| BackgroundCholestasis, also called cholestasis syndrome, refers to levels in the liver cells or bile duct, the bile generation, secretion, or movement disorders, and lead to bile into the duodenum unnormally, and cause the syndrome like jaundice and pruritus. Cholestasis including extrahepatic cholestasis and intrahepatic cholestasis, is a common basic disease of a lot of hepatobiliary diseases, its can causes include primary biliary cirrhosis, viral infection(PBC), primary sclerosing cholangitis (PSC), drug-induced liver damage, intrahepatic cholestasis of pregnancy (ICP) and many other factors, so the cholestasis is a common syndrome in clinical department of internal medicine on the incidence of very high (e. g., intrahepatic cholestasis of pregnancy incidence rate reaches as high as 13%), a serious threat to human health.Taraxacum has heat clearing and detoxification, detumescences fight and diuresis effect. In clinical, valids on chronic gall bladder spasm and stone disease. Based on the previous research, the Taraxacum can promote bile secretion in normal rats, protect the pharmacological activity of liver cells, is expected to be developed into a new medicine clinical treatment of cholestasis. But its mechanism has not been reported, pending further study. Therefore, to explore the signaling pathway of Chinese herbal medicine on choleretic effect such as dandelion, using modern analysis methods and molecular biology technology, and further research of the molecular mechanism of dandelion liver and gallbladder, not only to provide new research ideas of the development of safe and effective anti cholestatic drug, but also can guide the clinical scientific medicine, has important research significance and economic value.ObjectiveThe purpose of this study was to explore the ethyl acetate extract of Taraxacum(TEE) and caffeic acid induced effect and mechanism of liver and gallbladder acute intrahepatic cholestasis in rats by FXR and its target genes (CYP7A1 and Bsep)acting on alpha naphthyl isothiocyanate (a-naphthylisothiocyanate, ANIT).Methods1. Sprague-Dawley rats were randomly divided into 6 groups (n=6), namely: blank control group, Taraxacum-butanol extract group, Taraxacum-ethyl acetate extract group and Taraxacum-petroleum ether extract group. The model group were intragastrically administered (100.0 mg/kg body wt) to cause acute intrahepatic cholestasis model. Taraxacum-butanol extract group were intragastrically administered Taraxacum-butanol extract and perfusion of ANIT (100.0 mg/kg)as a model. Taraxacum-ethyl acetate extract group were intragastrically administered dandelion ethyl acetate extract and perfusion of ANIT (100.0 mg/kg) as a model. Taraxacum-petroleum ether extract group were administered intragastrically with petroleum ether extract of dandelion and perfusion of ANIT (100.0 mg/kg)as a model. The positive control group were intragastrically administered ursodeoxycholic acid(50mg/kg) and perfusion of ANIT (100.0 mg/kg) as a model. The model control group were intragastrically administered 0.5% CMC-Na solution and peanut oil as a control model.2h after the last administration, The rats were intraperitoneal injected 20% urethane (1 g/kg) to anesthetize, then bile duct intubated and operated, finally collected bile, systemic blood and liver.2. The acute toxicity evaluation of Taraxacum. The 100 KM mice were randomly divided into 10 groups according to the maximum dose to administrate with half male and half female, then declined to 0.75 times, They were feeded for 4 h after administration normally, then were observed for 7d.3. Pharmacokinetic comparison of cholestasis in rat model of acute liver Taraxacum-ethyl acetate extract in normal rats and ANIT induced rats.To establish a simple, rapid and sensitive LC-MS/MS method for determination of caffeic acid in biological samples, and the specificity, sensitivity, linear range, precision, recovery, matrix effect and stability of the method are confirmed. Normal rats and ANIT induced rats were treated with TEE. The blood concentrations wrer determinated by HPLC at each time point. The pharmacokinetic parameters were calculated, and then the difference of blood concentration of TEE in normal rats were compared to TEE in ANIT induced rats plasma.4. After the observation of cholestasis model rats were treating with TEE and caffeic acid of the three dose, the bile excretion, liver function and other biochemical indexes in bile pool, liver FXR, CYP7Aland Bsep gene expression and its protein expression effect. The model group were intragastrically administered (100 mg/kg body wt) to cause acute intrahepatic cholestasis as a model. TEE (high, middle, low) group were intragastrically administered different doses of TEE and perfusion of ANIT (100 mg/kg) as a model. Caffeic acid group were intragastrically administered corresponding dose of caffeic acid and perfusion of ANIT (100 mg/kg) as a model. The blank group were intragastrically administered ursodeoxycholic acid (50mg/Kg) and perfusion of ANIT (100 mg/kg) as a model. The model control group were intragastrically administered 0.3% CMC-Na solution and peanut oil as a control model. Animals were anesthetized with a single dose of urethane (1 g/kg body wt, intraperitoneally), Bile samples were collected and bile flow rate was also measured. The biochemical parameter (including ALT, AST, ALP, TBA TB) from plasma, bile and liver were determined using assay kits. The bile poor was determined by LC-MS/MS. H&E of liver sections, RT-PCR analysis of FXR, CYP7A1, BSEP mRNA expression and protein levels to explore and compare the different experimental groups rat. Results1. Compared with the model group, the bile flow and the biliary concentrations of TB, TBA in model group were decreased (P<0.05), specific gravity of liver was increased (P<0.05), the serum concentrations of ALT, AST, ALP, TB, TBA were obviously decreased (P<0.05). The TBA and TB were increased in liver homogenate (P<0.05). We found that among the Taraxacum-butanol extract, Taraxacum ethyl acetate extract and Taraxacum-petroleum ether extract group, compared with model group, the bile excretion were significantly increased in Taraxacum-ethyl acetate extract group (P<0.05), the serum concentrations of ALT, AST, ALP, TB, TBA and liver TB, TBA were decreased (P<0.05), and the concentrations of TBA, TB were increased significantly in bile (P<0.05).2. The median lethal dose(LD50) of Taraxacum-ethyl acetate extract was 306.25mg/kg,95% confidence limit was 249.75-376.99 mg/kg. The mice to be death may be the reason for liver ischemic necrosis.3. The average correlation coefficient (r) was> 0.9994, exhibited to be linear over the calibration range in 0.1～10μg/mL for Coffee acid. With the methodology like intra-day and inter-day accuracy and precision conforms to the requirements, respectively. No endogenous matrix interference was in plasma and with a good stability. Compared with the model group, the AUC (33.15±2.97vs9.61±0.76,) of TEE in rat acute cholestasis model group was larger, but the clearance rate (1.97±0.26 vs 5.73±0.37) was smaller.4. Compared with the model group, the bile flow and the biliary concentrations of TB, TBA in model group were increased (P<0.05), the serum concentrations of ALT, AST, ALP, TB, TBA were decreased (P<0.05), the TBA and TB were decreased in liver homogenate (P<0.05), the liver pathological damage was significantly reduced. Analysis of gene and protein expression in livers revealed that TEE can increase the expression of FXR and CYP7A1, Bsep gene and protein (P<0.05). The concentrations of TB, TBA in bile flow, bile pool and the biliary in Coffee acid were increased (P<0.05), the serum concentrations of ALT, AST, ALP, TB, TBA were decreased (P<0.05). the TBA and TB were decreased in liver homogenate (P<0.05), but no significant improvement was for the liver pathological damage. Analysis of gene and protein expression in livers revealed that TEE can increase the expression of FXR and CYP7A1, Bsep gene and protein (P<0.05). Efficacy was proportional to dose ConelusionTaraxacum-ethyl acetate extract and caffeic acid could ease the bile abnormal accumulation and the lesions of reflux into blood which were induced by the ANIT, then reduced jaundice and improved liver function; TEE can stimulate the increase in FXR expression, enhanced expression of FXR had a feedback inhibition to bile acid synthase CYP7A1 and decreased bile acid synthesis, activatied Bsep transcription of liver cells membrane,acceleratied bile acid efflux transporting from the liver cells to bile canalicular; That may be one of the mechanism of TEE activation of FXR in regulation and controlling bile acid synthesis and metabolism to cholagogue and hepatoprotect liver, demonstrated its hepatoprotective and choleretic, anti cholestatic effect. |
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