The prophylactic effect of silymarin on hepatic damage and IL-1β, IL-6 and TNF-α expression in rats with hepatic fibrosis

Naglaa Kamal Madkour 1,2 and Amal Attia El-Morsy Ibrahim 1,3 . 1. Dept. of Zoology, Faculty of Girls’ for Arts, Education and Science, Ain Shams University, Cairo, Egypt. 2. Biology department, University College of Umluj, Tabuk University, KSA. 3. Dept. of Biological Sciences, Faculty of Science for Girls, Northern Border University, KSA. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The present study aimed to evaluate the protective effect of silymarin (Sy) on liver fibrosis induced by carbon tetrachloride (CCl 4 ). Rats were distributed as follows: 1 st group is the normal control group administered only with olive oil. 2 nd group is the positive control group administered with Sy. 3 rd group of rats intoxicated with CCl 4 to induce hepatic fibrosis. 4 th group of rats treated with Sy+CCl 4 . CCl 4 intoxication were followed the Sy oral protection treatments by 2h. Hepatic fibrosis, augmentation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total protein (TP), IL-1β, IL-6, TNF-α expression and decreased levels in reduced glutathione (GSH) and oxidized glutathione disulfide (GSSH) contents; occurred as a result of CCl 4 intoxication. Pre-administration of Sy reversed liver injury by reversing AST, ALT, TB, ALB, improving antioxidant status, and pro-inflammatory cytokine expression.

Introduction:-
Hepatic fibrosis represents the final common pathological outcome for the majority of chronic liver insults. Its final stage is cirrhosis. Liver cirrhosis, the irreversible terminal stage of chronic liver disease, characterized by widespread fibrous scaring, and it is considered as a major cause of morbidity and mortality worldwide, with no effective therapy (Huang et al., 2006). The liver regulates many important metabolic functions, so the hepatic injury is associated with distortion of these functions (Wolf, 1999). Liver damage ranges from acute hepatitis to hepatocellular carcinoma, through apoptosis, necrosis, inflammation, immune response, fibrosis, ischemia, altered gene expression and regeneration. Loguercio and Federico (2003) stated that all processes that involve hepatocyte, Kupffer, stellate and endothelial cells which induce liver disease are related to the crucial role of reactive oxygen and nitrogen species. The main sources of free radicals are represented by hepatocyte mitochondria and cytochrome P450 enzymes, by endotoxin-activated macrophages (Kupffer cells) and by neutrophils.
The extracts of the flowers and leaves of Silybum marianum (St. Mary's thistle, milk thistle, or silymarin) has been used for centuries to treat liver, spleen and gallbladder disorders (Rainone, 2005). One of the important issues about this plant that it has accepted as a safe herbal product, since no health hazards or side effects are known in conjunction with the proper administration of designing therapeutic dosages (Medical Economic Company, 2000).

Drugs and Chemicals:-
Carbon tetrachloride (BDH Chemicals, England), the dose of CCl 4 was chosen according to the study of (Yao et al., 2004) to induce acute hepatitis model in rats. Silymarin (SEDICO, Cairo) was dissolved in saline to obtain the necessary doses. The dose of silymarin used in the study was chosen based on other studies in which the drugs produced beneficial effects in models of hepatic injury (Shaker et al. 2010). Biochemical and Antioxidant Analysis Blood samples were collected by cardiac puncture, allowed to clot and then centrifuged at 3,000 rpm for 15 minutes to separate serum. Serum kept at -20 ̊ C until required. The activities of aspartate aminotransferase (AST), and alanine aminotransferase (ALT), by using commercial kinetic kits (Prolabo, France). Levels of albumin (ALB), and total protein (TP) were measured coloremetrically according to Lowery et al. (1951) and Dumas and Biggs (1972), respectively.
Determination of GSH and GSSG Contents :-Reduced glutathione (GSH) and oxidized glutathione disulfide (GSSH) contents were determined by a modification of the method of Hissin and Hilf (1976). For GSH; 0.5 ml of the 10,000 g supernatant, 4.5 ml of the phosphate-EDTA buffer, pH 8.0, was added. The final assay mixture (2.0 ml) contained 100 μl of the diluted tissue supernatant, 1.8 ml of phosphate-EDTA buffer, and 100 μl of the OPT solution, containing 100 μg of OPT. After thorough mixing and incubation at room temperature for 15 min, the solution was transferred to a quartz cuvette. Fluorescence at 420 nm was determined with by the activation at 350 nm.
For GSSH; 0.5 ml portion of the original 10,000 g supernatant was incubated at room temperature with 200 μl of 0.04 M NEM for 30 min to interact with GSH present in the tissue. To this mixture, 4.3 ml of 0.1 N NaOH was added. A portion of this mixture (100 μl) was taken for measurement of GSSG, using the procedure outlined above for GSH assay, except that 0.1 N NaOH was employed as diluent rather than phosphate EDTA buffer.

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Cytokines Activity:-Cytokine activities of IL-1β, IL-6, and TNF-α in serum were measured via a highly sensitive commercial ELISA (Sandwich Immunoassay Technique) specific kit for rats (Immuno-Biological Laboratories Co., Ltd. USA). Briefly, 96-well microplates were coated with IL-1β, IL-6, and TNF-α antibodies and incubated overnight at room temperature. The plates were washed with PBS containing 0.05% Tween 20 and then blocked with PBS with 1% bovine serum albumin and 5% sucrose. After the addition of diluted samples and standard IL-1β, IL-6, and TNF-α dilutions, plates were incubated for 2h at room temperature. Biotinylated goat anti-rat was used as the detection antibody, and streptavidin-HRP was added as the conjugate to each well. Equal proportions of hydrogen peroxide and tetramethylbenzidine were used as the substrate solution, and the reaction was stopped by adding 2N sulfuric acid. All samples and standards were run in duplicate, and the optical density was determined with a microplate reader at a wavelength of 450nm. The values of plasma cytokine concentration were expressed as pm/ml.

Histopathological Examination:-
Autopsy samples were taken from the liver of rats from different groups and fixed at 10% neutral buffered formalin for 24h and subsequently embedded in paraffin. Paraffin tissue blocks were prepared for sectioning at a 4μm thickness by slide microtome. The obtained tissue sections were collected on glass slides, deparaffinized and stained with hematoxylin and eosin stain for histopathological examination through the light microscope (Banchroft et al., 1996).

Statistical Analysis:-
Mean data were calculated and SD was measured for each mean number. The obtained data were statistically analyzed by using the program analysis of variance (ANOVA) followed by Duncan's multiple range test according to Duncan (1955) and Snedecor & Cochran (1982) using a computer program (Costate). Values of P<0.05 were considered statistically significant.

Results:-Biochemical and Antioxidant Analysis:-
Biochemical data for all the studied parameters showed non-significant differences between the control group and Sy treated group. The liver-specific marker AST and ALT increased significantly (173.47 U/l) and (75.27 U/l) after the induction of fibrosis by CCl 4 , whereas the TP and ALB values of this group were reduced, thus indicating that liver cell damage was significantly induced; causing liver dysfunction. On the other hand, pre-administration of Sy diminishes hepatic damage by preventing the augmentation of AST, ALT, and by preventing the inhibition of TP, ALB values as appeared in table (1). The antioxidant status of liver tissue reduced significantly after establishment of hepatic fibrosis GSH (3.15 μM/mg) and GSSG (0.18 μM/mg). Sy administration restored these levels near to normal levels as reported in table 1. Histological Examination:-Liver sections from control rats illustrated preserved architecture with hexagonal hepatic lobules, each is formed of cords of hepatocytes radiating from the central vein to the periphery of the lobule (Fig.1). The hepatic cords were separated by narrow blood sinusoids lined by endothelial cells and kupffer cells. The acidophilic cytoplasm around a pale stained nucleus could be seen. Sections from liver tissue of rats treated with Sy showed no histopathological changes when compared with control animals (Fig.2).
Consistent with biochemical findings, intoxication of rats with CCl 4 induced moderate fibrosis without formation of septa (Fig. 3). Hepatocytes appeared with focal necrosis and fatty changes (steatosis); beside the increased number of mitotic figures (Fig. 4) and clear vacuolation of the hepatocytes were seen (Fig.). Hepatocyte degeneration and necrosis, lymphocyte infiltration (Fig. 5) and collagen deposition, eosinophilic hepatocytes were detected. Dilated central vein stuffed with RBCs (Fig. 6).
The microscopic examination of liver sections from rats pretreated with Sy followed by CCl 4 showed protective effects. The hepatic tissue revealed the general hepatic architecture with normal arrangement of hepatic cords and narrow hepatic sinusoids and bi-nucleated hepatic cells as a sign of regeneration. The degree of hepatocyte necrosis, degeneration was decreased markedly, and diminution of fibrosis and fatty changes; when compared to the liver sections of rats intoxicated with CCl 4 . However, there were a few RBCs infiltrates inside the sinusoids (Fig.7).

Discussion:-
Liver is the key organ of metabolism and excretion is continuously exposed to xenobiotics because of its strategic placement in the body. Toxins absorb from the intestinal tract gain access first to the liver resulting in a variety of liver problems (Wolf, 1999). Liver disorders are one of the common recent problems affects on the human health, resulted from the exposure to the environmental polluted sources ( Glutathione exists in reduced (GSH) and oxidized (GSSG) states. GSH prevent formation of reactive oxygen species (ROS) and their damaging effects. GSH effectively scavenges free radicals and other ROS and oxidized to form GSSG, then glutathione reductase (GR) recycles GSSG to GSH. In addition, GSH reacts with various electrophiles, physiological metabolites and xenobiotics to form mercapturates, which are catalyzed by GST (a family of Phase II detoxification enzymes) (Wu et al., 2004). In healthy cells and tissues, more than 90% of the total glutathione pool is in the reduced form (GSH) and less than 10% exists in the disulfide form (GSSG). An increased GSSG-to-GSH ratio is considered as the indicative of oxidative stress (Pompella et al., 2003). The present investigation indicated that Sy could restore the antioxidant status in the rat liver tissues.
The present work showed that CCl4 intoxication caused an increase in TNF-α, IL-1β, and IL-6 production. ROS upregulates NF-κB, which is required for the induction of pro-inflammatory cytokines, such as IL-1ß, TNF-α and IL-6 (Rocha et al., 2014). TNF-α is a key mediator of the immune and inflammatory responses and controls the expression of the inflammatory gene network. Therefore, the overproduction of TNF-α contributes significantly to the pathological complications observed in many inflammatory diseases. Hepatic injury is associated with the upregulation of TNF-α gene expression that was observed in the CCl 4 group. Consequently, the over-production of TNF-α contributed to the manifestation of the systemic inflammatory response and ultimately to the development of organ failure (Chehl et al., 2009). Also; Ebaid et al. (2013) found that the up-regulation of TNF-α expression was accompanied by the up-regulation of the Fas genes in CCl4-induced liver injury. The Fas protein is a type I membrane receptor that belongs to the TNF-receptor superfamily. While; Mita et al. (2005) found that the expression of FasL by macrophages plays a role in their pathogenesis. TNF-α is a pro-inflammation cytokine and a major endogenous mediator of hepatotoxicity. TNF-α is expressed in chronic liver injuries by both infiltrating inflammatory cells and hepatocytes and plays an important role in tissue damage (Hernandez-Munoz, et al., 1997).

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The significant increase in TNF-α, IL-1β, and IL-6 production, inhibited by Sy pre-administration. These results were confirmed in a study of Reyes-Gordillo et al. (2007). Sy markedly suppressed the expression of TNF-α in liver, suggesting it's exerts its inhibitory effect on hepatic fibrosis by blocking the release of inflammatory mediators such as TNF-α and preventing hepatic fibrosis. A similar result has been reported by Nakamuta et al. (2001) and Issa et al. (2004) who studied a model of cirrhosis to determine the mechanisms mediating and limiting spontaneous recovery, and found that micronodular cirrhosis undergoes remodeling to macronodular cirrhosis; and reverse hepatic fibrosis gradually (Lee et al., 2001). Intoxication of CCl 4 induced the translocation of NF-κB to the nucleus; CCl 4 induced NF-κB DNA binding activity was blocked by Sy; which prevents acute liver damage by at least two mechanisms: acting as an antioxidant and by inhibiting NF-κB activation and thus production of pro-inflammatory cytokines (Reyes-Gordillo et al., 2007).
In the present study, CCl4 intoxication; induced moderate fibrosis without formation of septa, architectural distortion, which in accordance with other Studies ( . Mechanism of action for Sy conducted mainly to the antiradical and anti-carcinogenic roles. Ethyl acetate (100 mg/kg bw) and ethanol seed extracts for S. marianum (100 mg/kg bw) were tested against the injection by CCl 4 (2 ml/kg bw). Their activity was compared with standard hepatic drug hepaticum (100 mg/kg bw) for 10 days. Ethanolic extract showed the most significantly decrease in the liver enzymes (Medical Economics Company, 2000). Sy has metabolic and cell-regulating effects at concentrations found in clinical conditions, namely carrier-mediated regulation of cell membrane permeability, inhibition of the 5-lipoxygenase pathway, scavenging of ROS and an action on DNA-expression, for example, via suppression of nuclear factor (NF)-kappaB (Saller, et al., 2001); beside its effect on cell proliferation (Tyagi et al., 2004) suggesting that Sy may be a useful additive therapy in patients with chronic liver disease.

Conclusion:-
Increasing requirements for natural plant products could modify the biological harmful molecules by the antioxidant potential. Pre-administration of Sy could mask the harmful effect of CCl 4 on hepatic fibrosis, blocking the free radical formation, preserving the cellular integrity, and thus elicit a reduction in the inflammatory response, restoration of cytokine expression.