The Molecular Mechanism Of MRP2 Post-transcriptional Down-regulation In Human Obstructive Cholestasis And The Hepatoprotective Study Of Swertianlarin In Common Bile Duct-Ligated Rats

Background:Bile acid transporters play an important role for maintenance of the enterohepatic BA circulation.Genetic defects and acquired BA transporters involved such as cholestasis, gallstones, fatty liver, liver cancer and other hepatic duct disease.Human BSEP(ABCB11) mutations are at least three kinds of molecular mechanism of liver disease, BRIC2(benign recurrent intrahepatic cholestasis type Ⅱ), PFIC2(progressive familial hereditary intrahepatic cholestasis type Ⅱ), ICP(intrahepatic cholestasis during pregnancy).MRP2 mutations cause DJS(Dubin- Johnson syndrome).Except the Nuclear receptors are closely balancing for BA transporter protein, post-transcriptional regulation machanism also play an important role.Transcriptional together with post-transcriptional regulation plasma membrane transporter expression and activity. Ubiquitin is a reversible post-transcriptional modification, recent studies have shown that Ub modification has wider areas and have more features in the process of cell.Kensuke Aida et al. found ABC transporters expression in canalicular membrane, such as bile salt export pump(BSEP) and multiple drug resistance associated proteins 2(MRP2) degradation by the ubiquitin-proteasome, and our previous study found that in obstructed cholestasis, MRP2 transporters in total protein levels also decreased.So we suspect that in obstructed cholestasis, MRP2 through endocytosis, the final destination is through ubiquitin to proteasome degradation. Swertianlarin is an herbal agent abundantly distributed in Swertiamussotii Franch, a Chinese traditional herb used for treatment of jaundice. Ursodeoxycholic acid(UDCA) and its analogs can inhibit inflammation and enhance elimination of toxic bile acids. It is the only approved drug that is widely clinically used to treat cholestatic patients such as primary biliary cirrhosis(PBC). However, one-third totwo-thirds of cholestatic patientswith PBC do not completely respond to UDCA. INT747, an FXR agonist, is a Phase II studies drug that exerts anticholestatic effects by altering bile acids metabolism in experiment models.Therefore, the search for potential drugs that target inflammation and bile acids pools for the treatment of cholestasis is important.Methods:1. We collected 30 obstructive cholestasis liver samples and 14 normal liver samples.All samples were immediately cut into pieces store in liquid nitrogen until used. 2. The m RNA expression levels of Ubiquitin ligase(GP78,HRD1,TEB4),and canalicular membrane transporter(MRP2),nuclear receptor(RXRα) in human obstructive cholestasis and normal liver were measured by Real time real-time PCR. 3. The protein expertion levels of Ubiquitin ligase(GP78,HRD1,TEB4),and canalicular membrane transporter(MRP2),nuclear receptor(RXRα) in human obstructive cholestasis and normal liver were measured by Western-Blot. 4. The protein-protein interaction among MRP2 and Ubquitin were analyzed by CO-IP(co-immunoprecipitation). 5. Use GP78 sh RNA to knock down GP78 in Hep G2 cells in order to test whether GP78 play an important role in MRP2 degradation. 6. Rats were randomly divided into three groups(n = 7 per group): sham saline,BDL without swertianlarin, and BDL with swertianlarin with each containing from five to seven animals.In the BDL swertianlarin group, rats were pretreated with swertianlarin 100mg/(kg/d) dissolved in 1%Tween-20 by gavage for 1 d and then underwent BDL followed by continued administration of swertianlarin for 3, 7, and 14 d. 7. The concentrations of alanine aminotransferase(ALT), aspartate aminotransferase(AST),alkaline phosphatase(ALP), total bile acids(TBA), total bilirubin(TBIL), and direct bilirubin(DBIL) in serum samples were analyzed by standard enzymatic assays using commercial kits. 8. Serum samples from sham operated rats and bile duct-ligated rats with and without swertianlarin were collected before sacrifice(n = 7 per each group) and stored at-80℃ untilanalysis. Serum cytokines, tumor necrosis factor alpha(TNFα), interleukin-1beta(IL-1 β), and IL-6 levels were determined by ELISA Kits. 9. The concentrations of serum bile acids chenodeoxycholic acid(CDCA), taurochenodeoxycholic acid(TCDCA), cholic acid(CA), taurocholic acid(TCA), deoxycholic acid(DCA),taurodeoxycholic acid(TDCA), tauroursodeoxycholic acid(TUDCA), tauro-alpha/beta-muricholic acid(Tα/βMCA),alpha-muricholic acid(αMCA), and beta-muricholic acid(βMCA) levels, serum lipid triglyceride hydrolase(Tgh), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C) weremeasured by standard enzymatic assays using commercial kits. 10. Rat liver samples were fixed in 4% formalin and subjected to standard histological procedures and paraffin embedding. Liver sections(5 um in thickness)were stained with hematoxylin and eosin and evaluated for histological lesions.Results1. real-time PCR confirmed that in human obstructive cholestasis livers GP78 m RNA expression levels are2.1 fold(P< 0.01) than normal control liver,while other ubiquitin ligases have no significant difference. 2. Western blotting confirmed that in human obstructive cholestasis livers GP78 protein expression levels are 4.2 fold(P< 0.05) than normal control liver,while other ubiquitin ligases have no significant difference. 3. The co-immunoprecipitation results show that in human obstructive cholestasis livers MRP2 ubiquitination were significantly than normal control livers. 4. Use ubquition-e GFP and ubquition-ko e GFP plasmid to transfection HEK293 cells 24 h then collected the total protein for pull down(co-immunoprecipitation).The results show that transfection ubquition-e GFP HEK293 cells have more MRP2 ubiquitination than ubquition-ko e GFP transfected HEK293 cells. 5. Hep G2 cells transfected GP78 sh RNA incressed MRP2 m RNA expression levels 1.5 fold than control plasmid(P< 0.05).While RXRa m RNA expression levels have no significant difference. MRP2 and RXRa protein expression levels are significantly increased(P< 0.05). 6. Serum ALT and AST levels for 14 d were markedly decreased in the BDL rats withswertianlarin(54% and 52% of saline BDL group, respectively.; P < 0.05). 7. The levels of serum TNFα in cholestatic rats with BDL for 3 and 7 d were significantly lower with swertianlarin treatment, 63% and 64% of saline BDL group, respectively(P < 0.05).The serum IL-1β levels for 3 and 14 d were noticeably lower in the swertianlarin BDL group(43% and 42% of BDL group without swertianlarin, resp.; P < 0.05).Serum IL-6 in the swertianlarin BDL group for 14 d was 61% of saline BDL group(P < 0.05) 8. The administration of swertianlarin decreased CDCA levels after 7 and 14 d and reduced TCDCA levels after 7 d in BDL rats(P < 0.05).The serum CA levels were decreased in BDL rats with swertianlarin for 3(P < 0.05),7and 14 d(P< 0.05).Serum TCA was significantly lower in BDL rats with swertianlarin after 3 and 14 d(P < 0.05) when compared to BDL rats without swertianlarin. TDCA levels were lower in BDL rats with swertianlarin after 7 d, compared with BDL rats without swertianlarin(P <0.05). Administration of swertianlarin decreased TUDCA levels in BDL rats after 14 d(P< 0.05) compared to BDL rats without swertianlarin.SerumαMCA levels were decreased in the BDL group with swertianlarin for 3, 7, and 14 d(P <0.05), compared with the BDL group without swertianlarin.Administration of swertianlarin for 14 d significantly decreased serumβMCA levels in the BDL rats(P < 0.05).Conclusion:1. In human obstructive cholestasis livers GP78 m RNA expression levels significantly increased than normal control livers. 2. In human obstructive cholestasis livers GP78 protein expression levels significantly increased than normal control livers. 3. In human obstructive cholestasis MRP2 ubiquitination were significantly increased than normal control livers. 4. Konck down of GP78 increase the expression of MRP2 m RNA levels and protein levels significant increased.While RXRαonly protein levels increased. 5. Swertianlarin Attenuates Liver Injury in Common Bile Duct-Ligated Rats. 6. Swertianlarin Has Anti-Inflammatory Effect on BDL Rats. 7. Swertianlarin Alters the Concentration of Serum Bile Salts in Rats with Bile Duct Ligation.8. Swertianlarin Does Not Affect Lipids Levels in BDL Rats.