Protective Effect Of Shuanghuanglian On Acetaminophen-Injuried Canine Hepatocytes

Shuanghuanglian preparation is composed by honeysuckble, baical skullcap root and forsythia suspense, with the efficacy of heat-clearing and detoxicating, acrid-cold herbs relieving superficies. The drug metabolism was involved by enzymes. The main enzyme with drug metabolism was cytochrome P450 system, which located in hepatic microsome. Objective: Establish a method for isolation and cultivation of primary cultured canine hepatocytes, explore the molecular mechanism of Chinese drug preparation SHL and interactions of each single drug, as well as the protection against hepatic injury, provide theory basement in clinic.Methods: Canine hepatocytes were obtained by two-step collagenase digestion method, cultured in Williams'E medium with multiple helper factors were added, the growth situation of the primary cultured canine hepatocytes was observed everyday and the images were took. At the same time acetaminophen(AP) was applied to induce the damage of hepatocyte, the supernatant of cell culture in differert period was collected, the survival rate of hepatocytes was measured by MTT method, the content of alanine aminotransferase(ALT), aspartate aminotransferase(AST) , malonaldehyde(MDA) and lactic acid dehydrogenase(LDH) in the supernatant were measured by spectrophotometric method, the injuried canine hepatocytes was treated by gradient concentration of SHL and the single herbs for 24-48h, the expression of CYP450 in canine hepatocytes was detected by Westen-blot.Results: 7.2×10~9 hepatocytes per dog was obtained by two-step collagenase digestion method, the survival rate of canine hepatocytes was 94.6%. Hepatocytes grew well, had a good morphology and could be used for investigation of drug metabolism. The optimal concentration of AP to injury the hepatocytes was 16mM. With gradient concentration of SHL treated for 24h, the expression of CYP450 in the injuried canine hepatocytes increased, but decreased when the drug concentration reached to 25mM. With 48h treatment, the expression of CYP450 decreased along with the increase of SHL contration, the expression of CYP450 enhanced obviously in group 1mM, 2mM and 5mM. It showed that the best effective dosage of SHL was 1-5mM. Different concentration of honeysuckble, baical skullcap root and forsythia suspense were administrated into the injuried Canine hepatocytes, the result showed that the expression of CYP450 was not as regular as that by SHL.Conclusion: SHL preparation had the ability of alleviating the AP-injuried Canine hepatocytes, it had protective effect to Canine hepatic injury.