the effect of S mansoni parasitic infection, upregulation of some inflammatory risk factors and complications in both spleen and kidney was observed. This was indicated by the marked increment of XO and NO in tissues of different organs (liver, spleen and kidney, tables 3, 4& 5 respectively), fructosamine and total IgE levels in serum (table 6), TNF-[alpha] in both serum and tissues of different organs (Tables 3, 4, 5, &6) in infected animals in relation to normal ones. In addition, the present data showed that infection with S mansoni induced significant elevation in the levels of serum GGT, AST, ALT, ALP and a reduction in albumin level (indices of liver function) as well as in serum uric acid and creatinine (markers of kidney function) compared to normal mice (table 6). Oral administration of N sativa seeds to S mansoni infected animals significantly modulated the alteration in the above biomarkers compared to infected mice. The best results were obtained in Gs 3 and 4 where most of these markers reached to near normal levels.

[FIGURE 1 OMITTED]

Discussion

The current study demonstrated that administration of N sativa crushed seeds to mice harboring S mansoni have shown potential antischistosomal activity against the different developmental stages of thee parasites, immature larveae (schistosomula, < 14 days Pi, G3), during schistosomula maturation (14-21 days PI,G4), at the time of schistosomula maturation (>21days PI,G5) and after the beginning of egg laying (>35 days PI,G6) (Davies et al. 2004; Lescano et al. 2004) which is evidence by significant reduction in the number of worm burden (solitary worms, male and female, and coupled) in comparison to infected untreated animals. The decrease in the number of worms was more notable among the coupled followed by the females which was coincided with a decrease in the total egg burden from liver. This effect was pronounced in animals treated with sativa seeds either simultaneously at the time of infestation (G3) or ten days after infection (G4), where fewer worms were recovered confirmed by the lower number of eggs, indicating that these phases is more susceptible to the drug attack. These data are in line with previous in vivo study showed that sativa oil was effective in reducing worm and egg burden (Mostafa, 2001, Mahmoud et al. 2002), while in vitro study, the crushed seeds showed strong antischistosomal potency against S mansoni different stages (miracidia, cercariae and adult worms) as well as the inhibition in egg laying by the female adult worms was also documented (Mohamed et al. 2005). The obvious decrease in the number of coupled worms among the different sativa seeds-treated groups presented in the current study may imply that this drug affects the ability of both male and female worms to couple and consequently inhibit egg output by female adult worms (Mohamed et al. 2005). The possible mechanism which may explain this beneficial antischistosomal effect of sativa seeds is that the seeds contain active constituents which may have a direct effect on the vitality of schistosome different stages as well as the fecundity of the remaining female adult worms. This is documented by our previous in vitro studies which revealed that the seeds strongly affect some antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathione reductase) of adult worms which have an important role in the protection of the parasite against host oxidant killing as well as some enzymes of glucose metabolism (hexokinase and glucose-6-phosphate dehydrogenase) which have a crucial role in the survival of the parasite inside its host (Mohamed et. al. 2005).

One of the major pathological features of S mansoni parasites is their ability to persist and establish a chronic infection. This leads to chronic inflammation, which in turn can lead to severe fibrotic modification of infected tissues and organs (Meneghin and Hogaboam, 2007). Previous studies revealed that the intensity of schistosomal infection which represented by the worm burden and egg count increase the degree of liver fibrosis and granulomatous reaction (El-Lakkany et al. 2004). This is in agreement with the present histopathological findings of schistosomal liver which confirmed by increased number and diameter of granuloma, liver eggs and extensive fibrous tissue accumulation. Treatment of infected mice with sativa seeds effectively improved the histopathological picture of liver. This was ensured by diminution in number and diameters of granulomas, reduction in their fibrotic content accompanied with a reduction in total area of infection. This finding was more evidence in Gs 3 and 4 followed by G 5. In group 6 (in which sativa seeds administered after the beginning of egg laying), non-significant change in granuloma diameters was noticed in relation to infected untreated animals, however significant reduction in the number of granulomas and total area of infection was observed, and this coincided with the decrease in the total egg burden proved in the current study, suggesting that the used drug may has a protective and therapeutic beneficial effects in reducing the intensity of schistosomal infection.

Fibrosis resulting from infection with S. mansoni is predominantly caused in liver by the host immune response to parasite eggs that are laid in the portal venous system and then become trapped in hepatic sinusoids and sequestered within granulomatous lesions (Chiaramonte et al. 2003).

The current investigation revealed that liver fibrosis in response to S. mansoni parasitic infection was documented by marked increase in the levels of liver hydroxyproline and serum AFP in group of infected mice versus normal healthy ones. Because collagen is the main element of ECMPs, hydroxyproline, is an amino acid charcteristic of collagen metabolism, used as a marker to express the extent of liver fibrosis, as it is the major alteration associated with morbidity (Souza et al. 2005). Similar result was obtained by some authors who emphasized that elevated liver hydroxyproline content was associated with S mansoni infection (Marck, 2005; Metwally, 2006) and this may be attributed to that S mansoni egg granulomas contain factors responsible for the elevation of free L-hydroxyproline content in the fibrotic liver (Adewusi et al. 1996; Potter et al. 2003).

AFP is a glycoprotein, of unknown function, normally produced during neonatal development by the liver and in small concentrations by the gastrointestinal tract (Abelev et al. 1963). Abnormal elevated serum level of AFP has been reported in patients with liver cirrhosis and hepatocellular carcinoma (Gupta et al. 2003). Elevated level of AFP in sera of S mansoni infected animals presented in the current study may be considered as an index for liver fibrosis related to schistosomiasis.

Administration of N. sativa crushed seeds down-modulate the alteration in liver hydroxyproline and serum AFP induced under the effect of S mansoni infection, indicating their strong antifibrotic corrective action which may attributed to that the seeds are effective in reducing granulomas formation which are associated with liver fibrosis. The best results were obtained in Gs 3 and 4 which is confirmed by the histopathological picture of liver. Our results are in harmony with previous published data revealed that treatment with N sativa oil prevented liver fibrosis induced by S mansoni infection in mice (Mahmoud et al. 2002) and by C[Cl.sub.4] in rabbits (Turkdogan et al. 2001). Two mechanisms may explain this good effect of the used drug, the first is that the seed oil induce modulation of the immune response to the fewer remaining eggs that trapped in the liver (Mahmoud et al. 2002). The second is that the seed active constituents have the ability to inhibit the pathways of the inflammatory mediators, which have the central role in tissue fibrosis (Mansour and Tomhamre, 2004). The production of such mediators, which induced oxidative stress and inflict tissue damage, have been implicated in the toxicity-induced complications and pathological events of different organs including spleen and kidney in S mansoni parasitic infection (El-Sokkary et al. 2002; Booth et al. 2004a & b).

In line with previous authors, the present study showed that S mansoni parasitic infection induces the production of inflammatory mediators which are indicated by significant increase of fructosamine in serum, XO activity in tissue of different organs (liver, spleen and kidney) and TNF-[alpha] level in both serum and different organs with concomitant increase in NO level in different organs in comparison to control healthy ones, the highest levels of these mediators was observed in liver as it is the target organ affected by S mansoni infection (Adewusi et al. 1996; Haseeb et al. 2001; deJesus et al. 2002; Davies et al. 2004; Metwally, 2006).

Some publications described in vitro evidence of the direct role of glucose and its abnormal metabolism in the development of tissue fibrosis (Huang et al. 1999). Serum fructosamine, one of the markers of abnormal glucose metabolism, is a glycated protein resulting from spontaneous nonenzymatic condensation of glucose and proteins such as plasma protein, which is generally referred to fructosamine due to its structural similarities to fructose (Huang et al. 1999; Misciagna et al, 2004). As albumin is the most abundant protein in serum and contains multiple lysine residues, measurement of fructosamine is mainly the determination of glycated albumin (Lapolla et al, 2005). Fructosamine has been recognized as a major cause of tissue fibrosis, as it has a main role in increasing the expression of ECMPs and activating of protein kinase C which has a central role in tissue fibrosis (Hattori et al. 2001). This nonenzymatic modification of protein is considered by several authors as a possible common mechanism involved in the progression of many pathological conditions (Vijayan et al., 2007) including myocardial and renal fibrosis (Roy et al. 1990, Khaidar et al. 1994; Chowdhury and Lasker, 1998), colorectal adenoma, and chronic renal failure (Sabater et al. 1991, Misciagna et al, 2004).

Depending on the above information and from the present study, it can be suggest that increased serum fructosamine, as it is the first study investigating it in relation to schistosomiasis, may be used as a useful marker for liver fibrosis and associated complications of other organs related with S. mansoni parasitic infection.

XO is an enzyme catalyzes the oxidation of hypoxanthine to xanthine and the later to uric acid. It was reported that XO is an endogenous source of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as NO that can induce oxidative stress and inflect tissue injury (Winterbourn and Sutton, 1986; Mohamed et al. 2001; Harrison, 2002). On the other hand, the production of NO, was found associated also with the elevated level of TNF-[alpha] in response to the activation of Th1 cells as an early response to parasitic infection (James et al. 1998).

Excessive NO production was reported to exert various influences on the pathogenesis of tissues (Mohamed et al, 2001). The direct toxicity of NO is enhanced by reacting with superoxide radical to give powerful secondary toxic oxidizing species, such as peroxynitrite (ONO[??]) which is capable of oxidizing cellular structure and causes lipid peroxidation (Beckman and Koppenol, 1996; El-Sokkary et al. 2002; Eboumbou et al. 2005), a process leads to membrane damage and correlates positively with tissue fibrosis through inducing fibrogenic cytokines and increasing collagen synthesis (Parola and Robino, 2001). In addition, the bioactive NO is known to have a vasodilatory effect (Napoli and Ignarro, 2001) and its inhibition leads to vasoconstriction and atherosclerosis. Thus, transformation of NO by [??]2 radical to peroxinitrite under the effect of S. mansoni infection may diminish the capacity of endothelial cells to generate bioactive useful NO, which is important in maintenance of normal blood pressure, thereby decreases in NO bioavailability, causing endothelial sclerosis and subsequently hypertension (Stokes et al, 2002). So, elevated level of tissue NO may be considered one of the important factors responsible for the pathological events induced in kidney and spleen as a consequence complications of liver fibrosis induced by S mansoni infection. This is supported by other studies revealed that liver fibrosis leads to blockage of blood flow into the liver causes portal hypertension and ascites as well as into the spleen leading to hyperplasia, congestion, hardening and enlargement of the spleen, referring to spleenomegaly (Booth et al. 2004 a). In addition, it was reported that patients with chronic S mansoni infection involving portal hypertension and an enlarged spleen have an increased frequency of renal disease and eventually leads to end-stage renal failure (Barsoum, 1993; van Velthuysen and Florquin, 2000).

Beside, it was reported that TNF-[alpha] essentially functions as a trophic factor for maintaining adult schistosome viability, it is expressed during egg deposition and has a crucial role in the modulation of granulomatous reaction induced by the eggs (Joseph and Boros, 1993; Haseeb et al. 2001). Torben and Hailu (2007) stated that increased level of this inflammatory cytokine after egg excretion may an indication of its effect in complications of schistosomiasis, it capable of inducing tissue injury and fibrosis through inducing ROS production, lipid peroxidation (Poli, 2000), collagen synthesis, other fibrogenic risk factors (Booth et al. 2004a) and inhibiting matrix metalloproteinases production, the key enzyme in the degradation of collagens (Pender et al. 1998). Chronic exposure to TNF-[alpha] was found to be associated with high risk of periportal fibrosis (Henri et al 2002, Booth et al. 2004b), ascites accumulation and splenomegaly (Vassalli, 1992).

As parasite eggs induce a strong type-2 response, Th2 cytokine, IL-4, has been demonstrated to play an important role in promoting B-cells proliferation and the isotype class switch to IgE (Finkelman et al. 19998). In consistent with this finding, increasing serum level of inflammatory antibody, total IgE in group of infected mice was observed in the present study. This is supported by previous studies stated that increasing circulating IgE level is a humoral response to egg and adult worm antigens suggesting that this mechanism might be involved in hepatic pathological patterns (Silva et al. 2004). IgE was reported to have the major role in mast cells stimulation which has a central role in the induction of chronic inflammation (Jayapal et al. 2006) and the progression of hepatic fibrosis by producing fibrogenic inflammatory mediators as well as the components of the ECMPs (Gruber 2003; Shen, 2008).

Accordingly, modulation of the inflammatory risk factors and reducing oxidative stress may be considered as targets for pharamacological or molecular interventions for the treatment of liver fibrosis and consequence complications in murine schistosomiasis (Lucey et al. 1996).

Supplmentation of N sativa crushed seeds to infected mice markedly corrected the infection caused elevation in the inflammatory risk factors (fructosamine, XO, NO, TNF-[alpha] and total IgE) presented in the current study, implying their potential antiglycating, antioxidant, anti-inflammatory and immunomodulatory capabilities. These beneficial effects of sativa seeds were previously reported by some authors who attributed these good actions to their active constituents such as the volatile oil, thymoquinone (TQ), as well as the crude fixed oil and its fractions (neutral lipids, glycolipids and phospholipids) (Houghton et al. 1995; El-Dakhakhny et al. 2000; Turkdogan et al. 2001; Mahmoud et al. 2002; Ramadan et al. 2003; Ali, 2004; El-Gazzar et al. 2006) and this may explain in part the mechanism of their useful action in ameliorating schistosomal liver fibrosis and the consequence oxidative complications induced in both spleen and kidney presented in the current investigation. Recent studies revealed that antioxidants, particularly those of plant origin could attenuate hepatic fibrosis in rodent and may exert beneficial effects in patients with chronic liver diseases (Wang et al. 2005).

Serum biochemical profiles in terms of GGT, AST, ALT, ALP, albumin, uric acid and creatinin provided supportive evidence to the pathological alterations observed in both liver and kidney in response to schistosomiasis.

In consistant with previous studies, the present study showed that the inflammatory reactions induced in livers of S. mansoni infected mice are ensured by marked increase in serum GGT, AST, ALT and ALP levels and a decrease in albumin level in infected animals (El-Sokkary et al. 2002; Mahmoud et al. 2002; Metwally, 2006). The increment of such enzymes in serum may be due to the destruction of hepatocytes by the action of toxins of the parasite eggs leading to their release into the circulation (Cheever and Anderson, 1971). The decrease in serum albumin may be due to its glycation by glucose forming fructosamine. This is emphasized in the current data by the elevated serum fructosamine level. Hypoalbuminemia is one of the factors responsible for the onset of ascites related to liver fibrosis (Horie et al. 1998). Ascites formation related to liver fibrosis is generally attributed to promotion of water reabsorption in renal tubules and plasma colloidal osmotic pressure reduction (Schrier et al. 1988) and albumin was found to have a crucial role in increasing urinary excretion (Horie et al. 1998).

According to some authors, the marked increment of serum creatinine and uric acid levels in the infected group presented in this study are well indicators of schistosomal renopathy (Johansen et al. 1994, Mostafa, 2001). Elevated serum uric acid level may attribute to the increased XO activity, which is documented in the current investigation in response to parasitic infection, together with a reduction in urate excretion.

Supplementation of sativa seeds, effectively ameliorated the above serum marker levels characteristic of schistosomal hepato- and renopathy, the best results was observed in the third and fourth groups as evidenced by the normalization of most of these markers. These results are in consistent with previous published data showed that administration of the seed oil to S. mansoni infected mice ameliorates the elevation in serum ALT and ALP (Mahmoud et al. 2002) and improved kidney function (Mostafa, 2001). This positive response obtained by the used drug may attributed to the ability of sativa seeds to protect and stabilize cellular membranes by mainipulating the inflammatory mediators induced liver fibrosis and tissue damage and hence can modify the intensity of the inflammation induced in the tissues of different organs in response to S. mansoni infection. Also it was found that treatment of diabetic rats with sativa seeds induced elevation in serum albumin (Hasanain and Hassan, 1996). Amelioration of serum albumin level may attenuate schistosomal ascites formation in response to liver fibrosis

In conclusion, the present study demonstrated that N. sativa seeds supplementation in infected mice were shown to have divergent effects and using them as immunoprotective and therapeutic agent may be effective in augmenting the reduction of immunopathology, granuloma formation, hepatic fibrosis, spleen and renal pathology related to S mansoni parasitic infection.

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Mohamed AM (1), Mahmoud SS(2) and Abdel Razik A. Farrag (3)

(1) Department of Medicinal Chemistry, National Research Center, Dokki, Egypt

(2) Department of Parasitology, Theodor Bilharz Research Institute, Giza, Egypt

(2) Department of Pathology, National Research Center, Dokki, Egypt
Table 1: Worm burden and ova count in infected and N sativa
seeds-treated groups.

                           Infected            Infected-N sativa
                          untreated             treated groups
    Parameters               (G2)                     G3

   Worm burden        19.33 [+ or -] 5.3      6.7 [+ or -] 3.7 *
  Solitary male           (3,4,5,6)                (2,5,6)
       LSD                                          65.33
   % Reduction
 Solitary female     21.45 [+ or -]  2.2     4.0 [+ or -] 0.79 *
       LSD                (3,4,5,6)                (2,5,6)
   % Reduction                                      81.35
     Coupled           16 [+ or -] 5.8       2.2  [+ or -] 1.4 *
       LSD                (3,4,5,6)                (2,5,6)
   % Reduction                                      86.25
   Ova count x        14.6 [+ or -] 3.2      3.1 [+ or -] 0.30 *
    [10.sup.3]            (3,4,5,6)                (2,5,6)
       LSD
   % Reduction                                      78.71

                           Infected-N sativa treated groups

    Parameters                G4                      G5

   Worm burden        6.7 [+ or -] 2.2 *     10.3  [+ or -] 3.7 *
  Solitary male            (2,5,6)                  (2,3,4)
       LSD                  65.33                    46.71
   % Reduction
 Solitary female     4.8 [+ or -] 0.24 *      10.5 [+ or -] 1.8 *
       LSD                 (2,5,6)                  (2,3,4)
   % Reduction              77.62                    51.04
     Coupled          2.5 [+ or -] 1.1 *      7.7 [+ or -] 1.2 *
       LSD                 (2,5,6)                  (2,3,4)
   % Reduction              84.37                    51.87
   Ova count x       4.3 [+ or -] 0.62 *      6.9 [+ or -] 0.62 *
    [10.sup.3]             (2,5,6)                  (2,3,4)
       LSD
   % Reduction               70.5                    52.73

                      Infected-N sativa       ANOVA
                       treated groups

    Parameters                G6               P<

   Worm burden        10.7 [+ or -] 3.8 *    0.0001
  Solitary male             (2,3,4)
       LSD                   44.64
   % Reduction
 Solitary female      16.6 [+ or -] 2.7 *    0.0001
       LSD                  (2,3,4)
   % Reduction               22.61
     Coupled          12.5 [+ or -] 2.6 *    0.0001
       LSD                  (2,3,4)
   % Reduction               21.88
   Ova count x        9.8 [+ or -] 1.5 *     0.0001
    [10.sup.3]              (2,3,4)
       LSD
   % Reduction              32.877

Data are mean [+ or -] SD of eight mice in each group;
Numbers between brackets indicate significant correlation.
* P <0.0001 compared with infected untreated group.

Table 2: Granuloma diameter and count in infected and sativa
seeds treated groups.

                             Infected          Infected-N sativa
    Parameters              untreated          treated groups
                               (G2)                  G3

    Granuloma            9.6 [+ or -] 0.5        1.8 [+ or -] 0.5 *
    count/LPF
       LSD                  (3,4,5,6)                 (2,5,6)
   % Reduction                                         81.25
    Granuloma          252.3 [+ or -] 15.7      87.6 [+ or -] 7.9 *
  diameter ([micro]m)
       LSD                   (3,4,5)                  (2,5,6)
   % Reduction                                         65.28
  Total area of        2422.08 [+ or -] 80.2   157.68 [+ or -] 6.32 *
    infection
   ([micro]m)/LPF            (3,4,5,6)                (2,5,6)
       LSD                                              93.5
% Reduction

    Parameters            Infected-N sativa      Infected-N sativa
                           treated groups         treated groups
                                 G4                    G5

    Granuloma            2.1 [+ or -] 0.8 *      4.9 [+ or -] 0.7 *
    count/LPF
       LSD                    (2,5,6)                 (2,3,4,6)
   % Reduction                 78.13                   48.9
    Granuloma           112.25 [+ or -] 28.3 *  179.7 [+ or -] 10.1 **
  diameter ([micro]m)
       LSD                    (2,5,6)                  (2,3,4,6)
   % Reduction                 55.48                    28.78
  Total area of         235.73 [+ or -] 10.5 *  880.5 [+ or -] 15.5 *
    infection
   ([micro]m)/LPF             (2,5,6)                  (2,3,4,6)
       LSD                      90.3                    63.65
% Reduction

                         Infected-N sativa          ANOVA
    Parameters             treated groups
                                 G6                  P<

    Granuloma            7.5 [+ or -] 0.4 **        0.0001
    count/LPF
       LSD                    (2,3,4,5)
   % Reduction                   21.9
    Granuloma           224.32 [+ or -] 24.4 (n)    0.0001
  diameter ([micro]m)
       LSD                     (3,4,5)
   % Reduction                  11.09
  Total area of         1682.4 [+ or -] 60.67 *     0.0001
    infection
   ([micro]m)/LPF             (2,3,4,5)
       LSD                      30.54
% Reduction

Data are mean [+ or -] SD of eight mice in each group; LPF:
Low power field of the microscope. Total area of infection
= number of granulomata x size of one granuloma.
Numbers between brackets indicate significant correlation.

* P <0.0001, ** P <0.001, compared with untreated infected group.
n : not significant compared with untreated infected group.

Table 3: Liver biochemical parameters in different experimental
groups.

                             Normal                 Infected
   Parameters                                       untreated
                               G1                      G2

  Hydroxypoline
  ([micro]g g)       103.60 [+ or -] 11.77 *    728.42 [+ or -] 77.69
       LSD                  (2,4,5,6)                 (1,3,4,5,6)
       XO
  (nmole/min/mg       2.32 [+ or -] 0.098 *       6.80 [+ or -] 0.20
    protein)
       LSD                   (2,5,6)                   (1,3,4,5,6)
       NO
  ([micro]mole       17.84 [+ or -] 2.02 *       86.60 [+ or -] 9.045
   /g tissue)
       LSD                   (2,5,6)                   (1,3,4,5,6)
   TNF-[alpha]        16.65 [+ or -] 0.91 *      50.59 [+ or -] 2.11
 S (pg/g tissue)
       LSD                   (2,5,6)                   (1,3,4,5,6)

   Parameters           Infected-N sativa          Infected-N sativa
                         treated groups             treated groups
                               G3                        G4

  Hydroxypoline
  ([micro]g g)       140.87 [+ or -] 7.48 *     210.21 [+ or -] 8.1 *
       LSD                  (2,4,5,6)                  (1,2,3,5,6)
       XO
  (nmole/min/mg       2.45 [+ or -] 0.10 *        2.55 [+ or -] 0.11 *
    protein)
       LSD                   (2,5,6)                    (2,5,6)
       NO
  ([micro]mole        20.38 [+ or -] 2.21 *      23.23 [+ or -] 2.57 *
   /g tissue)
       LSD                   (2,5,6)                    (2,5,6)
   TNF-[alpha]       18.34 [+ or -]  1.89 *      21.47 [+ or -]  3.2 *
 S (pg/g tissue)
       LSD                   (2,5,6)                    (2,5,6)

   Parameters          Infected-N sativa
                         treated groups
                              G5
  Hydroxypoline
  ([micro]g g)       431.12 [+ or -] 16.87 *
       LSD                 (1,2,3,4,5)
       XO
  (nmole/min/mg       3.17 [+ or -] 0.13 *
    protein)
       LSD                  (1,2,3,4)
       NO
  ([micro]mole        45.44 [+ or -] 5.04 *
   /g tissue)
       LSD                  (1,2,3,4)
   TNF-[alpha]       30.72 [+ or -]  3.96 *
 S (pg/g tissue)
       LSD                  (1,2,3,4)

                       Infected-N sativa        ANOVA
                         treated groups
    Parameters                G6                 P<

  Hydroxypoline
  ([micro]g g)       492.01 [+ or -] 12.45 *    0.0001
       LSD                 (1,2,3,4,6)
       XO
  (nmole/min/mg       3.24 [+ or -] 0.17 *      0.0001
    protein)
       LSD                  (1,2,3,4)
       NO
  ([micro]mole        50.98 [+ or -] 7.42 *     0.0001
   /g tissue)
       LSD                  (1,2,3,4)
   TNF-[alpha]        35.06 [+ or -] 2.56 *
 S (pg/g tissue)                                0.0001
       LSD                  (1,2,3,4)

Data are presented as mean  [+ or -]  S.D. of 5 independent
experiments. Numbers between brackets indicate significant
correlation.

* P <0.0001 compared with untreated infected group.

Table 4: Effect of N. Sativa seeds on spleen biochemical parameters
in different experimental groups.

  Parameters              Normal                   Infected
                                                  untreated
                            G1                        G2

      XO           1.2 [+ or -] 0.034 *       2.5 [+ or -] 0.082
 (nmol/min/mg
   protein)
      LSD                (2,4,5,6)               (1,3,4,5,6)
      NO           18.78 [+ or -] 1.36 *     53.98 [+ or -] 7.59
 ([micro]mole
  /g.tissue)
      LSD                (2, 5, 6)               (1,3,4,5,6)
      TNF          4.21 [+ or -] 0.33 *       9.89 [+ or -] 0.32
 (Pg/g tissue)              (2)                  (1,3,4,5,6)
      LSD

  Parameters            Infected-N sativa treated groups
                            G3                         G4

      XO           1.22 [+ or -] 0.042 *      1.30 [+ or -] 0.03 *
 (nmol/min/mg
   protein)
      LSD                (2,4,5,6)                 (1,2,3,5,6)
      NO           19.96 [+ or -] 1.59 *      22.40 [+ or -] 1.71 *
 ([micro]mole
  /g.tissue)
      LSD                 (2,5,6)                      (2)
      TNF          3.74 [+ or -] 0.43 *       4.35 [+ or -] 0.16 *
 (Pg/g tissue)              (2)                        (2)
      LSD

  Parameters             Infected-N sativa treated groups        ANOVA
                            G5                        G6          P <

      XO          1.46 [+ or -] 0.048 *  1.52 [+ or -] 0.05 *
 (nmol/min/mg
   protein)                                                      0.0001
      LSD               (1,2,3,4)             (1,2,3,4)
      NO          27.53 [+ or -] 2.90 *  30.53 [+ or -] 5.12 *
 ([micro]mole
  /g.tissue)                                                     0.0001
      LSD                (1, 2,3)              (1,2,3)
      TNF         4.30 [+ or -] 0.39 *   4.48 [+ or -] 0.22 *
 (Pg/g tissue)             (2)                   (2)             0.0001
      LSD

Data are presented as mean [+ or -] S.D. of 5 independent
experiments, Numbers between brackets indicate significant
correlation, * P <0.0001 compared with untreated infected
group.

Table 5: Effect of N. Sativa seeds on kidney biochemical parameters
in control, infected and treated groups.

   Parameters               Normal                   Infected
                                                     untreated
                              G1                        G2

       XO           2.352 [+ or -] 0.075 *       4.9 [+ or -] 0.35
   (nmol/min/
  mg protein)
      LSD                  (2,5,6)                  (1,3,4,5,6)
       NO           17.84 [+ or -] 2.015 *     40.61 [+ or -] 5.045
  ([micro]mole
   /g tissue)
      LSD                  (2,5,6)                  (1,3,4,5,6)
  TNF-[alpha]       6.44 [+ or -] 1.178 *       15.58 [+ or -] 1.26
 (pg/ g tissue)
      LSD                  (2,5,6)                  (1,3,4,5,6)

   Parameters                 Infected-N sativa treated groups
                               G3                          G4

       XO           2.44.50 [+ or -] 0.03 *      2.52 [+ or -] 0.08 *
   (nmol/min/
  mg protein)
      LSD                   (2,5,6)                    (2,5,6)
       NO            20.38 [+ or -] 2.21 *      23.23 [+ or -] 3.52 *
  ([micro]mole
   /g tissue)
      LSD                   (2,5,6)                     (2,6)
  TNF-[alpha]        8.53 [+ or -] 1.00 *        8.92 [+ or -] 1.08 *
 (pg/ g tissue)
      LSD                     (2)                        (2)

   Parameters              Infected-N sativa treated groups      ANOVA
                              G5                        G6        P<

       XO           3.13 [+ or -] 0.18 *  3.23 [+ or -] 0.12 *
   (nmol/min/
  mg protein)                                                    0.0001
      LSD                (1,2,3,4)             (1,2,3,4)
       NO           29.31 [+ or -] 3.0 *  34.98 [+ or -] 5.4 *
  ([micro]mole
   /g tissue)                                                    0.0001
      LSD                 (1,2,3)              (1,2,3,4)
  TNF-[alpha]      10.35 [+ or -] 1.77 *  11.52 [+ or -] 1.92 *
 (pg/ g tissue)                                                  0.0001
      LSD                  (1,2)                 (1,2)

Data are expressed as mean  [+ or -]  S.D. of 5 independent
experiments. Numbers between brackets indicate significant correlation.

* P < 0.0001 compared with infected untreated group.

Table 6: Effect of N. Sativa seeds on serum biochemical
parameters in different experimental groups.

Parameters                       Normal                Infected
                                                      untreated
                                   G1                     G2

AFP (ng/ml)               8.32 [+ or -] 0.92 *    60.56 [+ or -] 10.015
LSD                           (2,3,4,5,6)               (1,3,4,5,6)
Fructosamine              0.25 [+ or -] 0.086 *    0.96 [+ or -] 0.17
(mmol/L)                        (2,5,6)                 (1,3,4,5,6)
LSD
TNF-[alpha] (pg/ml)      14.51 [+ or -] 1.2 *     68.21 [+ or -] 9.21
LSD                             (2,5,6)                 (1,3,4,5,6)
IgE (total, Iu/ml)       36.98 [+ or -] 2.47 *   112.94 [+ or -] 7.02
LSD                             (2,5,6)                 (1,3,4,5,6)
GGT (U/L)                18.42 [+ or -] 1.32 *    88.59 [+ or -] 12.74
LSD                             (2,5,6)                 (1,3,4,5,6)
AST (U/L)                33.49 [+ or -] 3.65 *   152.12 [+ or -] 15.21
LSD                             (2,5,6)                 (1,3,4,5,6)
ALT (U/L)                35.01 [+ or -] 3.32 *   142.11 [+ or -] 10.65
LSD                             (2,5,6)                 (1,3,4,5,6)
ALP (U/L)                33.12 [+ or -] 2.55 *   174.79 [+ or -] 20.95
LSD                             (2,5,6)                 (1,3,4,5,6)
Albumin (g/dl)            4.66 [+ or -] 1.31 *     1.45 [+ or -] 0.21
LSD                             (2,5,6)                 (1,3,4,5,6)
Uric Acid (mg/dL)         3.46 [+ or -] 1.6 *     12.79 [+ or -] 2.69
LSD                             (2,5,6)                 (1,3,4,5,6)
Creatinine                1.12 [+ or -] 0.13 *     3.79 [+ or -] 0.19
(mg/dL)                         (2,5,6)                 (1,3,4,5,6)
LSD

Parameters                  Infected-N sativa treated groups
                                   G3                     G4

AFP (ng/ml)              13.03 [+ or -] 1.82 *   15.66 [+ or -] 1.44 *
LSD                            (1,2,5,6)               (1,2,5,6)
Fructosamine              0.27 [+ or -] 0.014 *   0.32 [+ or -] 0.066 *
(mmol/L)                       (2,5,6)                  (2,6)
LSD
TNF-[alpha] (pg/ml)      16.98 [+ or -] 1.5 *    18.21 [+ or -] 2.3 *
LSD                             (2,5,6)                 (2,6)
IgE (total, Iu/ml)       39.59 [+ or -] 2.94 *   41.49 [+ or -] 2.85 *
LSD                             (2,5,6)                 (2,6)
GGT (U/L)                20.74 [+ or -] 1.16 *   22.63 [+ or -] 1.93 *
LSD                             (2,5,6)                 (2,6)
AST (U/L)                35.33 [+ or -] 3.38 *   38.45 [+ or -] 2.12 *
LSD                             (2,5,6)                 (2,6)
ALT (U/L)                38.52 [+ or -] 4.79 *   43.23 [+ or -] 5.66 *
LSD                             (2,5,6)                 (2,5,6)
ALP (U/L)                36.66 [+ or -] 3.24 *   40.69 [+ or -] 3.95 *
LSD                             (2,5,6)                 (2,5,6)
Albumin (g/dl)            4.19 [+ or -] 0.24 *    3.90 [+ or -] 0.27 *
LSD                             (2,5,6)                 (2,5,6)
Uric Acid (mg/dL)         4.17 [+ or -] 0.78 *    5.33 [+ or -] 1.45 *
LSD                             (2,5,6)                 (2,5,6)
Creatinine                1.21 [+ or -] 0.18 *    1.29 [+ or -] 0.14 *
(mg/dL)                         (2,5,6)                 (2,5,6)
LSD

Parameters                  Infected-N sativa
                             treated groups
                                   G5

AFP (ng/ml)               36.93 [+ or -] 2.54 *
LSD                            (1, 2,3,4)
Fructosamine              0.41 [+ or -] 0.03 *
(mmol/L)                        (1,2,3)
LSD
TNF-[alpha] (pg/ml)       32.56 [+ or -] 2.3 *
LSD                            (1,2,3,6)
IgE (total, Iu/ml)       47.87 [+ or -] 2.54 *
LSD                             (1,2,3)
GGT (U/L)                46.34 [+ or -] 3.98 *
LSD                             (1,2,3)
AST (U/L)                52.49 [+ or -] 4.84 *
LSD                             (1,2,3)
ALT (U/L)                55.21 [+ or -] 3.99 *
LSD                            (1,2,3,4)
ALP (U/L)                 3.44 [+ or -] 5.19 *
LSD                            (1,2,3,4)
Albumin (g/dl)            2.51 [+ or -] 0.35 *
LSD                            (1,2,3,4)
Uric Acid (mg/dL)         6.51 [+ or -] 0.81 *
LSD                             (1,2,3)
Creatinine                1.85 [+ or -] 0.19 *
(mg/dL)                        (1,2,3,4)
LSD

Parameters                 Infected-N sativa       ANOVA
                            treated groups           P<
                                   G6

AFP (ng/ml)               40.71 [+ or -] 1.31*     0.0001
LSD                            (1, 2,3,4)
Fructosamine              0.51 [+ or -] 0.025*
(mmol/L)                       (1,2,3,4)           0.0001
LSD
TNF-[alpha] (pg/ml)       40.54 [+ or -] 2.5 *     0.0001
LSD                           (1,2,3,4,5)
IgE (total, Iu/ml)       51.25 [+ or -] 3.38 *
LSD                            (1,2,3,4)           0.0001
GGT (U/L)                50.63 [+ or -] 5.93 *     0.0001
LSD                            (1,2,3,4)
AST (U/L)                66.41 [+ or -] 2.89 *     0.0001
LSD                            (1,2,3,4)
ALT (U/L)                57.38 [+ or -] 4.43 *     0.0001
LSD                            (1,2,3,4)
ALP (U/L)                 74.95 [+ or -] 7.6 *     0.0001
LSD                            (1,2,3,4)
Albumin (g/dl)            2.19 [+ or -] 0.25 *     0.0001
LSD                            (1,2,3,4)
Uric Acid (mg/dL)         7.44 [+ or -] 1.51 *
LSD                             (1,2,3)            0.0001
Creatinine                1.98 [+ or -] 0.16 *
(mg/dL)                        (1,2,3,4)          0.000 1
LSD

Data are represented as mean [+ or -] S.D. of 5 independent
experiments. Numbers between brackets indicate significant
correlation. * P <0.0001 compared with infected untreated
group.
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Article Details
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Author:Mohamed, A.M.; Mahmoud, S.S.; Farrag, Abdel Razik A.
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Date:Dec 1, 2008
Words:10047
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