The Up-regulation Of PC By FXR And The Preliminary Study On The Cooresponding Mechanism

Background: Protein C is a glucoprotein mainly originated from liver,on the surface of endothelium cell membrane,converted into activated protein C which acted as an anticoagulant and anti-inflammatory molecule during Sepsis. Recombinant activated human protein used in clinical treatment can significantly improved the prognosis of Sepsis patients. The farnesoid X receptor (FXR) is a nuclear receptor that plays key roles in hepatoprotection by maintaining the homeostasis of liver metabolism. FXR null mice display strong hepatic inflammation and develop spontaneous liver tumors. The ligand of FXR including various primary and secondary bile acids,and some of polyunsaturated fatty acids in physiological condition. chenodexycholic acid(CDCA)is the natural ligand of FXR in vivo. Recent researches strongly indicate FXR exert an anti-inflammatory function, and is a potential candidate of anti inflammatory therapy. Since FXR and PC both are highly expressed in liver, whether FXR could regulate PC to inhibit inflammation? The study on the effect of FXR on PC will provide some useful information with regarding to the physiological and pathological roles that FXR and PC played in the process of inflammation,and help us to find a potential drug target.Objective: the present study investigated the effect of FXR on PC expression and the mechanism by which FXR enhances the expression of the PCMethods: hepatocyte cell lines (HepG2) was employed for investigation, presented with the stimulus of different concentrations of FXR agonist (CDCA), and detected the PC expression levels by the techniques of RT-PCR, Real-time PCR and ELISA.Bioinformatics analysis (http://www.nubiscan.unibas.ch/) indicated that there are some potential FXR binding sites within PC promoter region (-3000～+150bp). According to the information revealed,amplify 3 truncated DNA fragments of upstream 5'noncoding sequences of PC gene from human genomic DNA, construct plasmids that fused these DNA fragments with PGL3-Basic vector. PC promoter activity was detected with the transient cotransfection of constitutively activated FXR expression plasmid(VP-FXR) and constructed recombinant plasmids by Luciferase reporter assay.Real-time PCR and ELISA were then performed to analyze the PC expression under the treatment of the combination of GW9662 with CDCA on HepG2, as well as treating with PPARγagonist troglitazone.Results:(1) In HepG2 cells, treating with FXR agonist CDCA could up-regulate PC expression in concentration-dependent manner(2) In L02 cells, the activation of FXR enhanced PC promoter activity.(3) In HepG2 cells, the activation of PPARγby troglitazone could up-regulate PC expression in concentration-dependent manner(4) Confirmed"FXR→PPARγ→PC"pathway in HepG2 cells, the further research on the mechanisms involved in"PPARγup-regulates PC and FXR enhances PC promoter activity"are still in need.Conclusion: The up-regulation of PC expression through the activation of FXR and PPARγ, provide useful information to explore the physiological and pathological role of FXR played in the process of aoritic plaque formation and inflammation.