The Effect Of Swertia Mussitii Alcohol Extract On Expression Of Hepatocyte Membrane Transporters MRP3and BSEP

Background&objective:There are many important physiological functions of bile acid. Bile acid involves inseveral material metabolism and transportation. However, bile may also accumulate in liver(called cholestasis) when there is hepatocyte injury. In normal physiological conditions，some hepatocyte membrane transporters involved in the steady-state of intrahepatic bileacid. This precision adjustment mainly includes：Distributed in the basal membrane surfacesuch as MRP3, MRP4; in bile duct membrane surface such as MRP2,BSEP. The multidrugresistance-associated protein3(MRP3) is a member of the ATP-binding cassette (ABC)transporter superfamily. It is expressed in many tissues, including liver, kidney, bladder,intestine, and adrenal gland in humans and rodents. MRP3is localized on the basolateralmembrane of cells and excretes sulfated, glycine and taurine-conjugated bile salts, bilirubinglucuronides,17α-glucuronosy lestradiol, leukotrienes, and a number of drugs. Theexpression of hepatic MRP3is low under normal physiological condition, but its expressionis significantly up-regulated as an adaptive protective response in cholestasis asdemonstrated in rodents following bile duct ligation (BDL) or LPS-treatment. The bile saltexport pump (BSEP) belongs to the MDR/TAP subfamily of ATP binding cassettetransporter, BSEP are canalicular transporter proteins. BSEP is responsible for the bilesalt-dependent bile flow. It mainly transports monovalent bile acids including taurin andglycine conjugates of the primary bile acids cholic acid (CA) and chenodeoxycholic acid(CDCA) as well as the secondary bile acid deoxycholic acid (DCA). Mutations of BSEP areassociated with cholestatic liver diseases of varying severity including progressive familialintrahepatic cholestasis type2and genetic polymorphisms are linked to intrahepaticcholestasis of pregnancy (ICP) and drug-induced liver injury.Literature report，a variety of nuclear receptor/nuclear transcription factors involved in the gene transcription regulation/transcription of MRP3and BSEP. At present, many drugssuch as UDCA，rifampicin and phenobarbital used as standard treatments for cholestasis actvia NRs and stimulation of their target genes. Some studies shows that SP1、PXR、CPF canup-regulates the expression of MRP3. The expression of BSEP is closely related to RXRα.RXRα not only can be directly used as ligands of BSEP upregulates its expression，alsowith other nuclear receptors plays an indirect effect in clustering formation.Unfortunately, current therapy for cholestasis is suboptimal and challenging to theclinician, as there are limited drugs to treat it. Medical therapy has recommendedursodeoxycholic acid, however, the effect is not good enough. Accordingly, it is a highlypressing concern to find a safe and effective method of treatment for the cholestasis.Swertia mussitii is the plant of gentianaceae section and grows mainly in high altitude area.Swertia mussitii is widely used as a traditional Chinese medicine for treatment of jaundicehepatitis，the mechanism is not clearly demonstrated. Whether Swertia mussitii regulate theexpression of hepatocyte membrane transporters MRP3and BSEP，What kind of nuclearreceptors are involved in it are the focus of this study. The optimal concentration of Swertiamussitii used to stimulate HepG2cells was determined by MTT method. Then，Total RNA,membrane protein and nuclear protein were extracted from HepG2cells at0h，12h，24h，48h and72h post co-cultured with Swertia mussitii. The expression of MRP3and BSEP，SP1，PXR,CPF and RXRα at mRNA and protein level were detected using real-timequantitative PCR and Western blotting，respectively. The paper provides a new theory totreat cholestasis in clinic.Methods:1. The liver cancer cells (HepG2) was treated by Swertia mussitii under differentconcentrations and times, the proliferation concentrations was measured by MTT.2. The liver cancer cells (HepG2) were stimulated with Swertia mussitii in vitro and indifferent time point(0h、12h、24h、48h、72h)，DMSO as an negative control and UDCAas an positive control. RT-PCR was carried out to detect MRP3and BSEP，nuclear receptorPXR、CPF、RXRα，Nuclear transcription factors SP1mRNA expression in different timepoints.Results:1. Determined by MTT experiment results show that the concentration of25~50uM of Swertia mussitii treatments had no effect on cells proliferation.2. The result of RT-PCR indicated that the expression of MRP3、PXR、CPF、RXRαwas significantly induced by Swertia mussitii at mRNA. However, mRNA levels of BSEPhave little effect probably is regulated possibly after transcription.3. Western blot results show that the drug group can obviously stimulate membranetransporters MRP3, BSEP and related nuclear receptor PXR, CPF, RXR α proteinexpression. Among them, the increase of MRP3、 BSEP on protein levels were better thanUDCA group.Conclusion：1. The results showed that Swertia mussitii could up-regulation both mRNA andprotein expression of MRP3in liver cancer cells (HepG2).And the increased expressionmay be related to the increased of nuclear transcription factor SP1and nuclear receptorPXR, CPF.2. The expression of BSEP was up-regulation by treatment with Swertia mussitii onprotein level. And the increased expression may be related to the increased of nuclearreceptor RXRα. However, mRNA levels of BSEP have little effect，probably is regulatedpossibly after transcription.