Although chemotherapy eliminates matured worms effectively and prevents the accumulation of schistosome eggs, less effective drugs are directed to reversing the existing hepatic fibrosis, especially at the chronic and advanced stages of schistosomiasis. Therefore, treatment targeting hepatic fibrosis of schistosomiasis remains a challenging proposition . Because the mouse model has a similar course to human schistosomiasis , we, thus, established the mouse schistosomiasis model through Schistosoma mansoni cercariae infection, and then treated them with either silymarin alone or plus praziquantel to diminish liver fibrosis in the current study. In this study, all mice received S. mansoni infection in addition to the presence of hepatic granulomas around deposited schistosome eggs affecting hepatocellular function. The observed elevation in ALT reflects either acute active or chronic liver damage , and hypoalbuminemia in chronic infection occurs simultaneously with the increase in collagen deposition. This could be associated with malabsorption due to damaged intestinal mucosa resulting from the extrusion of large numbers of eggs, or could be due to decreased synthesis which may result from parasitic injury to hepatic cells . In addition, a significant depletion of GSH, which constitutes the first line of defence against free radicals and is a critical determinant of tissue susceptibility to oxidative damage, was observed. In the present study, hydroxyproline was chosen because it is a sensitive marker that increases significantly during liver fibrosis. The increase in this amino acid reflects an increase in the de novo synthesis of liver collagen and an increase in the amount of hydroxyproline . Here, there was a linked increase in serum MMP-2, TGF-β1 and the HYP content in both acute and chronic infections. This could be due to the activated HSCs during the acute stage of fibrosis producing more MMPs to counteract the overproduction of collagen-I, which was dominant from the onset of the fibrotic response and peaked at the chronic phase of infection . Singh et al. reported that there was a strong rise in collagen I and III expression in the liver seven weeks post infection indicating that induction of collagen production was associated with the early development of the granulomatous response. In addition, MMP-2 and TGF-β1 levels continued to rise reaching the highest level at the 18th week post infection. Loebermann et al. reported that the expression of MMP-2 correlated well with the onset and progression of fibrosis.
In the current study, treatment of S. mansoni-infected mice with silymarin alone at the 4th and 12th weeks PI resulted in remarkable worm and egg reduction accompanied with a partial increase in the percentage of dead eggs 10 and 18 weeks PI. This was associated with healing of hepatic granulomatous lesions as evidenced histopathologically with reduction of granuloma size, more granuloma circumscription, more ova degeneration and fewer inflammatory cells. This may be attributed to the antioxidative properties of silymarin , therefore, it is possible that silymarin eliminates the products of oxidative reactions and assists in the immune-mediated destruction of worms and eggs. Also, treatment with silymarin, whether in acute or chronic infection, significantly reduced the hepatic HYP content, tissue expression of TGF-β1 and MMP-2 and the number of mast cells. This is consistent with previous studies, which showed that administration of silymarin reduced the hepatic collagen content in diethylnitrosamine  and CCl4 administered rats , and exerts its anti-fibrotic properties by reducing TGF-β induced de novo synthesis of collagen type I . Fuchs et al. reported that prolonged treatment with silymarin resulted in its accumulation in HSCs and down regulated TGF-β1 expression; since this growth factor has the ability to induce its own production, silymarin broke the so-called fibrogenesis loop, or the self-perpetuation of hepatic fibrosis. In this study, the number of mast cells in portal areas and granulomas decreased significantly in the silymarin-treated group compared to those of the infected group. Mast cells secrete various mediators, which promote fibroblast growth, stimulate production of the extracellular matrix by fibroblasts of hepatic stellate cells, and produce components of the extramedullary matrix themselves . However, it is unclear whether they play a central role in its development. Thus, silymarin has been histopathologically shown to have a significant anti-inflammatory effect on hepatic tissue, including mast cell stabilization.
In the current study, silymarin alone significantly reduced the elevated ALT and restored the depletion of GSH with no alteration in serum albumin level 10 and 18 weeks PI. Silymarin restored the elevated level of serum ALT in CCl4 intoxicated rats  and in diethylnitrosamine administered rats  by preventing liver damage through maintaining the integrity of the plasma membrane, thereby suppressing the leakage of enzymes. The increase in the hepatic GSH content may be attributed to the well established antioxidant actions of silymarin , which was able to dramatically reduce the generation of intracellular ROS in response to different pro-oxidant stimuli . Furthermore, silymarin is capable of protecting liver cells directly by stabilizing the membrane permeability through inhibiting lipid peroxidation and preventing liver glutathione depletion, thus offering the synergistic benefit of sparing liver cells from destruction .
Reversal of schistosome-induced pathology after treatment with PZQ has been previously described . In contrast to that, our findings show that PZQ alone seemed to be effective in reducing hepatic fibrosis as shown by a clear reduction in the serum levels and tissue expression of TGF-β1 and MMP-2, the number of mast cells and hepatic HYP content at 10 and 18 weeks PI. This was accompanied by a significant reduction in ALT with significant restoration of albumin and GSH. The main explanations for these results are presumed to be a removal of schistosomal worms, and subsequent reduction of egg deposition and granuloma size. Researchers showed that patients in Ethiopia and Uganda had improvement or resolution of schistosomal periportal thickening/fibrosis after parasitologic cure with PZQ [48, 49]. Singh et al. reported that with elimination of eggs and subsidence of the inflammatory response, collagen gene expression was minimal and that collagen and MMP-2 gene expression diminished in a linked fashion. The authors added that, the weakening of the inflammatory signals, may lead to a decrease in the activation of HSCs and diminished the production of IL-13 by monocytes and macrophages, which in turn reduced the stimulation of TGF-β1. In addition, in the face of decreased collagen production and deposition, expression of MMP-2 genes also decreased because the bulk of the deposited fibrous tissue had been desorbed and consequently the curtailed gene expression of MMP-2 is appropriate . In a recent study by Liang et al. on the anti-fibrotic effects of PZQ in mice with both chronic and advanced schistosomiasis as well as in CCl4 induced liver fibrosis mice, they mentioned that the significant amelioration of hepatic fibrosis by praziquantel treatment validates it as a promising anti-fibrosis drug and offers the potential for new chemotherapy for hepatic fibrosis resulting from schistosomiasis.
Administration of silymarin in addition to PZQ also showed complete eradication of worms, no viable eggs with reduction in the hepatic tissue egg load and more healing of hepatic granulomatous lesions revealing that the use of silymarin along with PZQ did not interfere with, or affect, the antischistosomal activity of PZQ. The highest reduction observed in TGF-β1, MMP-2 and mast cells was in the group treated with PZQ plus silymarin. In addition, the improvement of liver function and histopathology whether in acute or chronic infections may be due to blocking liver fibrosis through killing parasites and complete eradication of eggs and their toxins by PZQ to alleviate liver inflammation, and to the anti-inflammatory, anti-fibrotic actions, in addition to the antioxidative properties of silymarin. Although the hepatoprotective properties of silymarin have been reported from both in vitro and in vivo studies, its mechanism of action still has not been established . Velebný et al. showed that combined treatment with PZQ and silymarin in mice infected with Mesocestoides vogae (Cestoda) tetrathyridia was able to ameliorate or suppress fibrogenesis in the liver, protect liver cells from oxidative damage and, possibly, stimulate regeneration of the parenchyma. Interestingly, the reduction in HYP content in the group treated with silymarin plus PZQ did not reach a significant level compared with the PZQ treated group. Silymarin is thought to be dose-dependent and studies vary considerably in duration without agreement on the minimum duration needed to see the effect [54, 55]. Therefore, further studies are clearly needed to investigate the efficacy of higher doses and a longer duration of treatment with silymarin in this model of liver fibrosis.
In conclusion, silymarin has partially toxic effects on worms and eggs. Furthermore, the anti-fibrotic and anti-inflammatory effects of silymarin/PZQ alone were observed in the acute and chronic fibrogenesis induced by S. mansoni infection. This was evident in diminishing hepatic content of HYP, serum levels and hepatic expression of TGF-β1 and MMP-2 and the number of mast cells. These effects were more obvious in most parameters studied, when silymarin was used with the removal of the causative agent by PZQ. Accordingly, our results point to silymarin as a convenient and promising therapeutic agent in the treatment of schistosomal liver fibrosis. Further studies on mechanisms of action of silymarin and praziquantel during schistosomal liver fibrosis or other chronic liver diseases may shed some light on developing therapeutic methods in clinical practice.