Study On Cholagogic Actions Of Chinese Drugs By Regulating The Flow Of Qi To Alleviate Depression And Cholagogic Mechanisms Of Chai Yu Decoction

Objective: To study the cholagogic actions of Chaiyu Decoction on experimental rats and provide experimental basis for clinical use, we observed the influence of Chaiyu Decoction, Shaogan Decoction and Tongxie prescription on ANIT-induced cholestasic rats and investigated the protective effect of Chaiyu Decoction on primary cultured hepatocytes injured by CCl₄.Method: Experiment 1. The cholagogic actions of three traditional Chinese prescriptions on ANIT-induced cholestasic rats: 96 healthy Wistar rats were divided into 12 groups, namely normal control group, model group, Chaiyu Decoction groups of low, middle and high dose, Shaogan Decoction groups of low, middle and high dose, and Ursodeoxycholic acid tablet group. On day 1, rats were treated with bile duct intubation. On day 2, rats of normal control group were administered bean oil, and other groups were treated with 4 % ANIT. On day 4, each group was administered corresponding drug after being colleceted bile for 1 hour in the morning, and was treated accordingly again at night, then collected bile for 1 hour. On day 5 , treatment was same as day 4 except rats were not collected bile in the morning. Experiment 2. Establishing the method of primary cultured rat hepatocytes: The rat was anesthetized and the abdomen was opened. The portal vein was cannulated with a No. 19 Rochester needle and inferior vena cava was snipped. The liver was perfused via portal vein in situ with 39 ℃ preheated calcicum and magnesium-free perfusate, until the liver turns to grey, then with 39 ℃ preheated perfusate containing 0.025 % collagenase, until the liver turns to yellowish brown. The liver was transfered to a beaker containing RPMI1640 culture medium with serum, then liver was wobbled with sterile forceps. Thus cell suspension was prepared, then the beaker was shaken at 37 ℃ for 15 minutes and cooled at 0 ℃ for 10 minutes. Thesuspension was filtered through stainless steel mesh and the cell were separated from debris by centrifuging at 50 g for 2 minutes 3 times. The supernatant was removed and the cells were resuspended with culture medium. Then morphology of hepatocytes was observed and growth curve was described.Experiment 3. Preparation of Chinese herb contained serum: Wistar rats were divided into normal control group, Chaiyu Decoction groups of low, middle and high dose, and Ursodeoxycholic acid tablet group. Each rat was administered corresponding drug continuously for 3 days and 2 times a day. After last administration all rats were killed and the blood was withdrawn from inferior vena cava. The blood was centrifuged 3000 r/min for 15 minutes to separate the serum. Protective effect of Chaiyu Decoction on primary cultured rat hepatocytes injured by CCI4: The hepatocytes were inoculated at a density of 4 X 104 cells ml"1 onto 24 hole and 96 hole plates. The plates were cultured in incubaton at the condition of 37 °C and 5 % CO2 for 48 hours. Then the hepatocytes were divided into normal control group, Chaiyu Decoction groups of low, middle and high dose, and Ursodeoxycholic acid tablet group, and continued to be cultured for 6, 18 and 30 hours. Subsequently each group were treated with 10 mmol/1 CCI4 for 6 hours except normal control group. After treatment, MTT> ALI\ CG and Na+-K+-ATPase were measured respectively.Result: Experiment 1. After treated with three Chinese traditional prescriptions bile of rats on day 2 was remarkably increased compared with group of non-treatment. Experiment 2. Rat hepatocytes were isolated by modified two-step method described by Seglen. The yield of rat hepatocytes was 3 X 107 cells with viability of 90 %. Experiment 3. Normal control serum and drug-contained serum were preparedsuccessfully. Drug-contained serum remarkably decreased ALT concentration and increased CG concentration in cell culture medium. In addition, activity of Na+-K+-ATPase was remarkably heightened compared with model group. Conclusion: Three traditional Chinese prescriptions has cholagogic actions on ANIT-induced cholestasic rats. Chaiyu Decoction has protective effect on primary cultured rat hepatocytes injuried by CCI4 and probably by heightening activity of Na+-K+-ATPase.