Research articles
 

By Dr. Rehab M Elghrabawy
Corresponding Author Dr. Rehab M Elghrabawy
Faculty of Pharmacy, Pharmacology Department, - Egypt
Submitting Author Dr. Rehab M Elghrabawy
PHARMACOLOGY

Schistosoma Mansoni, Hepatic Fibrosis, Praziquantel, Rosiglitazone, Propolis.

Elghrabawy RM. Protection Against Schistosomiasis-Induced Hepatic Fibrosis By Modulating The Immune System. WebmedCentral PHARMACOLOGY 2011;2(1):WMC001498
doi: 10.9754/journal.wmc.2011.001498
No
Submitted on: 22 Jan 2011 08:44:44 PM GMT
Published on: 31 Jan 2011 01:20:00 PM GMT

Abstract


Objectives: Hepatic Schistosomiasis resulted in the morbidity from infection due to its complications of liver fibrosis. However, there are few medicines or means available to control and treat fibrosis in Schistosomiasis. The aim of this study was to assess the beneficial effects of immunomodulating agents in hepatic schistosomal fibrosis.
Methods: Three hundred male Swiss albino mice infected with Schistosoma mansoni live cercariae were divided into seven groups: Control; infected (Schistosoma mansoni live cercariae); Praziquantel (500 mg/kg/day); Rosiglitazone (4 mg/kg/day); Propolis (250 mg/kg/day); Bisphenol A diglycidyl ether (BADGE) (30 mg/kg/day); combination of Praziquantel plus Propolis, Praziquantel plus Rosiglitazone, Praziquantel plus BADGE, Rosiglitazone plus BADGE. Blood samples, Liver and intestine were taken for determination of serum interleukin-2, interleukin-10, immunoglobulin E and G, Alanine aminotransferas, Aspartate aminotransferase, hepatic hydroxyproline, immunohistochemical examination of liver tissues and tissue egg count (in liver and colon).
RESULTS: Serum level of IL-2 showed significant increase in mice treated with Praziquantel and Praziquantel plus Propolis, plus Rosiglitazone or plus BADGE and decrease in Rosiglitazone group. Serum IL-10 showed significant decrease in all treated groups except Rosiglitazone and BADGE showed no significant change. Serum IL-10 showed significant decrease in all treated groups except Rosiglitazone and BADGE showed no significant change. Serum IgE, IgG, ALT, AST and hepatic hydroxyproline, showed significant decrease in all treated groups except BADGE showed no significant change.
Conclusions: We can concluded that combination of chemotherapy plus immunomodulating agents modulate cellular and humoral immune responses led to significant reduction in hepatic hydroxyproline content and liver pathology in schistosomal infected mice.


Introduction


Schistosomiasis is one of the most important public health problems affecting Egyptians, especially the rural inhabitants of the Nile Delta [1].
Praziquantel has become the drug of choice against Schistosomiasis. Indeed, it has effectively become the only antischistosomal drug that is commercially available. Praziquantel has activity against all schistosome species with minimal adverse effects, and it is also effective against other trematode and cestode infections [2].
Eggs of Schistosoma mansoni embolize to liver, where the granulomatous inflammatory response induces presinusoidal inflammation and periportal fibrosis. T cells and B cells have also been implicated as playing a regulatory role in schistosome granuloma formation [3].
Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occur in many patients with chronic disease and involves the abnormal accumulation of extracellular matrix (ECM) and hepatic stellate cells (HSCs) [4].
HSCs, is the central event in hepatic fibrogenesis, can undergo the process of activation and transdifferentiate to a myofibroblast-like phenotype characterized by an increase in cell proliferation, loss of vitamin A-storing capability, expression of α- SMA (α-smooth muscle actin) and overproduction of ECM components, especially type I collagen [5].
Peroxisome proliferator activated receptor γ (PPARγ) activation has been found to affect diverse physiological and pathophysiological events, including stimulation of adipocyte differentiation, regulation of lipid metabolism, inhibition of cell proliferation and induction of apoptosis [6].
Propolis also exhibits anti-inflammatory effects against acute and chronic models of inflammation (formaldehyde-and adjuvant-induced arthritis, carrageenan-and prostaglandin (PGE)-induced paw oedema, cotton pallet granuloma).The exact mechanism of the anti-inflammatory actions of propolis is still unclear [7].
Among the compounds tested, only caffeic acid phenethyl ester (CAPE) and galangin contributed to the anti-inflammatory activity of propolis; however, the contribution of CAPE was greater. Propolis also exhibits immunostimulatory and immunomodulatory effects in vitro on macrophages, while in vivo it increases the ratio of CD4 and CD8 cells in mice [8].
AIM OF THE WORK
The aim of this study was to assess the possible disturbance in the immune system and mechanism through which immunomodulating agents can produce their beneficial effects in Schistosomiasis induced hepatic fibrosis.

Methods


Experimental Animals:
Male Swiss albino mice of CD strain, weighing 18-20 gm were used in this study. Mice were obtained from Schistosome biological supply center, Theador Bilharz Research Institute (TBRI, Imbaba, Giza). Schistosoma mansoni (John Bruce Egyptian strain) cercariae were obtained from Biomphalaria alexandrina snails (TBRI). The monthly meeting of pharmacology department approved the study and it is done under standard ethical measures for experimental study. Mice infection:
Mice were infected with Schistosoma mansoni live cercariae.
Classification of Animals:
Mice were divided randomly into seven groups:
1.The first group (Control group, n=8):
In this group, mice were not infected or immunomodulated. This group is served as a control group.
2. The second group (control Infected untreated group, n=8):
In this group, mice were infected with Schistosoma mansoni live cercariae.
3. The third group (Praziquantel group, n=8):
In this group, mice were infected with Schistosoma mansoni live cercariae then treated by Praziquantel (500 mg/kg/day for 2 days by intra-gastric administration 4 weeks after infection) [9]. It was dissolved in water as suspension.
4. The fourth group (Rosiglitazone group, n=8):
In this group, mice infected with Schistosoma mansoni live cercariae then treated by PPAR-γ modulating agent (Rosiglitazone, 4 mg/kg/day by intra-gastric administration from 5th to 12th week after infection) [10].
5. The fifth group (Propolis group, n=8):
In this group, mice were infected with Schistosoma mansoni live cercariae then treated by Propolis (250 mg/kg/day by intra-gastric administration from 5th to 12th week after infection) [11].
6. The sixth group (Praziquantel plus Propolis group, n=8):
In this group, mice were infected with Schistosoma mansoni live cercariae then treated by combination of Praziquantel (500 mg/kg/day for 2 days by intra-gastric administration 4 weeks after infection) plus Propolis (250 mg/kg/day by intra-gastric administration from 5th to 12th week after infection).
7. The seventh group (Praziquantel plus Rosiglitazone group, n=8):
In this group, mice were infected with Schistosoma mansoni live cercariae then treated by Praziquantel (500 mg/kg/day for 2 days by intra-gastric administration 4 weeks after infection) plus (Rosiglitazone, 4 mg/kg/day by intra-gastric administration from 5th to 12th week after infection).
Determination of total Serum mouse Interleukin-2 (IL-2) and Interleukin-10 (IL-10): Sandwich enzyme linked immunosorbent (ELISA) was used to measure serum IL-2 level. It was determined with commercially available reagents and ELISA kits purchased from Biosource International (Camarillo CA , USA).
Determination of total Serum mouse immunoglobulin E (IgE): Enzyme-linked immunosorbent assays (ELISA) was used to measure serum IgE level. It was determined with commercially available reagents and ELISA kits purchased from the BD OptEIA™ Set, USA .
Determination of total Serum mouse immunoglobulin G (IgG): Enzyme-linked immunosorbent assays (ELISA) was used to measure serum IgG level. It was determined with commercially available reagents and ELISA kits purchased from the the Life Diagnostics, Inc. kit, USA.
Determination of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST):
Serum alanine aminotransferase and aspartate aminotransferase was determined colorimetrically according to the method of Reitman and Frankel, using Diamond kit.
Determination of hepatic Hydroxyproline content:
The total collagen present in the liver was determined by estimating hydroxyproline content using base hydrolysis for the dissolution of tissue as described previously [12].
Immunohistochemical examination:
Liver tissues were fixed in buffered neutral formalin (10%) and processed to make paraffin blocks and examined under light microscope.
Tissue egg count (in liver and colon):
The number of schistosoma mansoni eggs in the perfused liver and in the intestine (from duodenum to rectum) of mice was estimated after alkali digestion as described by Cheever (1968) [13] and the percentage of egg/gm tissues was calculated.
Statistical Analysis:
Results were expressed as the mean ± SD. Regression analysis and correlation coefficient were done for standard curves. Comparison between different groups was carried out by one way analysis of variance (ANOVA). The level of significance was set as P


Results


Mice infected with Schistosoma mansoni showed a significant time dependent decrease in serum IL-2 level compared with the control group. The highest decrease in the serum IL-2 was found after 12 weeks. It showed that treatment of infected mice with Propolis, BADGE and Rosiglitazone plus BADGE showed no significant change in the serum IL-2 at all time points determined up to 12 weeks compared with their respective infected groups. However, treatment of infected mice with Praziquantel alone or in combination with Propolis, Rosiglitazone and BADGE showed a significant time dependant increase in the serum IL-2 when compared with their respective infected groups. On the other hand, treatment of infected mice with Rosiglitazone alone revealed a significant time dependent decrease in the serum IL-2. The maximum decrease was after 12 weeks when compared with their respective infected groups (Table 1, p < 0.05).
The present study showed that mice infected with Schistosoma mansoni showed a significant time dependent increase in serum IL-10 level compared with the control group. The highest increase in the serum IL-10 was found after 12 weeks. Treatment of infected mice with Praziquantel, Propolis, Praziquantel plus Propolis, Praziquantel plus Rosiglitazone and Praziquantel plus BADGE showed significant decrease in the serum IL-10 at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were after 12 weeks. However, treatment of infected mice with Rosiglitazone, BADGE and combination of Rosiglitazone plus BADGE showed no significant change in the serum IL-10 at all time points determined up to 12 weeks compared with their respective infected groups (Table 2, p > 0.05).
Mice infected with Schistosoma mansoni live cercariae showed a significant time dependent increase in serum IgE level compared with the control group. The highest increase in the serum IgE was found after 12 weeks. It showed that treatment of infected mice with Praziquantel, Propolis, Rosiglitazone, and Praziquantel plus Propolis, Praziquantel plus Rosiglitazone and Praziquantel plus BADGE showed significant decrease in the serum IgE at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were after 12 weeks. However, treatment of infected mice with BADGE and Rosiglitazone plus BADGE showed no significant change in the serum IgE at all time points determined up to 12 weeks compared with their respective infected groups (Table 3, p > 0.05).
Infection with Schistosoma mansoni showed a significant time dependent increase in serum IgG level compared with the control group. The highest increase in the serum IgG was found after 12 weeks. Treatment of infected mice with Praziquantel, Propolis, Rosiglitazone, and combination of Praziquantel plus Propolis, plus Rosiglitazone or plus BADGE resulted significant decrease in the serum IgG at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were after 12 weeks. While treatment of infected mice with BADGE and Rosiglitazone plus BADGE showed no significant change in the serum IgG at all time points determined up to 12 weeks compared with their respective infected groups (Table 4, p > 0.05).
In this study, mice infected with Schistosoma mansoni showed a significant increase at all time points in serum ALT level compared with their corresponding control group. The increase in the serum ALT was found after 12 weeks. However treatment of infected mice with Praziquantel, Propolis, Rosiglitazone and combination of Praziquantel plus Propolis, plus Rosiglitazone or plus BADGE showed significant decrease in the serum ALT at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks. On the contrary treatment of infected mice with BADGE and Rosiglitazone plus BADGE showed no significant change in the serum ALT at all time points determined up to 12 weeks compared with their respective infected groups (Table 5, p > 0.05).
Table 6, showed that mice infected with Schistosoma mansoni live cercariae resulted a significant increase at all time points in serum AST level compared with their corresponding control group. The increase in the serum AST was found after 12 weeks. While treatment of infected mice with Praziquantel, Propolis, Rosiglitazone and combination of Praziquantel plus Propolis, plus Rosiglitazone or plus BADGE showed significant decrease in the serum AST at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks. However treatment of infected mice with BADGE and Rosiglitazone plus BADGE showed no significant change in the serum AST at all time points determined up to 12 weeks compared with their respective infected groups (Table 6, p > 0.05).
In this study, mice infected with Schistosoma mansoni showed a time dependent increase in the hepatic hydroxyproline content compared with the control group. The highest increase in the hepatic hydroxyproline was found after 12 weeks. While treatment of infected mice with Praziquantel, Propolis, Rosiglitazone and combination of Praziquantel plus Propolis, plus Rosiglitazone or BADGE showed significant decrease in the hepatic hydroxyproline content after 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks. On the contrary treatment of infected mice with BADGE and Rosiglitazone plus BADGE showed no significant change in the hepatic hydroxyproline content after 12 weeks compared with their respective infected groups (Table 7, p > 0.05).
Table 8, showed that treatment of infected mice with BADGE, Rosiglitazone and Rosiglitazone plus BADGE showed no significant change in the hepatic egg count after 12 weeks compared with their respective infected groups. However in Praziquantel, Propolis, combination of Praziquantel plus Propolis, combination of Praziquantel plus Rosiglitazone and combination of Praziquantel plus BADGE groups, the mean number of Schistosoma mansoni ova per gram liver was significantly decrease compared with their respective infected groups (Table 8, p < 0.05).
Also table 8, showed that the mean number of Schistosoma mansoni ova per gram intestine in the infected group was 4841.00 ± 311.06. Treatment of infected mice with BADGE, Rosiglitazone and Rosiglitazone plus BADGE showed no significant change in the intestinal egg count after 12 weeks compared with their respective infected groups. While treatment of infected mice with Praziquantel, Propolis, combination of Praziquantel plus Propolis, combination of Praziquantel plus Rosiglitazone, and combination of Praziquantel plus BADGE showed that the mean number of Schistosoma mansoni ova per gram intestine was significantly decreased compared with their respective infected groups (Table 8, p < 0.05).
Under the light microscope, the liver from animals infected with schistosoma mansoni scarified after 12 weeks showed strong positively for collagen fibre deposition (Photomicrograph 1) and minimal or absent collagen type IV reaction (Photomicrograph 2). Treatment of infected mice with Praziquantel showed significant increased positively for collagen cells in the mouse liver section in comparison with normal control group (Photomicrograph 3). Similarly, treatment of infected mice with Rosiglitazone showed dense positively for collagen type IV in the mouse liver sections in compared with infected control group (Photomicrograph 4). However, animals treated with the combination of Praziquantel plus Rosiglitazone showed dramatic decrease in positively for type IV collagen in comparison with infected liver sections (Photomicrograph 5). On the other hand, treatment of infected mice with BADGE alone or in combination with Rosiglitazone or Praziquantel showed dense deposition of positive collagen bundles (Photomicrograph 6,7,8). In contrast, treatment of infected mice with Propolis showed very thin collagen within the granuloma (Photomicrograph 9). The combination of Praziquantel plus Propolis showed that most of granulomas in Praziquantel plus Propolis treated mice contained very fine collagen fibres with a minimal interstitial and perivascular collagen deposition (Photomicrograph 10).

Discussion


Schistosomiasis is the major public health problem in rural Egypt. The main agent of human schistosomiasis is Schistosoma mansoni [14]. Previous study reported that the severity of the disease has been shown to be a consequence of immunopathological processes that led to the development of fibrosis caused by delayed type inflammatory granulomatous reaction to the schistosome eggs that are trapped in the small vessels of the liver. Granuloma formation around entrapped eggs is generally regarded as the key pathological event in this disease. However, granulomas also exert protective effects, since mice which fail to develop granulomas experience extensive hepatotoxic effects around the parasite eggs [15].
The present study was designed to assess the possible disturbances in the immune system that is associated with Schistosomiasis induced hepatic fibrosis and the possible mechanism through which the different immunomodulating agents can produce their beneficial effects in Schistosomiasis induced hepatic fibrosis. This was achieved by determination of cellular immunity (Serum IL-2 and IL-10) and humoral immunity (IgG and IgE), ALT, AST, hepatic hydroxyproline content as a fibrotic markers and egg count in tissues (liver and intestine) as well as the histopathological and immunohistochemical examination of the liver.
In this study, mice infected with Schistosoma mansoni live cercariae showed a time dependent decrease in serum IL-2 level compared with the control group. The highest decrease in the serum IL-2 was found after 12 weeks. On the other hand mice infected with Schistosoma mansoni live cercariae showed a time dependent increase in serum IL-10, IgE, IgG, ALT, AST and hepatic hydroxyproline levels compared with the control group. The highest increase was found after 12 weeks. Also, our results revealed that the mean number of Schistosoma mansoni ova per gram liver and intestine in the infected group were 4447.38 ± 376.24 and 4841.00 ± 311.06, respectively. This results supported by histopathological examination of slice of the liver from fibrotic model (infected group) revealed the presence of granulomatous reactions within the portal tracts. Each granuloma were found around Schistosoma ovum and composed of cellular collection of eosinophils, histocytes, lymphocytes and plasma cells. Peripheral fibroblasts with variable amounts of collagen deposition were seen in fibrocellular and fibrous types. The segregation of liver by collagen fibers, necrotic lesions in the granulomas and inflammatory cells was found extensively in the periphery of granulomas in comparison with normal control group. Moreover, immunohistochemical examination of liver section showed large number of positive collagen cells distributed not only in the central venule pericytes and the linkage region of lobules but also in fibrous proliferation and Disec cavities.
These results in agreement with Cheever et al., 1994 [16], who found that the magnitude of the granulomatous fibrosis and the severity of the fibrotic response appear to be dependent on the strain of host involved. While other investigation have been found that cytokines regulate the fibrotic process, in both human [17] and murine infections [18]. Finally, the outcome of fibrosis is also influenced by the degradative matrix metalloproteinases [19], which break down the collagens produced, and by the tissue inhibitors of these metalloproteinases, which enhance fibrosis. Metalloproteinases have been localized within the infected livers [20]. Yamashita et al., 1987 [21] observed an 80-90 % reduction in the production of IL-2 produced by infected mice compared with controls, which was not accounted for by a reduction in the number of IL-2 producing T cells, suppressive macrophages. On the contrary Khalil et al., 1995[22], revealed that IL-2 level was higher in all patients infected with schistosomiasis compared to normal control group and the level was higher in lightly infected group than heavily infected ones with no respect to age.
Praziquantel is an effective, yet safe non hepatotoxic schistosomicides drug that may reverse schistosomal induced liver pathology and eliminate the parasite worms from infected host [22]. Also, it was able to reduce the number of ova either in liver or intestine with complete absence of immature stages and increase of the number of dead ova [23]. After antischistosomal therapy with Praziquantel IL-2 became up to normal within 3 months [22]. These results are in accordance with the finding reported in this study since; treatment with Praziquantel showed significant increase in the serum IL-2 when compared with their respective infected groups. The maximum increase was found after 12 weeks. On the other hand, treatment of infected mice with Praziquantel revealed significant decrease in the serum IL-10 when compared with their respective infected group. The maximum decrease was found after 12 weeks. IL-10 involved in the regulation of T-cell responses in cases of human schistosomiasis, IL-10 undoubtedly plays a key role in controlling infection [24]. Martins-Leite et al., 2008 [25] observed a significant reduction in the level of IL-10 following treatment with Praziquantel in individuals with and without hepatic fibrosis. Previous research involving subjects with chronic schistosomiasis had suggested that the levels of IL-10 secretion are dependent on the intensity of infection, as determined by the number of eggs in the stool [26].
The present study showed that administration of Praziquantel induced significant decrease in the serum IgE and IgG at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks. Stevens et al., 1983 [27] found that, the concentration of total serum IgE and IgG were lowest in patients mono-infected with Schistosoma haematobium and highest in those with a mixed infection. Schistosoma mansoni may be considered to release more soluble antigenic material into circulation than Schistosoma haematobium, stimulating more intensively IgE and IgG producing B lymphocytes. This should consequently lead to higher concentrations of IgE and IgG. The rapid reduction of serum IgE after specific chemotherapy has already been noted by Ito et al., 1972 and Kojima et al., 1972 [28,29] who found that with Schistosoma japonicum IgE concentrations reduced approximately by 50 % 2-4 months after chemotherapy. The noted similar tendency of IgG to fall after efficient chemotherapy has been confirmed in a study of more heavily infected patients.
In the present study, mice infected with Schistosoma mansoni showed a time dependent increase in serum ALT and AST levels compared with their control group. The highest increase in the serum ALT and AST were found after 12 weeks. The elevation of serum ALT and AST of infected mice seems to be a consequence of the damage of hepatic cells and/or impaired permeability of cell membranes, or may be due to heavy schistosome egg deposition. Treatment of infected mice with Praziquantel revealed significant decrease in the serum ALT and AST when compared with their respective infected group. The maximum decreases were found after 12 weeks. These results are in accordance with Mahmoud et al., 2002 [23], who found that mice infected with Schistosoma mansoni live cercariae showed elevation of serum ALT and AST levels compared with the normal control group and treatment of infected mice with Praziquantel revealed reduction in the serum ALT and AST nearly to the normal values at 16 weeks post infection.
Praziquantel treatment led to highly significant decrease in eggs per gram tissue (liver and intestine). This reduction is manifested in egg deposition in the liver and large intestine. These results are in basic agreement with the finding of Issa, 2007 [11]. The main possible explanations for these results are that, Praziquantel may reduces the Schistosoma mansoni worm and/or regulates the adhesion molecules playing a key role in egg trapping and increase the mobilization of eggs from liver and intestine to the exterior of the body in the feces. These data may suggest an anti-fibrotic effect of Praziquantel in the early stage of liver fibrosis. Praziquantel might able to block liver fibrosis through killing parasite to alleviate liver inflammation [11,23].
Also, the present study was designed to assay the total collagen contents of granulomatous livers, measured as hydroxyproline content. Infected mice treated with Praziquantel showed significant decrease in the hepatic hydroxyproline content. The maximum decrease was found after 12 weeks. It was interesting to find that, Praziquantel affect the level of hepatic hydroxyproline content but did not affect histopathological changes in the liver section. This finding was confirmed histopathologically and immunohitochemically as liver section from Praziquantel treated mice preserved non-significant changes in the fibroplasias, hepatocellular necrosis and inflammatory cell infiltration in the liver sections stained with H & E when compared with infected fibrotic group. In agreement with this result, Chen et al., 2008 was found that, treatment of mice with Praziquantel alone did not show any significant changes in the liver sections compared with infected non-treated mice.
In this study, treatment of infected mice with Propolis alone showed no significant change in the serum IL-2 at all time points determined up to 12 weeks compared with their respective infected groups. On the other hand, treatment of infected mice with Praziquantel plus Propolis showed a time dependant increase in the serum IL-2 when compared with their respective infected groups. The maximum increases were found after 12 weeks. These results could be explained by Popova et al., 2004 [30], who reported that several types of flavonols (contents of Propolis) stimulate human peripheral blood leukocyte proliferation. They significantly increase the activity of helper T cells, cytokines, interleukin-2, interferon-γ and macrophages and are thereby useful in the treatment of several diseases caused by immune dysfunction [31]. It is thus apparent that the immunostimulatory effect produced by the Propolis may be due to cell mediated and humoral antibody mediated immune response. So that, administration of Propolis and its extract increase IL-2 [32].
In our study, treatment of infected mice with Propolis and combination of Praziquantel plus Propolis showed significant decrease in the serum IL-10, IgE and IgG at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks. In agreement with these results, Sy et al., 2006 [33] found that, administration of Propolis and its extract strongly inhibit IL-10 secretion and the serum levels of IgG and IgE and exhibit anti-inflammatory effects.
In the present study, all infected mice showed a significant increase in serum ALT, AST which are measures of liver affection. This seems consequent with hepatic cell damage and impaired cell membrane permeability or due to heavy schistosome egg deposition and these in agreement with Giboda et al., 1994 [34].
While treatment with Propolis and combination of Praziquantel plus Propolis in the present study demonstrates that it produced an effective action against the hepatosplenic damaging effect, caused by Schistosoma mansoni infection, as shown by reducing the number of ova count in the liver and in the intestine and reduced the granuloma size. Eventually, the liver functions were improved as evidenced by a decrease of the elevated serum levels of ALT and AST when compared with their respective infected group. The maximum decreases were found after 12 weeks. The effect of Propolis could, at least partly, be attributed to drug-induced modulation of the immune response to schistosome eggs trapped in the liver. In murine schistosomiasis, a variety of cytokines and lymphokines are implicated as mediators of the granulomatous inflammatory response [11].
The present study demonstrated that Propolis administration alone or in combination with Praziquantel significantly decrease tissue load compared with infected untreated mice. This reduction was manifested in egg deposition in the liver and large intestine. This reduction was significantly different from infected untreated mice. These results could be explained by Ibrahim et al., 2000 [35] who found that, the reduction in the number of eggs in intestine and liver of mice infected with Schistosoma mansoni, after administration of Propolis, is most probably secondary to that occurring in the worm load and its direct effects on the eggs, due to the enhancement of the immunological reactions. It has been recorded that when treatment was done with a daily dose of 250 mg/kg, starting from the 1st day of infection and with sacrifice at 16 weeks post-infection, there was a reduction in worm load compared to control, but the worms were not completely eradicated. Other study found that administration of Propolis to infected mice with Schistosoma mansoni produced direct effect on the eggs due to the enhancement of the immunological reactions. These findings explained by reduction in worm load compared to control group, but the worms were not completely eradicated [11].
This study found that, treatment of infected mice with Propolis alone or in combination with Praziquantel in this study showed significant decrease in the hepatic hydroxyproline content after 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks. The combination of Praziquantel plus Propolis was the best combination to decrease the value of hepatic hydroxyproline content. In agreement with these results, Chen et al., 2008 [10], found that administration of Propolis inhibits tTG activation and prevented the development of Thioacetamide (TAA)-induced liver cirrhosis. As Propolis is safe for consumption by humans, it may have a beneficial role in chronic liver diseases caused by ongoing hepatic damages.
Immunohistochemical examination of liver section from mice treated with Propolis alone or in combination with Praziquantel showed few expression of type IV collagen which found only in the central venule pericytes and the linkage region of lobules from the mouse liver and these results explained by Fitzpatrick et al., 2001[36], who found that caffeic acid phenylethyl ester (CAPE) is an active component of Propolis from honeybee hives and is widely known for its antiviral, anti-inflammatory, and immunomodulatory properties. Also, CAPE can suppress NF-κB activation and thereby inhibits inflammatory responses and significantly reduces the level of pro-inflammatory cytokines (TNF-α and IL-1β) [37].
In the present study, it was found that, treatment of infected mice with Rosiglitazone alone revealed a time dependent decrease in the serum IL-2. The maximum decrease was found after 12 weeks. While, treatment of infected mice with Praziquantel in combination with Rosiglitazone showed a time dependant increase in the serum IL-2 when compared with their respective infected groups. The maximum increases were found after 12 weeks. On the other hand, treatment of infected mice with Rosiglitazone alone showed no significant change in the serum IL-10 at all time points determined up to 12 weeks compared with their respective infected groups. Treatment of infected mice with Praziquantel plus Rosiglitazone showed significant decrease in the serum IL-10 at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 weeks.
These results in agreement with Clark et al., 2000 [38] who suggested an immunoregulatory role of PPAR in macrophages and monocytes, but recently also on lymphocyte function. It has been shown that PPAR ligands inhibit T helper cell responses in terms of inhibition of interleukin (IL)-2 production by T cell clones, while not inhibiting proliferation of such clones. Similarly, data from murine splenocytes showed that PPAR-α and PPAR-γ ligands decreased IL-2 and interferon (IFN)-γ production in mitogen-activated cells, but had modest ejects on proliferation [39]. While IL-10 production and leukocyte infiltration remained unchanged [40].
Rühl et al., 2003 [41] demonstrated that the PPAR-α ligand and, to a greater extent, the PPAR-γ ligand inhibit IgE production and also the production of other isotypes (IgG and IgM) in vitro and in vivo. Co-culture experiments of B cells and monocytes showed that the PPAR ligand-associated inhibition of IgE production is not directly mediated through activated B lymphocytes, but rather primarily indirectly mediated via regulatory signal pathways of monocytes. The analysis of supernatants from peripheral blood monocyte (PBMC) in the presence of PPAR ligands reveals that inhibition of IgE synthesis is most likely related to the reduced secretion of several cytokines. In this study treatment of infected mice with Rosiglitazone and Praziquantel plus Rosiglitazone showed significant decrease in the serum IgE and IgG at all time points determined up to 12 weeks compared with their respective infected groups. The maximum decreases were found after 12 week.
Our experiments showed that treatment of infected mice with Rosiglitazone, ameliorated hepatocyte degeneration, necrosis and infiltration of inflammatory cells and reduced the scores of necroinflammation significantly compared with model group. Liver functions (ALT, AST) were also improved apparently, treatment of infected mice with Rosiglitazone and combination of Praziquantel plus Rosiglitazone revealed significant decrease in the serum ALT and AST when compared with their respective infected group. The maximum decreases were found after 12 weeks.
These results demonstrated that PPAR-γ agonists also had anti-inflammatory effects, and subsequently retarded the progression of hepatic fibrosis in mice. Currently two TZDs, rosiglitazone and pioglitazone, have been approved by U.S. Food and Drug Administration (FDA) for treatment of type 2 diabetes. A wealth of clinical studies indicated that rosiglitazone and pioglitazone have no hepatotoxicity [42]. Our experiments also showed that after administration with Rosiglitazone for 5 weeks, no mice were observed to have hepatotoxicity; moreover, their liver functions (ALT, AST) were improved greatly compared with model mice. In conclusion Rosiglitazone, a PPAR-γ ligand, greatly retards the progression of experimental hepatic fibrosis through inhibition of HSC activation and amelioration of hepatocyte necroinflammation in mice. Therefore, it is a potential new antifibrotic drug for clinical application.
Immunohistochemical examination of liver sections from mice treated with Rosiglitazone alone or in combination with Praziquantel showed few collagens in the central venule pericytes and the linkage region of lobules from mouse liver. These results also suggest that PPAR-γ ligand; rosiglitazone can impede liver fibrosis after Schistosoma infection. In agreement with these results, Chen et al., 2008 [10] was found that treatment of mice with Rosiglitazone showed a significant reduction of liver fibrosis induced by Schistosoma japonicum. The effect of this compound on preventing liver fibrosis may be through down regulation of liver TGF-β1 and collagens.
In order to elucidate whether the anti-inflammatory effect of rosiglitazone observed here is related to activation of the PPAR-γ receptor, we also investigated the effect of a PPAR-γ antagonist, bisphenol diglycidyl ether (BADGE), on the anti-inflammatory effects of rosiglitazone. The present study demonstrated that, pre-treatment of animals with BADGE attenuates the protective effects of rosiglitazone. Thus, we propose that (i) activation of PPAR-γ reduces the development of inflammation, and (ii) that the activation of PPAR-γ contributes to the anti-inflammatory effects of rosiglitazone.
Treatment of infected mice with BADGE and Rosiglitazone plus BADGE showed no significant change in the serum IL-2, IL-10, IgE, IgG, ALT, AST, hepatic hydroxyproline content and egg count (in liver and intestine) at all time points determined up to 12 weeks compared with their respective infected groups. Histological and immunohistochemical examination showed no significant change compared with fibrotic untreated mice.
Treatment of monocytes and macrophages with high concentrations of PPAR-γ agonists reduced secretion of inflammatory cytokines and inhibited macrophage activation. In particular, treatment of monocytes with Thiazolidinedione (TZD) reduced release of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 [43]. In the present study, we found that the co-administration of BADGE and Rosiglitazone blocked these effects of the PPAR-γ agonist. These findings, therefore, confirm (i) that inflammation results in the activation and the subsequent expression of pro-inflammatory inflammatory cytokines, and suggest (ii) that rosiglitazone activates the PPAR-γ receptor resulting in the reduction of the release of these of inflammatory cytokines.

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Faculty of Pharmacy, Tanta University, Egypt

Competing Interests


Nil

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