الفهرس 
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Abstract
Hepatic schistosomiasis is one of the most common chronic liver disorders in the world, resulting in infection related morbidity and liver fibrosis consequences. The severity of the condition is due to fibrosis generated by a granulomatous response surrounding the schistosome eggs trapped in the liver’s tiny capillaries. Currently, the treatment of schistosomiasis relies on a single anti-parasitic drug praziquantel. However, PZQ possesses a limited effect on granulomatous inflammation and developed fibrosis. Nevertheless, morbidity and mortality in S. mansoni infection arises from the granulomatous inflammation and subsequent fibrosis. Plumbagin is a potent immunomodulatory agent that inhibits T cell proliferation and downregulates IL-2, IL-4, IL-6, and IFN-γ cytokines in vitro and in vivo by blocking cell cycle progression. Plumbagin also inhibits NF-қB activation. Thus, the inhibition of NF-κB activation by plumbagin may modulate functions of leukocytes of participating in various immune responses. Therefore, the present study is the first to report immunomodulatory and schistosomicidal activities of plumbagin in schistosomiasis. Mice were divided into five groups: non-infected untreated (C); infected untreated (IU); non-infected treated with plumbagin (P); infected treated with plumbagin (PI) and infected treated with praziquantel (PZ).
Mice were infected with schistosomes by tail emersion method and were injected intraperitoneally (i.p.) with the agent at a total dose of 20 mg/kg body weight divided equally into four injections, two injections/week for consecutive two weeks. PZQ control mice (PZ) were administered 500 mg/kg bw of PZQ by gavage for two consecutive days starting at the time of the first plumbagin dose. All mice were sacrificed at the end of week eight following infection (two weeks post-treatment). Worm burdens were estimated by portal perfusion. Numbers of S. mansoni eggs in perfused liver and intestine of infected mice were estimated after alkali (5% KOH) digestion. Fixed liver tissue was embedded in paraffin and cut into 4-μm sections. Sections were stained with hematoxylin and eosin (H&E) and used for the determination of granuloma size. Sections were also stained with Masson’s trichrome for evaluating collagen fiber deposition. The size of granulomas surrounding a single mature schistosome egg was measured using morphometric methods.
Sandwich enzyme-linked immunosorbent assays (ELISA) were used to measure serum cytokine levels. Cytokine concentrations for mouse IL-4, IL-10, IL-13, IL-17, IL-37, IFN-γ, TGF -β, and TNF-α were determined with commercially available reagents and ELISA kits. All activities of liver enzymes e.g. alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity homogenate supernatants, hepatic g-glutamyl transferase (g-GT) activity, in addition to measurement of the hepatic lactate dehydrogenase (LDH) activity.
The amount of collagen in the liver samples was determined from its hydroxyproline content. Briefly, defatted, and dried samples were hydrolyzed by autoclaving in 2 ml of 6 N HCl at 50 pressures. Protein hydrolysates were analyzed for hydroxyproline, and the percentage of collagen was calculated using the 7.46 factor.
Soluble worm antigen preparation (SWAP), soluble egg antigen (SEA), and cercarial antigen preparation (CAP) from S. mansoni were purchased from the Schistosome Biological Supply Center at Theodor Bilharz Research Institute, Imbaba, Giza, Egypt. The measurement of anti-SWAP, anti-SEA, and anti-CAP antibodies used indirect ELISA.
The present investigation showed that plumbagin treatment reduced both male and female worm burdens by 64.28 and 59.88%, respectively, and is thus effective against both sexes. Treatment of S. mansoni-infected mice with plumbagin markedly reduced liver and intestine egg numbers by more than half. In addition, eggs/g tissue per worm decreased by 40.75% in comparison with untreated mice.
Interestingly, the present investigation showed a 35.26% reduction in liver collagen content in infected and treated plumbagin mice. Conversely, the present investigation showed that PZQ does not display anti-fibrotic effects (11.21%) vis-à-vis plumbagin. Interestingly, the present investigation showed that plumbagin treatment reduced hepatic granuloma size by 62.5%. On the other hand, PZQ treatment reduced hepatic granuloma size by 41,18%.
Further, plumbagin treatment significantly reduced IL-4, IL-13, IL-17, IL-37, IFN-γ, TGF-β, and TNF-α levels and significantly upregulated IL-10. Plumbagin treatment restored hepatic enzymes activity to nearly normal levels and induced an increase in catalase, SOD, GSH, total thiol, and GST in liver tissue homogenate. NO and LPO content was, however, decreased. Moreover, serum IgG levels significantly increased.
In conclusion, antischistosomal effects of plumbagin are associated with reducing the parasitic burden. It reduces granulomatous inflammation and inhibits liver fibrosis. Upregulation of the anti-inflammatory cytokine, IL-10, and downregulation of inflammatory cytokines during acute S. mansoni infection underlie these actions. Plumbagin treatment also had a hepatoprotective effect through modulating granuloma formation in liver tissues and might attenuate S. mansoni-induced liver fibrosis by decreasing free radical production and collagen deposition. Thus, the compound might protect hepatocytes from damage, dysfunction, and demise caused by oxidative stress at sites of inflammation. These results provide an important basis for further investigation of plumbagin treatment. For example, a combination of plumbagin with an antischistosomal drug may be a focus for future work.