| Objective:1. To establish rat model of intrahepatic/extrahepatic cholestasis and obtain rat models with different expression of Mrp2;2. To investigate the pharmacokinetics behavior of cefoperazone (CPZ) in rat model of intrahepatic/extrahepatic cholestasis;3. To clarify the influence of hepatocyte membrane transporters Mrp2 on the pharmacokinetics of CPZ.Methods:1. Estradiol benzoate (EB,11 mg/kg/d) was used to replicate rat model of intrahepatic cholestasis. Rats were under ether anesthetized, a 3-cm incision was made just below the xiphoid process. A sterile PE-10 cannula was inserted into the proximal portion of the common bile duct and held in position with two silk sutures. The distal portion of the cannula was then occluded and brought out through the lower end of the midline incision and secured in the cervical part for 5 days. Then, cut the ligation of tube on the 6th day for 3 days, extrahepatic cholestasis rats were replicated.2. Wistar rats were divided into normal control group, intrahepatic cholestasis group, dissolvant control group (subcutaneous injection of olive oil), extrahepatic cholestasis group, sham operation group (rats opened abdomen, freed bile duct and closed abdomen), intrahepatic cholestatic group interfered with Yinzhihuang granules (gave Yin-zhi-huang granules to rat model of intrahepatic cholestatic for 3 d or 7 d), extrahepatic cholestatic group interfered with Yin-zhi-huang granules (gave Yin-zhi-huang granules to rat model of extrahepatic cholestatic for 7 d). Rats fasted for 12 h before experiment. Under ether anesthesia, the femoral artery was cannulated for the collection of blood samples (except for the extrahepatic cholestasis group). The bile duct was cannulated for the collection of bile samples. After the animals had recovered from anesthesia, CPZ (dissolved in saline) was intravenously injected at a dose of 20 mg/kg. Blood samples (0.25 mL) were collected from the femoral artery at different time points. Bile specimens were collected in fractions at different time period. The samples were determined by HPLC. Chromatographic conditions to determine of CPZ in plasma and bile (Reverse phase column:Cosmosil-ODS C18,5μm, 4.6×150mm, Kyoto, Japan) was been used. The mobile phase consisted of methanol-water containing 30 mM/L ammonium acetate buffer (20:80, v/v). Prior to use, the mobile phase was filtered through a 0.45μm hydrophilic membrane filter. The mobile phase was delivered at a flow rate of 1.0 mL/min. Detection was performed at a wavelength of 270 nm at 40℃. The sample injection volume was 10μL. Internal standard was puerarin). Plasma concentration-time data and bile concentration-time data for CPZ were analyzed by the DAS 1.0 statistical software for pharmacology.Results:1. The food intake, body weight and activity were decreased significantly in intrahepatic/extrahepatic cholestasis rats (P<0.01), and their biochemical indicators of AST, ALT, TBIL, DBIL, TBA and ALP significantly increased, while ALB and CHOL significantly reduced (P<0.01). In rat models of extrahepatic, the ears, eyes, tail and urine were yellowing, the liver was swelling, capsular tension of large, blunt the edge of thick, dull red color, and the histology of extrahepatic cholestasis rat was capillary duct dilatation, hepatic sinusoids become narrow and so on. After reconstruction surgery, liver tissue began to return to normal gradually, but some of the organizations had cirrhosis phenomenon. In rat models of intrahepatic, the liver was swelling, and yellow dye, a large number of intrahepatic bile duct hyperplasia, congestion phenomena, disordered arrangement of liver cells, lobular interstitial inflammation.2. The assay of CPZ in plasma samples and bile samples showed good linearity (0.9993 and 0.9996 respectivel) over a relatively wide concentration range from 0.16 to 160μg/mL. The standard linear equations were Y= 0.1703X+0.0019and Y'= 0.1404 X'+0.0011, respectively. The precision of CPZ at low to high concentrations were better than 9.55%and 8.06%for plasma samples and bile samples, respectively. The recovery of the method exceeded 88.3%and 92.42%for CPZ in plasma samples and bile samples.3. The pharmacokinetics behavior of CPZ in rat models of intrahepatic/ extrahepatic cholestasis were significant different from normal control rats. The concentrations of CPZ in plasma were significantly increased (P<0.01), but concentrations of CPZ in biliary were significantly decreased (P<0.01). The amounts of CPZ excreted by bile were reduced 28.58%and 59.23%, respectively. The t1/2, MRT and the CLsys were increased. However, CLbile were significant decreased (P<0.01).4. The pharmacokinetics behavior of CPZ in rat model of intrahepetic cholestasis rats treated by Yin-zhi-huang granules was significant different from intrahepatic cholestasis rats treated by physiologic saline. The t1/2 of them was 0.385±0.016 h and 0.526±0.17 h, respectively. The MRT of them was 0.472±0.098 h and 0.653±0.131 h, respectively. However, the pharmacokinetics behavior of CPZ was no significant difference between extrahepatic cholestasis rats treated by Yin-zhi-huang granules and extrahepatic cholestasis rats treated by physiologic saline (P>0.05). Conclusion:1. The rat models of intrahepatic cholestasis were replicated by injecting EB. Based on the bile duct obstruction-reconstruction, the rat models of extrahepatic cholestasis were replicated.2. The expression of efflux transporter Mrp2 are markedly reduced in cholestatic rat, which means that a large number of CPZ can not be smoothly excreted into the bile and cause concentration of CPZ in the blood increased significatly, the account of bile excretion was significantly reduced.3. Yin-zhi-huang granules commonly used in clinic as an adjuvant treatment of jaundice. Based on the pharmacokinetics of CPZ, the pharmcological effects of Yin-zhi-huang granules may be caused by adjusting the expression of Mrp2 or enhancing the transport function of Mrp2.New Ideas:1.To investigate the pharmacokinetics behavior in rats model of intrahepatic/extrahepatic Cholestasis;2. To interpret the pharmacokinetics behavior in cholestasis rats by Mrp2.
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