Methods
Chemicals
All chemicals were provided as detailed in (Additional file 1).
Animals
Male Wistar rats, weighing 100–130 g, were provided by the animal house colony of the National Research Centre, Dokki, Giza, Egypt. Animal-related procedures were all approved and operated in accordance with the Ethics Committee of the National Research Centre and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Experimental design
Rats were randomized and divided into six groups (n = 6). Each was subjected to different treatment as detailed in (Additional file 1). Briefly, Control, received water (5 ml/kg b.wt.) for 14 days then vehicle (1 ml corn oil/kg b.wt.). CCl4 group received water (5 ml/kg b.wt) then CCl4 (50% CCl4/corn oil; 1 ml/kg body weight). Silymarin (SIL) + CCL4 group received SIL (200 mg/kg b.wt.) [24] and CCl4 (50% CCl4/corn oil; 1 ml/kg b.wt. i.p.). The AES + CCl4 group received AES (3.6 mg/kg b.wt.) [16] and CCl4 (50% CCl4/corn oil; 1 ml/kg b.wt. ip). DIO + CCl4 group received DIO (100 mg/kg) [25] and CCL4 (50% CCl4/corn oil; 1 ml/kg b.wt. ip). The ASE + DIO + CCl4 group received AES (1.75 mg/kg) and DIO (50 mg/kg) and then CCL4 (50% CCl4/corn oil; 1 ml/kg b.wt. ip).
Sample collection and preparation
Rats were anesthetized using diethyl ether then samples of blood were withdrawn from the retro-orbital plexus. Post blood collection, animals were euthanized by cervical dislocation under 3% sodium pentobarbital anesthesia and livers were swiftly dissected out, washed with cold normal saline and weighted. Blood samples were centrifuged at 3000 rpm g for 10 min and serum was used for further biochemical analyses.
Assessment of serum biochemical parameters
Kits used were purchased from sources detailed in (Additional file 1).
Histological and histochemical assessment studies
After blood collection, rats of each group were euthanized by cervical dislocation under 3% sodium pentobarbital anesthesia, and their livers were collected, dissected immediately after the sacrifice and were used for the histopathological analysis as described in [26, 27] and detailed in (Additional file 1).
Assessments of morphometric area percentage and optical density
This assessment was carried out on periodic acid Schiff (PAS) & Mercuric bromophenol blue stained slides. Assessed areas were analyzed as detailed in (Additional file 1).
Statistical analyses
Using statistical package for social science (SPSS), collected data were statistically analyzed as detailed in (Additional file 1).
Results and discussion
AES, DIO and their combination attenuated liver lesions in CCl4-induced liver damage
Normal liver sections “control group” showed typical hepatic architecture with the central vein centrally located and normally thickened hepatic cords radiating with well-formed hepatocytes with centrally located nuclei and intact cell membrane (Fig. 1A). CCl4-induced group showed hepatocytes degenerative changes including pyknotic nuclei. In addition, central veins were massively enlarged and clogged with ductular cells hyperplasia, scattered inflammatory cells which were concentrated around hepatic vessels (Fig. 1B). Compared to the SIL + CCl4 group (Fig. 3C) and to the protected groups; treated with ASE (Fig. 3D), DIO (Fig. 1E), animals intoxicated with CCl4 and treated with both AES and DIO at low dose combination showed marked degree of improvement of hepatic tissue as the hepatic tissue restored its normal structure and architecture with centrally located central vein and intact hepatocytes (Fig. 1F). Currently, CCl4-induced liver injury is a model that is widely employed to screen for anti-hepatotoxic/hepatoprotective drugs. It is also a useful tool to study different liver illnesses, such as fatty liver, fibrosis, and cirrhosis [2, 28]. The magnitude of CCl4 hepatic impairment is evaluated by the enhanced serum level of cytoplasmic enzymes and by histopathologic changes in liver [29, 30].
Compared to control, CCl4-induced group had significantly higher levels of serum alanine aminotransferase (ALT) (130%), and aspartate aminotransferase (AST) (40%) (Fig. 1G, H). Levels of ALT and AST were significantly (P ≤ 0.05) improved upon treatment with SIL, AES and DIO. Pretreatment with SIL, AES, DIO and their combination reduced the elevated serum ALT by 28%, 34%, 31% and 21%, respectively, and AST levels by 31%, 33%, 29% and 32%, respectively, compared to animals in CCl4 group (Fig. 1G, H). Upregulation of ALT and AST have been ascribed to hepatotoxicity as they are released into blood serum upon hepatocyte degeneration [31]. The current study showed quite a restoration of almost normal levels especially when combined treatment was applied.
Glycogen and proteins distribution
Figure 2 shows the distribution of glycogen contents in normal liver tissue as reflected by PAS positivity area (Fig. 3A; 20.74 ± 1.65). CCl4 group showed severe depletion of glycogen 6.63 ± 1.6 with a reduction percent of 82.45% from the control group.
Groups treated with DIO, AES and both showed varying improvements in the glycogen content as reflected by the PAS area % of 15.84 ± 1.23, 16.54 ± 0.79 and 22.56 ± 1.36 respectively. Hepatotoxicity is always correlated with disruption in the synthesis and metabolism of glycogen and proteins. From the present findings, both protein and glycogen were reduced in CCl4 group. Poisonous effect on liver may mainly contribute to such reduction through the drug-induced necrosis of the plasma membrane, or via depleting energy sources needed for synthesizing protein and other metabolic events through obstructing the oxidative phosphorylation activity as documented elsewhere [32]. All tested preventive treatments successfully reversed such effect.
The control group showed the highest protein content (as reflected by Mercuric bromophenol blue positively stained area of the hepatocytes and its optical density of 26.75 ± 0.812 and 25.76 ± 1.66; Fig. 3). CCl4-intoxicated group showed the lowest comparable values of 2.29 ± 0.61 and 13.9 ± 0.98. Protected groups treated by AES and DIO showed varying degrees of protein content improvements; 16.037 ± 1.45 and 15.913 ± 1.7 and optical density of 17.88 ± 1.01 and 16.66 ± 0.08 respectively. The combined administration of AES and DIO at low dose, strengthened the stain reaction and showed marked improvement in the protein content of hepatocytes of 19.27 ± 0.8 and optical density of 20.59 ± 0.9. The present results of pre-administration of AES improved liver histology and function and significantly enhanced total hepatic glycogen and protein content. These results confirmed the hepatoprotective effect of AES as previously reported against liver damage induced by methyl parathion [15] and CCl4 [16, 22] in rats. Although treatment with DIO do positively contribute to liver histology and function, the protective effect of DIO was less evident than that of AES. These hepatoprotective effects of DIO coincide with those of [15, 19, 20]. Interestingly, livers of rats in CCl4 group treated with both AES and DIO showed minimal inflammation, eosinophilic staining and degenerations with significant enhancements in overall glycogen and protein contents in liver.
AES, DIO and their combination ameliorated oxidative stress and inflammation in CCl4-induced liver damage in rats
The administration of SIL, AES, DIO and AES/DIO combination significantly attenuated the increment in serum MDA and nitric oxide (NO) levels and partly attenuated the decrement in glutathione (GSH), Catalase and protein kinase C (PKC). Compared to SIL, AES, DIO and AES/DIO groups, the treatment with combined AES and DIO showed the best effects against all examined oxidative stress markers (Additional file 2). Abundant evidence suggests that reactive oxygen species (ROS) and other free radicals are induced following hepatic insults with drugs like CCl4. The enzymatic/non-enzymatic defense system is the natural protec-tor against free radicals accumulation [28, 30]. The accumulation of free radicals can cause decreased Catalase and GSH levels and a decline in capacity of scavenging free radicals. Consisting with previous report [30, 33], liver damage shown in CCl4-treated group was associated with enhanced MDA and NO as well as depletion of GSH and Catalase. Oxidative stress is known to cause activation of several stress kinases such as PKC and leads to exacerbation of cell toxicity [34]. The elevation of PKC in the present work was in line with the results of the previous studies which suggested that the hepatic oxidative stress and injury induced by bezafibrate [34] and acetaminophen [35] and CCl4 [36] was mediated by elevation of PKC. PKC induces upregulation of tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), and ROS, a major contributor to CCl4-induced liver injury [36]. Here, and in agreement with results of [16], AES ameliorated CCl4-induced hepatotoxicity by reducing MDA and NO and increasing GSH activity. Similarly, in this study, DIO mitigated CCl4-induced oxidative and nitrative stresses confirming previous studies that showed that DIO alleviates the oxidative stress caused by aflatoxin [22], methotrexate [20] by increasing GSH levels, lowering NO levels, and enhancing the activity of antioxidant enzymes. Most interestingly, much improvement in oxidative stress and antioxidants status was observed here when AES was combined with DIO.