Hepatic injury is a fundamental pathological process in most chronic hepatic diseases. Long-standing hepatic injury leads to hepatic fibrosis, liver cirrhosis, and even hepatocellular carcinoma.1 Previous clinical studies have indicated that some herbal extractives, such as silymarin,2 glycyrrhizin,3 and oxymatrine,4 can significantly inhibit these aforementioned pathologic processes and protect hepatocytes against the etiologies of chronic hepatic injury. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an herbal extractive with various biological effects. The previous experimental results in our lab had shown that emodin, which was respectively derived from Chinese herbs rhubarb, had some inhibitive effect on hepatitis B virus (HBV) replication in vitro.5 And astragalus polysaccharides (APS) is an active ingredient of Astragalus mongholicus which has immunoregulation effect. In this study, we investigated hepatoprotective effect of emodin and APS on carbon tetrachloride (CCl4)-induced chronic hepatic injury in rats, trying to elucidate hepatoprotective effect of these two compounds for experimental hepatic injury in vivo.
Emodin (purity >98%) was obtained from Tianxingjian Natural Bio-products Co. (Xi'an, China) and was resuspended in a 1.2% solution (w/v) in 1% sodium carboxymethycellulose just before gavage. APS (purity >65%) was purchased from Hongsheng Bio-products Co. (Xi'an, China) and was resuspended in a 6% solution (w/v) in distilled water just before gavage. Colchicine was purchased from Banna Products Co. (Kunming, China). CCl4 (reagent grade) and cholesterin (reagent grade) were from Xi'an Chemical Factory (Xi'an, China).
Animals and grouping
Ninety healthy male Sprague-Dawley (SD) rats with an average weight of (306±18)g were provided by the Experimental Animal Centre of Xi'an Jiaotong University, China. The rats were housed in conventional cages with free access to water and rodent chow at 20–22°C with a 12-hour light-dark cycle for seven days to allow for acclimatization before the experiments were performed. All procedures involving the use of laboratory animals were in accordance with the National Institutes of Health guidelines. Ninety male SD rats were randomly divided into the following six groups: normal group (n=10), simply model group (n=20), emodin group (n=15), APS group (n=15), combination group (n=15), and colchicine group (n=15).
Establishment of a rat model of hepatic injury and the experimental treatments
The experimental hepatic injury model was established using a previously described protocol.6 Briefly, the rats were given hypodermic injections of 40% CCl4 (dose of 2.0 ml/kg) in olive oil for the first dosing, followed by semiweekly hypodermic injections of 40% CCl4 (dose of 1.0 ml/kg) in olive oil. The rats in the normal group were injected with isovolumetric amounts of olive oil. The rats were fed a high fat diet (79.5% maizena, 20% fat, and 0.5% cholesterol) and 10% alcohol in the drinking water every day; except for the normal group, which was fed rodent chow and water. All the rats were sacrificed 12 weeks after the first injection.
When accustomed to the injections and the high fat diet, all the rats were administered the corresponding treatments in parallel by gavage once a day for 12 weeks (Table 1).
At the end of the experiment, animals were sacrificed by intraperitoneal injection (20% urethane, 0.5 ml/100 g) followed by orbital enucleation. Blood samples were collected using test tubes containing 1:500 of the anticoagulant sodium heparin (v/v). Serum was separated by centrifugation at 3000 r/min for 20 minutes and was analyzed for the various biochemical parameters including total bilirubin (TBIL), alanine transaminase (ALT), aspartate transaminase (AST), and albumin (ALB) using a Hitachi 7150 automatic biochemistry analyzer (Hitachi, Japan). The livers and spleens were removed, weighed and then rinsed in phosphate buffered saline followed by dissection into various portions. One portion was fixed in 4% paraformaldehyde for histological examination and the other portion was homogenized to determine the levels of superoxide dismutase (SOD) and malondialdehyde (MDA).
Determination of the levels of SOD and MDA
SOD activity and MDA content in the liver tissue homogenates were investigated using commercial kits (Nanjing Jiancheng Biotech, China) according to the supplier's protocols, and optical densities were determined using an automated spectrophotometer.
Paraformaldehyde-fixed and paraffin-embedded liver sections were cut into six-micrometer sections and placed on poly-L-lysine-coated glass slides. The sections were stained with hemotoxylin & eosin (H&E) and Van Gieson's (VG) stain. Liver pathology was classified according to the standard formula from the Chinese Medical Association in 2001. The criteria used for scoring fibrosis severity were the following: 0, normal; 1+, fibrosis present (collagen fibers present that extend from the portal triad or central vein to the peripheral region); 2+, mild fibrosis (some extended collagen fibers present without compartmental formation); 3+, moderate fibrosis (moderate amounts of collagen fibers present with some pseudolobe formation); and 4+, severe fibrosis (abundant collagen fibers present with a thickening of the partial compartments and frequent pseudolobe formation).
The data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was carried out by SPSS 12.0 software (SPSS Inc, US). Data from the liver histopathological examinations were analyzed by a Kruskal-Wallis H test and a Nemenyi test. A level of significance was set at P <0.05 in all cases.
At the end of the experiment, the weights of the rats in the simply model group significantly decreased compared with the normal control group (P <0.05). Specifically, the mean weight in the simply model group, only 379 g, was the lowest value observed and was significantly different compared to the other groups (P <0.05). The weights of the rats in the APS and combination groups significantly increased compared to the colchicine group (P <0.05) (Table 2).
Liver and spleen indexes
As shown in Table 3, there were significant increases in the liver and spleen indexes in the injured group compared to the normal control group (P <0.05), which suggests that the injection of CCl4 caused hepatomegaly in the rat. In addition, there were lower liver indexes observed in the emodin, colchicine, and combination groups compared to the simply model group (P <0.05), which suggests that emodin and colchicine could inhibit the hepatomegaly induced by the injection of CCl4. The liver and spleen indexes in the emodin, APS, and combination groups were similar to those observed in the normal control group (P >0.05). In Table 3, the numbers of rats in each group refered to the live experimental rats till 12 weeks, and the death rats were not counted.
Serum enzyme parameters
Serum levels of TBIL, ALT, and AST in the CCl4-induced model group were significantly increased, but ALB was decreased when compared to the normal control group (P <0.05) (Table 4). Serum levels of TBIL and ALT from the emodin, APS, combination, and colchicine groups significantly decreased when compared to the simply model group (P <0.05). Meanwhile, the serum levels of ALB were significantly increased in the APS and combination groups, but the levels of ALT were significantly decreased when compared to the colchicine group (P <0.05). In addition, univariate analysis indicted that there was a synergistic action resulting from the co-administration of emodin and APS in decreasing the serum levels of ALT and increasing the serum levels of ALB in this rat model of hepatic injury (P <0.05).
SOD activity and MDA content in liver
There was a dramatic decrease in SOD activity in all CCl4-induced model groups when compared to the normal control group (P <0.05). Interestingly, a higher SOD activity was observed in the combination group when compared to the colchicine group (P <0.05). In contrast, the MDA content in liver tissue homogenates significantly increased in all the CCl4-induced model groups. However, administration with emodin, APS, and emodin combined with APS prevented MDA elevation when compared to the simply model group (P <0.05). Furthermore, there was a negative correlation between the SOD activity and MDA levels in the liver homogenates (r=-0.700, P <0.01) (Table 5).
There was no significant pathological alteration in the normal control (Figure A). However, overt pathological changes were observed in the simply model group in the following manners: the margins of the liver were uneven; more fibrous tissues were formed and extended into the hepatic lobules to partially separate them; the liver structure was disordered with some displacement of central veins, and there were more necrotic and degenerated liver cells compared to the normal control group (Figure B).
After administration of emodin or colchicine, there was less fibrous tissue proliferation. The hepatic cell cords were arranged radially with a decreased displacement of the central veins. Meanwhile, less hepatic cell necrosis and pseudolobule formation were observed (Figure C). In the combination and APS groups, hepatocyte denaturation and necrosis were mild and pseudolobule formation was not observed when compared to the simply model group (Figure D). The fibrosis grades of all the groups were shown in Table 6. Administration with APS, or the combination of emodin and APS, significantly decreased the hepatic injury grade when compared to the simply model group (P <0.05).
It is well known that CCl4 is a hepatotoxic agent. This compound is rapidly metabolized in vivo to form the CCl3 free radical, which subsequently acts on liver cells to covalently conjugate membranous unsaturated lipids leading to lipid peroxidation.7,8
In this study, a rat model of chronic hepatic injury was established via exposure to CCl4, a high fat diet, and alcohol in the drinking water. Consequently, these animals developed significant hepatic damage and oxidative stress,9,10 which was confirmed by a decreased body weight, splenohepatomegaly, substantially increased serum levels of TBIL, ALT and AST, a decreased serum ALB level, and pseudolobule formation in the liver upon histological examination.
All of the above results indicate that there was cellular leakage and a reduction in the functional integrity of cell membranes in the liver.11 However, in the groups that were administered herbal extracts, treatments could significantly reduce the decrease of body weight, alleviate the splenohepatomegalia, and decrease the serum levels of TBIL and ALT during the hepatic injury process induced by CCl4. Meanwhile, higher ALB levels in the APS and combination groups were observed. Histological analysis showed that the APS and combination groups significantly alleviated the hepatic injury. Therefore, emodin and APS appeared to suppress the hepatic inflammatory responses leading to the increased serum levels of TBIL, ALT and AST. These results, coupled with the inhibitory effects on the histological changes, indicate that these herbal extracts protect against hepatic injury induced by CCl4.
SOD is an important antioxidant enzyme in cells and catalyzes the conversion of superoxide ions into oxygen and hydrogen peroxide.12 MDA is the final product of lipid peroxidation.13 Levels of SOD and MDA reflect the status of the free radical stress in vivo. During hepatic injury caused by CCl4, there is inflammation of the liver tissue, which can activate macrophages, neutrophils, and hepatic stellate cells.12 Consequently, large amounts of superoxide radicals are released, thereby increasing the consumption of SOD, and resulting in the decline of SOD levels. Meanwhile, superoxide radicals can increase lipid peroxidation, thus increasing the generation of reactive oxygen intermediates and MDA. In our study, the observed decreases in SOD activity were presumably associated with the increased oxidative stress caused by CCl4, and these decreases in SOD activity were reversed by all the treatments. In addition, administration of emodin can significantly increase SOD levels in the liver and reduce the content of the lipoperoxidiation product, MDA, when compared to the simply model group and colchicine group. Similar investigative findings also had been reported.14–18 Therefore, emodin and APS may prevent hepatic injury and protect hepatocytes through inhibition of lipid peroxidation and by limiting free radical production.
Furthermore, the effects resulting from the co-administration of emodin and APS should be noted. Compared to the emodin group, the combination group demonstrated synergistic actions in decreasing the serum levels of ALT and increasing the serum levels of ALB. In addition, SOD activity was significantly increased while MDA content was significantly decreased in the combination group when compared to the simply model group. Moreover, pathological changes observed in the combination group were milder than those of the CCl4-induced model group. Therefore, a combination of emodin and APS may have an enhanced protective effect on hepatic injury. However, the mechanism of this synergistic action is not clear to date. Previous studies suggest that these compounds may protect hepatocytes through eliminating toxic free radicals, inhibiting lipid peroxidation of the cytomembranes,19 reducing necrosis of hepatocytes, and by reducing inflammation.20 In addition, these protective effects may also be mediated through the strengthening of spleen function,18 reinforcing the body's immunological function,21,22 and by maintaining the equilibrium of hepatocyte Ca2+ levels.
In conclusion, emodin and APS provide significant protective effects during CCl4-induced hepatic injury through decreasing the serum levels of TBIL, ALT and AST, and enhancing SOD activity. The data from this study revealed that emodin combined with APS has a synergistic action in reducing ALT and restoring ALB levels, and was superior to the administration of a single dose of emodin. However, the hepatoprotective mechanisms of these medicinal herbs remain to be elucidated.
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