The Association Between Homocysteine And Hydrogen Sulfide And Venous Thrombosis And The Mechanism Research

Objective:1)To explore the risk factors of the deep venous thrombosis（DVT）in our hospital,lay the foundation for subsequent research of genetic risk factors and pathogenic mechanisms.2)Screening the gene polymorphism of metabolic enzyme of homocysteine（Hcy）and hydrogen sulfide（H₂S）and analysis the correlation of gene polymorphism and the level of H₂S and metabolic enzyme and DVT,analysis the role of H₂S in the process of venous thrombosis.3)Combined the results of first part and second part in vitro validation,explore the influence of H₂S and Hcy on Human Umbilical Vein Endothelial Cells（HUVEC）and the mechanisms of H₂S and Hcy regulating HUVEC via PI3K/AKT and eNOS pathway.Methods:Part One:A case-control study was carried on DVT patients and healthy population in People’s Hospital of Xinjiang Uygur Automous Region,discuss the risk factors for the disease through questionnaire and laboratory examination,synthetical analysis the risk factors by logistic regression model.Part Two:Detected the concentration of H₂S and the levels of Methylene Tetrahydrofolate Reductase（MTHFR）,cystathionine-β-synthase（CBS）and cystathionine-γ-lyase（CSE）in plasma of study population in part one.Screened the genetic polymorphisms of MTHFR,CBS and CSE by snapshot and direct-sequencing method,then analysis the effect of H₂S on DVT,the correlation of the levels of metabolic enzymes and the concentration of H₂S and Hcy and the genetic polymorphism and the association with the disease.Part Three:Cultured HUVECs with Hcy and H₂S,selected their concentration according to the results of MTT test.Cells can be divided into normal control group,Hcy group,Hcy+NaHS group,Hcy+NaHS+LY294002 group and Hcy+NaHS+L-NAME group.Detected the cells proliferation by MTT test,Annexin-V APC/7-AAD double staining method was used to detect apoptosis,Transwell method was used to detected cell migration,angiogenesis was observed under microscope,western blot was used to detected the PI3K,p-PI3K,AKT,p-AKT,eNOS and p-eNOS changes in protein levels,the concentration of NO was detected using chemical method.Comprehensive analysis the influence on PI3K/AKT pathway and the activation of eNOS by Hcy and H₂S.Results:Part One:1)Fruit intake（P<0.001）,pregnancy/puerperium（P<0.05）,AT-Ⅲ（P<0.05）,protein C（P<0.05）,Hcy（P<0.05）and folic acid（P<0.05）have statistic difference with the control group and were risk factors for DVT.2)After screening the discovery after multiariable logistic regression analysis,BMI（X²=5.59,P=0.018,OR=1.082,95%CI:1.014-1.154）,gestation（X²=9.002,P=0.003,OR=5.038,95%CI:1.752-14.490）,plasma levels of AT-Ⅲ（X²=54.135,P<0.001,OR=0.937,95%CI:0.920-0.953）and Hcy（X²=20.384,P<0.001,OR=1.084,95%CI:1.047-1.122）were independent risk factors for DVT.Part Two:1)The concentration of H₂S（Z=-2.344,P<0.05）and level of CSE（Z=-2.238,P<0.05）have statistic difference with the control group and may be the risk factors for DVT;plasma concentration of H₂S and Hcy were negatively correlated.2)The frequencies of TT genotype in MTHFR C677T and AG genotype in CSE A426G were significantly higher than in the controls and were significantly correlated with an increased risk of DVT（OR=3.0,95%CI:1.561-5.766,P<0.05;OR=2.011,95%CI:1.272-3.181,P<0.05）;there were no significant differences in genotype of MTHFR A1298C and CSE G1364T between the two groups;the genotype of CBS G919A,T833C and 844ins68 were wildtype homozygous in both two groups.3)Correlation analysis showed that:CSE A426G genotype was associated with plasma CSE protein level,MTHFR C677T and A1298C genotype were associated with plasma Hcy level,CSE A426G genotype was associated with plasma H₂S concentration.Part Three:1)HUVECs proliferation was inhibited when Hcy>15.625μmol/L,Na HS had no significant effect on HUVEC proliferation,500μmol/L for the cell inhibitory effect may be due to the too much drug concentration caused the cytotoxicity.2)Hcy can inhibit HUVECs proliferation,migration,angiogenesis and promote the apoptosis of HUVECs,H₂S can attenuate all these effects.3)Hcy can inhibit the PI3K/AKT signaling pathway,decrease the activity of e NOS and reduce endothelium synthesize NO;H₂S can stimulate the activity of the PI3K/AKT signaling pathway,enhance the activity of eNOS,increase endothelium synthesize NO,weaken the effect of Hcy on PI3K/AKT signaling pathway.4)The inhibitor of PI3K/AKT or eNOS can attenuate the protective effect of H₂S on HUVECs.Conclusion:Part One:The plasma concentration of AT-Ⅲand Hcy are closely related with DVT.Fruit intake,pregnancy/puerperium and serum folate concentration are the influence factors of DVT.Part Two:1)The plasma H₂S concentration is associated with DVT and a protective factor for DVT.2)There is a negative correlation between plasma Hcy and H₂S which is related to the decreased activity of metabolic enzymes.3)The genotype of MTHFR C677T and CSE A426G are associated with DVT,CSE A426G genotype man affect the plasma concentration of H₂S.Part Three:1)The mechanism of injury caused by Hcy on HUVEC is associated with the inhibition of PI3K/AKT signaling pathway,and make the downstream effector e NOS activity decreased.2)H₂S has an effect on protecting vascular endothelium via activating PI3K/AKT signaling pathway.3)The protection mechanism on HUVEC involves the interaction between H₂S and NO.