Study On The Implication Of The Gut Microbial Metabolite Trimethylamine-N-oxide On Venous Thrombosis

Part ? Effect of trimethylamine-N-oxide on the expression of TF and TFPI in human Umbilical Vein Endothelial CellsObjective: To investigate the effects of TMAO on the expression of TF and TFPI in primary HUVEC.Method: Primary HUVEC were cultured in medium ECM,containing 5% fetal bovine serum,5% penicillin and 5% streptomycin.CCK-8 was adopted to evaluate the influence of TMAO on cell viability;Reverse polymerase chain reaction and western blot were applied to evaluate TF/TFPI m RNA and TF/TFPI antigen respectively;TF procoagulant activity was determined by chromogenic assay.Result: Within the concentration range of 0-400?M,no significant difference of cell viability was observed between TMAO treated group and untreated controls;TMAO increased both TF m RNA and TF antigen in a dose and time-dependent manner,reaching the maximum level at 4 and 8 hours later respectively since HUVEC was treated with TMAO of 400 ?M;TMAO also induced an increased TF procoagulant activity in HUVEC.No significant difference of TFPI m RNA and antigen level was observed compared with the controls.Conclusion: TMAO induces TF expression in a dose and time-dependent manner while has no effect on the expression of TFPI.Part ? Mechanisms of TMAO on the regulation of TF expression in HUVECObjective: To investigate the potential mechanism of TMAO on the regulation of TF expression in HUVECMethod: Primary HUVEC were cultured in ECM,containing 5% fetal bovine serum,5% penicillin and 5% streptomycin.TF m RNA and activity was determined by reverse polymerase chain reaction and chromogenic assay respectively;western blot was adopted to assess phosphorylation of NF-?B?MAPK p38?ERK?JNK?c-jun and the level of I?B-?;immunofluorescence was applied to observe the localization of NF-?B in HUVEC.Result: The amount of I?B-?was decreased while the phosphorylation of NF-?B p65?c-jun?MAPK p38?ERK and JNK were increased by TMAO;NF-?B p65 translocated from cytoplasm into nucleus when stimulated by TMAO;the inducing effect mediated by TMAO was abolished by pretreatment with either PDTC or curcumin,the inhibitor of NF-?B p65 and c-jun respectively.Conclusion: the enhanced expression of TF by TMAO was mediated by transcription factors NF-?B and c-jun,both of which were regulated at least in part by the upstream MAPK signaling pathways.Part ? Effect of TMAO on the formation of venous thrombosis in mice modelsObjective: To observe the effect of dietary-origined TMAO on the formation of venous thrombosis in mice models.Method: C57 BL / 6J mice were fed with high choline diet and basic diet for 8 weeks respectively.The plasma TMAO levels were measured by HPLC tandem mass spectrometry(HPLC-MS / MS).The bleeding time was determined by cutting tail method and the plasma thrombin thrombin complex(TAT)level was detected by ELISA.The potential of thrombus formation was evaluated by deep vein thrombosis(DVT)mice model established by inferior vena cava stenosis method.Results: the plasma TMAO levels of hyper-choline group and control group were 21.40±2.65? M and 3.04±0.77?M 6.54 ? M respectively after feeding with different diets for 8 weeks.No significant difference was observed in body weight,liver and kidney function,blood glucose and lipid level.Compared with the normal diet,mice with hyper-choline diet manifested slightly shortened bleeding time,increased plasma TAT level and enhanced thrombus formation in the DVT model established by inferior vena cava stenosis method.Conclusion: The increase of plasma TMAO level induced by high choline diet can activate the coagulation process in vivo to some extent and promote the formation of venous thrombosis.Part ? Association of TMAO with venous thrombosisObjective: To study the association of plasma TMAO level and FMO3 gene polymorphism with venous thrombosis.Methods: Level of plasma TMAO from venous thrombosis group and control group was determined by LC / MS method.Competitive allele-specific PCR(KASP)was used to genotype the three polymorphism(rs1736557?rs2266782?and rs2266780)on the FMO3,which was crucial in the metabolism of TMAO.Results: The plasma TMAO concentrations in case and control group were 8.77 ±4.81?g/L vusus 6.50±4.07?g/ L.The difference was statistically significant(P < 0.026).The detection rates of rs1736557?rs2266782 and rs2266780 were 96.7598 and 97.7598,respectively.No significant correlation between VTE and rs226678 or rs2266780 polymorphism.The “A” allele of rs1736557 seems to be a protective factor of venous thrombosis.In the additive model and recessive model,the OR value of “A” allele for VTE were 0.32(95% CI: 0.11-0.92,p=0.027)and 0.34(95%CI: 0.12-0.96,p=0.033)respectively.Conclusion: High plasma level of TMAO may confer higher risk for venous thrombosis,but needs to be further confirmed.