Silymarin treatment reduces granuloma and hepatic fibrosis in experimental schistosomiasis
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- Mata-Santos, H.A., Lino, F.G., Rocha, C.C. et al. Parasitol Res (2010) 107: 1429. doi:10.1007/s00436-010-2014-8
The schistosomiasis is a parasitic infection with relevant social impact and an important health problem in many countries around world. The pathology of this infection is characterized by a granulomatous reaction around parasite eggs and by hepatic fibrosis. Silymarin, a complex compound isolated from Silybum marianum (L.) Gaertner, have been described as hepatoprotective, antioxidant, antifibrotic, immunomodulator, and anti-neoplastic agent. Some of these capacities could potentially protect against pathology in schistosomiasis. Herein, we evaluated the effects of silymarin on parasite burden, granuloma sizes, and liver fibrosis, which are associated with severity and morbidity of this disease. BALB/c mice treated intraperitoneally with 10, 20, or 25 doses of silymarin (10 mg kg−1) suspended in carboxymethylcellulose were analyzed at 55 days post-infection. Silymarin (1) did not affect parasite oviposition capacity; (2) reduced granulomatous peri-ovular reaction in the liver, and (3) decreased hepatic fibrosis in this infection. Taken together, these data suggest that treatment with silymarin at acute phase of schistosomiasis may result in a mild course of murine schistosomiasis and can be a promising complementary treatment reverting sequelae of this infection.
Schistosomiasis is a parasitic disease of great social impact due to its morbidity. This disease affects 200 million people and has a very wide distribution, mainly in third world countries (Hotez et al. 2007; Ross et al. 2002). The distribution of this infection is increasing as new dams for hydroelectricity or for irrigation have been constructed. Besides this fact, the big cities of the third world have grown with inadequate sanitation, which contributes to the expansion of this parasitic disease. The etiological agent is from genus Schistosoma, which has three important species: Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium. The adult parasites live inside veins where female start oviposition. Although morbidity is frequently associated with inflammatory granulomatous reaction and the subsequent liver fibrosis, drugs used to treat this disease are generally unable to reduce these pathological sequelae. The fibrosis evolves to portal hypertension and sometimes fatal bleeding. Therefore, the search for new strategies able to reduce granuloma formation and fibrosis or its sequelae is quite relevant for treatment of this disease.
Empiric use of silymarin for hepatic injuries has more than 2,000 years. It is composed mainly by flavonolignans including silychristin (27.9%), silydianin (2.9%), silybin A (19.3%), silybin B (31.3%), isosilybin A (8.2%), and one flavonoid taxifolin (4%) as determined by HPLC (He et al. 2002). Nowadays, it has been used as positive control for a screening of new drugs with hepatoprotective. This effect could be attributed to its role as antioxidant (Channabasavaraj et al. 2008), antifibrotic (Jia et al. 2001), and anti-inflammatory/immunomodulatory agent. Moreover, silymarin has been pointed as good candidate to anti-neoplastic treatment (Ramasamy and Agarwal 2008). The response on immune system could be attributed to its action on T lymphocytes (Gharagozloo et al. 2010; Johnson et al. 2003). Although silymarin mechanisms were not completely clarified, their anti-inflammatory activity could be at least partially attributed to its inhibitor role on pleiotropic NF-kB (Lin et al. 2008) and on reduction of TNF, IL-1α, IL-6 (Agarwal 2000), and IL-2 and IL-4 (Johnson et al. 2002).
In view of these properties of silymarin, we have used an experimental model to investigate its effects on the pathogenesis of schistosomiasis, in which exuberant granulomatous reaction and liver injuries are exhibited during infection. In a previous paper, we showed that treatment with anti-inflammatory drugs such as dexamethasone can cause a decrease in granuloma size, a reduction in peri-ovular fibrosis deposition and improved morphological and hematological parameters (Pyrrho et al. 2004; Pyrrho et al. 2002). Herein, we investigated the effects of silymarin on egg burden, granuloma sizes, and fibrosis. Our results show that although parasite egg oviposition is not affected, the treatment with silymarin leads to a decrease in hepatic granulomas area and to a reduction in liver fibrosis. These results indicate that silymarin treatment can minimize the deleterious effects of experimental S. mansoni infection.
Materials and methods
Animals, drug and infection
Adult BALB/c female mice (7–8 weeks) were infected with 80 cercariae of S. mansoni BH strain, by cutaneous route. Prior to the experiments, we used oral route for treatment, but both infected and non-infected mice suffered weight loss probably due to oral injuries caused by the high number of doses, so we chose the intraperitoneal route. The best dosage of silymarin was established using curve-response assays. We standardized 10 mg kg−1, which was also used by other authors (He et al. 2002; Johnson et al. 2003; Johnson et al. 2002).
Silymarin was purchased from Sigma-Aldrich (USA; batch no. 107K0762). It was suspended in 1% carboxymethylcellulose (CMC) (Sigma-Aldrich, USA) (Channabasavaraj et al. 2008; Huber et al. 2008) to avoid quick precipitation. The drug was administered by intraperitoneal route every 48 h, until the end of the experiment at 55 days post-infection (dpi). Non-infected controls (N) and infected (I) mice were divided in seven groups: non-treated (N and I), treated with 20 doses of CMC (N + CMC and I + CMC), and treated with 10 (I + SIL-10D), 20 (I + SIL-20D), or 25 doses (I + SIL-25D) of silymarin. The group that received 10 doses started treatment at 35 dpi; 20 doses, 15 dpi; and 25 doses, 5 dpi. Animals from all groups (at least eight per group) were maintained in controlled temperature and light conditions, fed with balanced diet and sterile water ad libitum, and submitted to euthanasia under anesthesia at 55 dpi. Livers, spleens, and intestines were removed, and their weights were determined. All experimental procedures were approved and conducted in accordance with guidelines for care and use of laboratory animals (CEUA) of Centro de Ciências da Saúde-UFRJ (protocol number DBFCICB032), which conform to the National Institute of Health (Bethesda, MD, USA) guidelines.
Hepatic and intestinal tissues were digested as described by Cheever (Cheever 1968). Briefly, tissues were maintained in 4% KOH at room temperature for approximately 12 h, followed by 1 h incubation at 37°C. Results were expressed as eggs per gram of liver. Eight independent samples were counted.
Transversal sections of all liver lobes were collected, fixed in 4% buffered formaldehyde solution and embedded in paraffin. Five micrometer sections were stained with hematoxylin–eosine (H–E) and read by bright field microscopy. The area of hepatic granuloma was determined in histological sections from 20 to 30 granulomas per animal, containing central viable eggs, randomly chosen. The granuloma area was manually delimited in H–E images, captured by CCD camera using bright field microscopy and automatically processed with Scion Image calibrated software (version 4.0.3, Scioncorp, http://www.scioncorp.com). All evaluations were performed by two different blinded observers.
Hydroxyproline quantification was determined to evaluate collagen amount from liver tissue. This determination was performed as described by Stegemann and Stalder (Stegemann and Stalder 1967). Briefly, liver were maintained in acetone at room temperature until complete dehydratation, followed by cloridric acid hydrolization for overnight incubation at 107°C. Colorimetric assay was then performed using chloramine-T buffer (Sigma, USA), Ehrlich’s reagent (Sigma, USA), and perchloric acid (Merck). The results were expressed as milligram of hydroxyproline per liver gram. Groups were represented by three independent pools of livers, each composed of three organs.
Statistical analysis was performed with the SigmaPlot® for Windows software, version 8.0 (SPSS Inc.). Comparison between groups was done by a one-way analysis of variance (ANOVA), followed by non-paired Student t test. Values of p < 0.05 were considered as significant.
Herein, we investigated the effects of silymarin treatment using the dosage 10 mg/kg in experimental schistosomiasis. Some clinical, parasitological, histological, and fibrotic parameters were evaluated.
Many effects of silymarin have been reported along the years. Herein, we demonstrated that treatment with silymarin in experimental schistosomiasis led to reduction of granuloma area and hepatic fibrosis, without interfering in splenomegaly. These data together with an observed reduction in hepatomegaly could reflect a decrease in experimental schistosomiasis morbidity.
As treatment started before complete maturation of parasite (28–30 dpi) in some groups (I + SIL-20D and I + SIL-25D), we investigated if silymarin could interfere in parasite maturation. We did not observe any alteration on parasite oviposition capacity, as could be seen by the same number of eggs among infected treated and non-treated mice in both hepatic and intestinal tissue (data not shown). Although, silymarin and its derivatives present anti-viral and antimicrobial action in some models (Ferenci et al. 2008; Lee et al. 2003), apparently it has no inhibitory effect in a metazoan such as S. mansoni. Certainly, as our experimental groups have similar egg burden, it is feasible to compare liver fibrosis and granuloma sizes among them. Different data was observed by Ibrahim et al. who showed that in C57BL/6 mice previously immunized with SEA and treated orally with silymarin, there was a significant reduction in parasite burden or oviposition capacity (Ibrahim et al. 2010), differences that could probably be explained by immunization.
As many other infectious disease, schistosomiasis induces hepatosplenomegaly. Prior works have correlated hepatosplenomegaly with schistosomiasis morbidity, and a reduction in both spleen and liver weights was associated with mild morbidity (Henderson et al. 1993). Herein, we showed that the group treated with 25 doses of silymarin had a reduction in hepatomegaly, but no differences were observed in splenomegaly. The reduction on hepatomegaly observed here (I + SIL-25D) was associated with decreased granuloma sizes and fibrosis, but no changes in spleen weights, indicating that it is probably related to its effects on the granulomatous reaction itself rather than on general lymphocyte activation. Thus, although granulomatous inflammation in schistosomiasis is essentially a cell-mediated hypersensitivity to parasite egg antigens that are lodged in hepatic tissue (Warren et al. 1967), their reduction could be explained by action of silymarin on the production of proinflammatory cytokines/chemokines in hepatic tissue and cellular accumulation (Cho et al. 2000; Kang et al. 2004; Schumann et al. 2003; Trappoliere et al. 2009). Indeed, silymarin was reported to be able to decrease IL-4 and increase IL-10 synthesis (Schumann et al. 2003). The organic damage caused by schistosomiasis depends on a T cell response, which is critical to the granuloma formation and morbidity outcome (Burke et al. 2009); however, the failure to establish a Th2 is correlated with an early mortality, while excessive Th2 leads to a high fibrotic response and morbidity (Hoffmann et al. 2000; Pearce and Macdonald 2002). Thus, the key to prevent morbidity in schistosomiasis may lie in interfering in fibrotic response itself, without compromising T cell response. The unaltered splenomegaly in face of decreased hepatomegaly, granuloma sizes, and fibrosis makes silymarin candidate as a drug to prevent morbidity.
As peri-ovular granulomas evolve, collagen fibers deposit around them, a process that leads to fibrosis (Lenzi et al. 1998). The hepatic granuloma area has been associated with hepatic fibrosis, as most of the collagen is deposited around eggs (Chesney et al. 1998; Fanning et al. 1981). Herein, we observed a reduction in granuloma area and in fibrosis, evaluated by hidroxyproline measurement, in all infected silymarin-treated groups, corroborating the association between liver fibrosis and granuloma area (Cheever et al. 1987). Praziquantel, the standard clinical treatment for schistosomiasis, also acts to reduce granuloma sizes (Mehlhorn et al. 1982), apparently due to reduction of cellular infiltrates. Though our results could be explained by the indirect anti-inflammatory capacities cited above, it could alternatively be due to a direct antifibrotic role of silymarin, described in other models (Boigk et al. 1997; Gebhardt 2002; Jia et al. 2001).
In murine schistosomiasis, the IL-13 deficiency is correlated with hepatic fibrosis reduction (Fallon et al. 2000), and a recent paper has demonstrated that silybin reduces the level of IL-13 (Tyagi et al. 2009). Moreover, silybin reduces activation of stellate cells, which are responsible for collagen deposition, by inhibiting Kupffer cells (Dehmlow et al. 1996). Confirming these data, IL-13 rather than IL-1β/IL-17 was found to be responsible for hepatic fibrosis in schistosomiasis (Wilson et al. 2010). No interplay between oxidative stress and IL-13 production has been shown so far. Thus, we believe that the previously demonstrated capacity to inhibit IL-13 production and stellate cell activation underlie the reduction in fibrosis produced by treatment with silymarin, a hypothesis currently under investigation in our laboratory.
Taken together, these results show that silymarin treatment of murine schistosomiasis could reduce peri-ovular granulomatous reaction and hepatic fibrosis. Although specific drugs are effective to destroy adult parasites in schistosomiasis, no drugs are used to accelerate the reversion of established damage in this infection. The uncovering of drugs that are related to the immunoresponse in schistosomiasis could be essential to reveal an alternative protocol to treat this important parasitic infection and its malefic consequences. Therefore, silymarin could be a promising complementary treatment to revert/minimize sequelae of this infection.
This work was supported by FAPERJ, CNPq, and FAF/FECD/OncoII. The authors are grateful to Laboratório de Malacologia (FIOCRUZ-RJ) for the S. mansoni cercariae.