Geniposidic Acid Protected Against ANIT Induced Hepatotoxity And Acute Intrahepatic Cholestasis Due To FXR Mediated Regulation Of BSEP And MRP2

ObjectiveCholestasis, is refers to hepatocytes or bile duct level of bile formation or excretion dysfunction, which cause to reduce the amount of bile to reach the duodenum, or bile reflux into the blood. Cholestasis is modern clinical common diseases, the pathogenesis is extremely complex, the lack of effective drug treatment. In traditional medicine, clearing heat and removing dampness herbs in anti cholestatic has its unique effect and advantages, especially rich in ring iridoids medicine Kyo, but its mechanism of action is no definite conclusion, this paper examines the iridoid compounds geniposide acid in the effectiveness and mechanism of hepatoprotective and choleretic action research, provide basic theoretical data for its further development.The investigation was designed to evaluate the hepatoprotection effect and potential mechanisms of GPA derived from Gardenia jasminoides Ellis (Rubiaceae) on fighting against α-naphthylisothiocyanate (ANIT) caused liver injury with acute intrahepatic cholestasis. GPA has been widely reported, but the underlying occurrence mechanism remains unclear.MethodsIn vivo study to establish a hplc method for determination the purity of geniposidic acid. Median lethal dose (LD50) of GPA in mice was determined by Bliss method. Sprague-Dawley (SD) rats were intragastrically (i.g.) administered with the GPA (100,50 and 25 mg/kg B. W. every 24 hours) for seven consecutive days, and then they were treated with ANIT (i.g.65 mg/kg once in the 5th day) which induced liver injury with acute intrahepatic cholestasis. Serum and bile biochemical analysis, bile flow rate and liver histopathology were measured to evaluate the protective effect of GPA fight against ANIT treatment. The protein and mRNA expression levels of farnesoid X receptor (FXR), bile salt export pump (BSEP), multidrug resistance associated protein2 (MRP2), were evaluated to study the effect of liver protection about GPA against ANIT induced hepatotoxicity and underlying mechanisms. Chromatographic conditions: Phenomenex C18(250 mm×4.6 mm,5μm) column with a mobile phase:acetonitrile: 0.5% acetic acid solution (28:72), flow rate:1 mL/min isocratic elution, column temperature was 30℃, determined under UV=238 nm, fitted with statal2.0 compartment model and calculate the pharmacokinetic parameters.In vitro, the cells were divided into normal control group without treatment, high, medium and low dose group (adding a final concentration of 4,1,0.25 mmol/L (mM)GPA induced by 48 h), after 48 h, RNA and protein samples of BRL-3A cell that interved by GPA were collected. The levels of protein and mRNA expression of samples about MRP2/BSEP/FXR were determined by WB and RT-PCR methods. The cells were also divided for normal group (routine cell culture), negative control group (blank transfection) and transfected siRNA-FXR group, transfected siRNA-FXR+GPA 4 mM group (i. e. after transfection siRNA-FXR with 4 mM GPA), siRNA-FXR+GPA 1 mM group (i.e. after transfection siRNA-FXR with 1 mM GPA intervention), intervention siFXR+GPA 0.25 mM group (i.e. after transfection siRNA-FXR with 0.25 mM GPA). After 6 h, changing the culture medium to normal cultured for 24 h, and then after 24 h in each group, according to the dosge of GPA added to the culture plate. The levels of protein and mRNA expression of siRNA-FXR transfected cells samples about MRP2/BSEP/FXR were determined by WB and RT-PCR methods.ResultsSome abnormalities were observed on ANIT treated rats including weight loss, reduced food intake and hair turned yellow. Obtained results demonstrated that at dose 100 and 50 mg/kg B. W. (P< 0.01) and 25 mg/kg B. W. (P< 0.05) of GPA pretreated dramatically prevented ANIT induced decreased in bile flow rate. Compared with ANIT treated group, the results of bile biochemical parameters about total bile acid (TBA) was increased by GPA at groups with any dose (P< 0.01), glutathione (GSH) was increased significantly at high dose (P<0.01) and medium dose (P<0.05), total bilirubin (TB) was increased at high and medium dose (P<0.05), direct bilirubin (DB) was only increased at high dose (P<0.01). Serum levels of glutamic-Oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), γ-glutamyltranspeptidase (γ-GT), TB, DB and TBA in comparison with ANIT treated group (P<0.01) were reduced by GPA (100 and 50 mg/kg B. W.) pretreatment. Histopathology of the liver tissue showed that pathological damages and hepatic portal area filled with bile were relieved after GPA pretreatment compared with ANIT treated group. The protein and mRNA expression of FXR, BSEP and MRP2 were decreased in ANIT treated group. On the contrary, the protein and mRNA of FXR, BSEP and MRP2 were up regulated significantly pretreatment by GPA at dose of high and medium groups. On protein level of BSEP and MRP2 the result shown no statistical difference in GPA (25 mg/kg B. W.), but it was not same shown in mRNA level.The level of protein and gene expression of FXR/MRP2/BSEP of BRL-3A cells treated by GPA were detected by WB and RT-PCR method, some doses of GPA on BRL-3A after 48 h treatment, compared with interfered by GPA in BRL-3A cells, the 4mM GPA obviously (P<0.01) increased the expression level of FXR/MRP2/BSEP protein and mRNA in BRL-3A cells; the lmM GPA significantly (P<0.01) upregulate the FXR/BSEP protein expression in BRL-3A cells (P<0.05), and significantly(P<0.05) upregulated MRP2 protein,1 mM GPA increased the expression of FXR/BSEP mRNA expression in BRL-3A cells, and significantly upregulated MRP2 mRNA (P<0.01); there was no difference in protein and mRNA levels about 0.25 mM GPA, GPA had a significant effect on the upregulation of MRP2 BRL-3A cells (P<0.05).The results of WB and RT-PCR showed that FXR gene silenced compared with the normal cultured BRL-3A cells; cell transfection siRNA-FXR, the FXR gene and protein expression levels were significantly decreased (P<0.01), gene and protein levels of BSEP and MRP2 also decreased significantly (P< 0.01); and negative siRNA transfection group (transfected with siRNA-conrol) was not influenced comparing with normal group. FXR, MRP2 and BSEP expression levels of transfection group were lower, with statistical significance (P< 0.01); and negative transfected group compared with the normal group, the FXR expression level no difference (P>0.05). Compared with each group of cells in the transfection siRNA-FXR, siRNA-FXR+4 mM GPA group, siRNA-FXR+1 mM GPA group, GPA siRNA-FXR+0.25 mM group FXR, MRP2 and BSEP gene and protein levels and were improved(P<0.01), and were positively related to the dose, which siFXR+4 mM GPA for the group with significantly difference (P<0.01).ConclusionsThe results of this investigation have demonstrated that the GPA exerts a dose dependent hepatoprotection effect on ANIT induced liver damage with acute intrahepatic cholestasis in rats, which may due to FXR mediated regulation of bile transporters like BSEP and MRP2. In vivo study, FXR gene has been silent by siRNA-FXR on BRL-3A cell, and its were found that GPA protected against ANIT-induced hepatotoxity and acute intrahepatic cholestasis, may due to FXR mediated regulation bile acid transporter BSEP and MRP2.