Hepatoprotective Role Of Milk Thistle On Acetaminophen-Induced Primary Cultured Rat Hepatocytes Injury

The development of livestock and poultry industry was seriously damaged and blocked by drug-induced liver injury. Acetaminophen was widely used as an analgesic and antipyretic drug, when overdose, could greatly hurt liver, and cause drug-induced liver disease or fulminant hepatitis. In research field of Pharmacology and Toxicology, acetaminophen is usually used in the model preparation of drug-induced liver injury, to investigate the mechanism of drug hepatitis.Primary cultures of rat hepatocytes were isolated from male Sprague-Dawley rats by two step collagenase digestion method, and treated with gradient doses of actaminophen. The viability was measured by MTT assay. The activities of ALT, AST and the levels of NO, MDA were determined by spectrophotometrical method, and the expression levels of cytochrome 2E1 were determined by Western-blot. The results as follows: the viability of hepatocytes treated with acetaminophen decreased, and decreased vertically with 16mM acetaminophen, the viability reduced to about 70% with 20mM acetaminophen treated for 24h. The activities of ALT, AST increased significantly with 10mM, 16mM acetaminophen treated for 12h. The concentration of NO increased, and there were greatly significance when 4mM, 10mM, 16mM acetaminophen treated groups compared with control. The concentreation of LDH increased significantly. The induction level of CYP 2E1 increased with the concentration enhancement of acetaminophen, but the induction level of 20mM acetaminophen was lower than that of 16mM acetaminophen. MDA changed less. Above all, we treated primary rat hepatocytes with 16mM acetaminophen to make a hepatocyte injury model. At the same time, with Milk Thistle to treat this model. We used the same methods before to investigate the best hepatoprotective effect of Milk Thistle in vitro, and select efficacious concentration of Milk Thistle. The results showed that the hepatocytes viability increased and the levels of ALT and AST decreased with Milk Thistle treated. There were correlation between MTT and the changement of ALT, AST. The concentration of NO decreased, the expression of CYP 2El increased after treated with Milk Thistle. The best hepatoprotective dose in vitro were: 1, 5μM silydianin, 0.5, 1, 5, 10μM silychristin, 5μM silybin, and 20, 50μg/L ethanol extract of Milk Thistle. Silydianin had the best hepatoprotective effect, and the ethanol extract of Milk Thistle was the weakest.We succeeded making hepatocyte injury model to investigate the mechanism of hepatocyte injury, and provided theoretic and statistical bases for selection of hepatoprotective drugs. Silydianin, Silychristin, Silybin had the same molecular formula, but their hepatoprotective effects were different. Maybe their different protective effects related with their space conformation, and need to be researched next.