Farnesoid X receptor (FXR) inhibits coagulation process via inducing hepatic antithrombin III expression in mice / 生理学报

Farnesoid X receptor (FXR) has been identified as an inhibitor of platelet function and an inducer of fibrinogen protein complex. However, the regulatory mechanism of FXR in hemostatic system remains incompletely understood. In this study, we aimed to investigate the functions of FXR in regulating antithrombin III (AT III). C57BL/6 mice and FXR knockout (FXR KO) mice were treated with or without GW4064 (30 mg/kg per day). FXR activation significantly prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT), lowered activity of activated factor X (FXa) and concentrations of thrombin-antithrombin complex (TAT) and activated factor II (FIIa), and increased level of AT III, whereas all of these effects were markedly reversed in FXR KO mice. In vivo, hepatic AT III mRNA and protein expression levels were up-regulated in wild-type mice after FXR activation, but down-regulated in FXR KO mice. In vitro study showed that FXR activation induced, while FXR knockdown inhibited, AT III expression in mouse primary hepatocytes. The luciferase assay and ChIP assay revealed that FXR can bind to the promoter region of AT III gene where FXR activation increased AT III transcription. These results suggest FXR activation inhibits coagulation process via inducing hepatic AT III expression in mice. The present study reveals a new role of FXR in hemostatic homeostasis and indicates that FXR might act as a potential therapeutic target for diseases related to hypercoagulation.

[Stable green fluorescent protein expression in Toxoplasma gondii mutant].

OBJECTIVE: To construct a Toxoplasma gondii mutant for stably expressing green fluorescent protein (GFP), and establish method to determine the rate of mutant-infected HeLa cells. METHODS: Freshly lysed-out tachyzoites of T. gondii RH strain were transfected with plasmid ptubP30-GFP/sag-CAT. Stable transformants were selected with chloramphenicol and limited dilution. The expression of GFP in mutant tachyzoite was determined by RT-PCR and fluorescence microscopy. When infected with 1 x 10(4)-1 x 10(7) mutant tachyzoites respectively for 24 h, the total number of HeLa cells with green fluorescence was determined by fluorescent microscope in 10 high-power fields, and the rate of HeLa cells with parasitophorous vacuole was determined by flow cytometry. RESULTS: Untransfected tachy-zoites were killed by chloramphenicol, while the stable transformants showed resistance to chloramphenicol. The expression of GFP gene was detected by RT-PCR. The P30-GFP transfectants displayed fluorescence outside the parasite. The rate of mutant-infected HeLa cells increased with the rise of the number of mutant for infection. When infected with 1 x 10(4)-1 x 10(7) tachyzoites, the numbers of HeLa cells with fluorescence were (14 +/- 6), (133 +/- 45), (332 +/- 93) and (443 +/- 90), and the rates of infected cells were (0.49 +/- 0.09)%, (8.76 +/- 0.50)%, (21.0 +/- 21.49)%, and (39.00 +/- 3.47)% by flow cytometry, respectively. CONCLUSION: T. gondii mutant with GFP tag is constructed, which provides a new method to determine the proliferation when cultured in host cells.