In Vitro And Vivo Hepatoprotective Effcts Of Asragalus Polysaccharide Against Carbon Tetrachloride-Induced Hepatocyte Damage In Common Carp(Cyprinus Carpio)

In aquatic environments, fish are directly exposed to various natural and man-made chemicals derived from agricultural and industrial pollution. Fish liver is the major target organ for the metabolism of most chemicals, an increasing number of chemicals have shown the potential to induce lesions in the liver of fish. Besides, heavy use of prophylactic antibiotics and other chemicals in intensive pond aquaculture systems to prevent and control the increased incidence of various kinds of fish diseases may also increased the chance of fish liver lesion. Recently, fish "liver and gall syndrome" has been frequently reported and caused dramatic loss in China. It is a non-infectious disease, pathogenic bacteria or viruses have not been identified, xenobiotic challenge due to drug abuse may be one of the important causes of the disease. So, it is important to develop different hepatoprotective agent for aquaculture. The present work is aiming at evaluating the hepatoprotective and antioxidant effects of Astragalus Polysaccharide (APS) on the carbon tetrachloride (CCl4)-induced hepatocyte and liver injury in common carp in vitro and in vivo. The main results were as follows:1. Primary culture of hepatocytes of common carp:In order to establish a stable primary culture model of hepatocytes in Cyprinus carpio,3different isolation methods (tissue culture, mechanical seperation and pancreatin digestion) and culture conditions of primary hepatocytes culture were compared in the present study. The survival rate was tested with trypan blue exclusion and the viability was assessed with WST-1; the morphological changes of cells in long-term culture were also observed. The results showed that in tissue culture, hepatocytes migrated from liver and proliferated after4-5days, compared with the other methods, it took much longer time for the hepatocytes to move out and it was difficult to collect the cells for further culture. The number and viability of the hepatocytes obtained by mechanical separation were obviously lower than by pancreatin digestion. The liver tissue digested by0.1%trypsin for30min yielded1.47×108cell/per g (liver weight) and the viability was91.43%. Both culture medium and concentration of serum influenced the attachment and proliferation of the hepatocytes. The cell cultivated in M199basal medium supplemented with10%fetal calf serum at28℃,4%CO2for8-9days can sub-cultured.2. Establishment of in vitro and in vivo hepatotoxicity models in Carp:The hepatotoxicity models in carp were established both in vivo and in vitro. In vitro, the primary cultured carp hepatocytes were treated with CCl4at concentrations of0,2,4,8,16&32mM/mL for4h. The result showed that effective concentration of CCl4was8mM/mL, and the viability of carp hapetocytes treated with CCl4(8,16&32mM/L) was significantly reduced and the contents of LDH, GPT, GOT, MDA and SOD were dramatically changed in the culture medium, compared to control; the best injury concentration of CC14was8mM/mL. In vivo, the fish were injected with0,12.5%,25%,30%and50%(v/v) of CCl4in arachis oil at a volume of5ml/kg of body weight and test biochemical parameters at24-hour intervals, the result showed that injection of12.5%of CC14didn’t cause significant differences, while injection of50%of CCl4led to death of fish, the fish injected with25%and30%CCl4showed significantly changes in serum GPT, GOT and LDH, compared with the control.3. The hepatoprotective effect of APS against CCI4induced liver (hepatocyte) injury:In vitro, APS (200,400&800μg/ml) was added to the carp primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre-and post-treatment) the incubation of the hepatocytes with CCl4(8mM/mL). APS at concentrations of200,400&800μg/ml significantly improved cell viability and inhibited the elevation of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH) and malondialdehyde (MDA), and significantly increased the reduced level of superoxide dismutase (SOD). In vivo, administration of APS at the doses of1.5and3g/kg in the diet for60days prior to CCl4intoxication significantly reduced the elevated activities of GPT, GOT and LDH and increased the reduced levels of total protein (TP) and albumin (Alb) in the serum, meanwhile, the reduced levels of SOD, glutathione (GSH) and total antioxidant capacity (T-AOC) were markedly increased and the MDA formation was significantly inhibited in liver tissue.