The Mechanism Research Of SIRT1 Protecting Endothelial Cells From Oxidative Damage And Inhibiting Expressions Of Pro-thrombosis Molecules In Deep Venous Thrombosis

Our previous research has established stable DVT animal models of C57 mice, SD rats and rabbits by inferior vena cava stenosis, and constructed H2O2 and thrombin induced human umbilical vein endothelial cells injury models in vitro. Using methods of RT-PCR、ELISA、Western blot、immunofluorescence、viral vector and gene chips, we have discovered and confirmed that KLF15、Rac1、MCP-1、IL-18 and NF-κB regulating oxidative stress genes played a crucial role in the pathogenesis of DVT, whose expression changes could affect the structure and function of endothelial cells, platelet and leukocyte, interrupt the balance of coagulation/anti-coagulation and fibrinolysis/anti-fibrinolysis system, promoting DVT. However, the mechanism of oxidative stress inducing endothelial cells injury and apoptosis is uncertain; the up and down regulation relationship of these genes are still unclear; whether Silent information regulator 1 (SIRT1) could inhibit DVT remains unreported. Therefore, this research aims to explore: ①Molecular mechanism of oxidative stress inducing endothelial cells injury and apoptosis; ②Protecting effect and regulating pathway of SIRT1 in OSI induced endothelial cells apoptosis; ③Effect and molecular mechanism of SIRT1 inhibiting secrection of pro-thrombosis molecules in endothelial cells. In animal experiment, preliminary researche is carried out to exlore expressions of SIRT1, Reactive oxygen species (ROS), Superoxide dismutase (SOD) and Malondialdehyde (MDA) in DVT mice models. In cells research, establishing hydrogen peroxide induced HUVECs injury model, by overexpressing and inhibiting SIRT1, explore the effect and mechanism of SIRT1 and PI3-K/Akt/GSK3β、MAPKs (c-Jun、ERK1/2、p38)、NF-κB、Nfr2/ARE signaling pathway regulating HUVECs apoptosis, secrection of pro-thrombosis molecules and influencing DVT. Providing new theoretical evidences for prevention and treatment of DVT.Objective:1. Establish DVT mice models by inferior vena cava stenosis method, the inferior vena cava wall tissues are obtained 24 hours after mdodel establishing. HE staining is used to observe the state of thrombosis. Detecting SIRT1, SOD, MDA and ROS levels in venous wall tissues, analyse the relationship between SIRT1 and oxidative stress reaction with the formation of DVT.2. Construct H2O2 induced HUVEC cells injury model in vitro, inspecting PI3-K/Akt/GSK3β、MAPKs、NF-κB and Nfr2/ARE signaling pathway in OSI mediated endothelial cells injury and apoptosis, exploring effects and molecular mechanism of SIRT1 protecting venous endothelial cells from oxidative injury and apoptosis.3. By overexpression and inhibition of SIRT1, explore its regulation effects on the secretions of pro-thrombotic molecular—P-Selectin, P-selectin glycoprotein ligand-1 (PSGL-1), Thrombomodulin (TM) and von Willebrand factor (vWF) in HUVECs and downstream key signaling pathways.Method:1. Ninety C57 mice are randomly divided into three groups:Control group (n=30), Sham group (n=30) and DVT group (n=30). Inferior vena cava stenosis method is used to establishe DVT model. The inferior vena cava wall tissues are obtained 24 hours after establishing model. HE staining is used to observe the thrombosis; SOD and MDA levels are detected by kits; Western blot is used to detect protein change of SIRT1; Flow cymetry is adopted to test the contents of ROS in inferior vena cava wall tissues.2. Human umbilical vein endothelial cells were treated with 0,100,200,400μmol/L concentrations of H2O2 4,12 and 24 hours, cell viability was measured by MTT, in order to determine the optimal concentration and time of H2O2 injury. HUVECs is pre-treated by 10,20,30μmol/L concentrations of SIRT1 agonist 2h before exposing to 200μmol/L H2O2 24h, cell viability was measured by MTT, in order to determine the protective effect of SIRT1 on HUVECs oxidative damage and its optimal concentration. HUVECs is pre-treated by 30μmol/L concentrations of SIRT1 agonist 2h before exposing to 200μmol/L H2O2 24h, DCFH-DA fluorescent probe capture, laser confocal microscopy, double hydrogen of DHR123 staining, flow cytometry technique is used to detect ROS levels in cells, in order to clear antioxidant effect of SIRT1. In situ end labeling (TUNEL) method, AnnexinV/PI staining, flow cytometry is used to detect the apoptosis rate; Western blot is used to detect changes of apoptosis related proteins Caspase-3 and Bcl-2, in order to confirm inhibition effect of SIRT1 in oxidative stress induced apoptosis of HUVECs. The HUVECs was exposed to 200μmol/L H2O2, at 0,15min,30min, lh, 2h,3h,4h,8h, Western blot is used to detect PI3-K/Akt and MAPKs pathway key protein expression changes in order to the oxidative stress response protein changes with time. Based on 30μmol/L SIRT1 HUVECs 2h after agonist pretreatment, the exposure to 200μmol/L H2O2,4h Real-time PCR, Western blot detection SIRT1, PI3-K/Akt/GSK3β,MAPKs, NF-kappa B, Nfr2/ARE signaling pathway key genes, the expression in regulation and molecular mechanism of SIRT1 mediated oxidative stress on endothelial cell injury and apoptosis3.①Immunofluorescence is used to locate the SIRT1 protein in HUVECs.②EX527 specific regulation of SIRT1 to 10,20,30μmol/L concentration of agonist pretreatment of HUVECs 2h after the exposure to 200μmol/L H2O2,24h after Western blot detection P-selectin PSGL-1, vWF, TM protein expression changes, to explore the SIRT1 on endothelial cells secrete prothrombotic molecular formation and concentration dependent effect. ③30μmol/L of SIRT1 agonist pretreatment of HUVECs 2h, with or without the SIRT1 inhibitor, and the exposure to 200μmol/L H2O2,24h after PCR and Western blot were used to detect P-selectin and PSGL-1, vWF, TM gene and protein expression changes, to verify the SIRT1 regulation of endothelial cell secretagogue thrombosis molecules, effect of DVT formation effect and mechanismResult:1. DVT group mortality was 0%, plug rate 83.3%, stenosis rate was 96.05%±0.62%, the residual lumen cross-sectional area percentage of 3.95%±0.62%, 5.57±0.32mm thrombus length, wet weight of thrombus in inferior vena cava diameter was 0.013±0.009g,1.52±0.03mm. SIRT1, SOD, MDA and ROS expression in normal group compared with the sham operation group no statistically significant difference (P> 0.05) group vein. DVT superoxide dismutase (SOD) was significantly lower than that in normal group and sham operation group, malondialdehyde (MDA) were significantly higher than those in normal group and sham operation group (P<0.05) lower than that in normal group and sham operation group. DVT group the expression of SIRT1 protein the reactive oxygen species (P<0.05) group. DVT (ROS) in venous wall were significantly higher than those in normal group and sham operation group (P< 0.05).2. ①Human umbilical vein endothelial cells were treated with 0、100、200、 400μmol/L concentration of H2O24,12,24 hours, cell viability showed significant concentration and time dependent, with the increase of H2O2 concentration and prolonging treatment time, cell viability decreased (P<0.01),200μmol/L group and 400μmol/L each time point group OD value had no statistical difference (P> 0.05), showed decreased cell viability to the platform stage, and decreased with the increase of H2O2 concentration. The classical literature reference results, selected 200μmol/L,24h is H2O2 the optimal concentration and time. ②The intervention with 10,20,30μmol/L concentration of SIRT1 agonist pretreatment HUVECs 2h, the cell viability gradually restored (P<0.05), and showed a concentration dependent, with the increase of SIRT1 agonist concentration, cell viability gradually restored (P><0.05), for 30μmol/L SIRT1 agonist effect was most significant, so we choose the 30μmol/L As the concentration of the follow-up experiments.③200μmol/L H2O2 treatment 24h of HUVECs, cell apoptosis rate, expression of apoptosis related proteins (Bcl-2, caspase-3), intracellular ROS content was significantly increased (P<0.05), and given 30μmol/L of SIRT1 agonist pretreatment for 2h, the index decreased significantly (P<0.05).④the HUVECs exposed to 200μmol/L H2O2, phosphorylation of protein pAkt p-c-jun and pERK1/2, p38 protein in 30min expression began to increase to 4-8h to peak (P<0.05), so select the 4h pathway protein detection time. ⑤By taking 200μmol/L H2O2 treated HUVECs 4h, c-jun, ERK1/2, p38, NF kappaB, Nrf2, GSK3βmRNA expression is upregulated (P<0.05), Akt, SIRT1 mRNA expression (P<0.05); p-c-jun and pERK1/2, p38, NF-kappaB, Nrf2, GSK3β protein expression (P<0.05), pAkt, SBRT1 mRNA and protein expression (P<0.05), while giving 30μmol/L of SIRT1 agonist pretreatment after 2h and H2O2 on the above gene and protein regulation was reversed (P<0.05).3. ①SIRT1 protein mainly located in the nucleus. (DHUVECs were exposed for 24h in 200μmol/L H2O2, P-selectin and PSGL-1, vWF and TM mRNA and protein expression increased significantly (P<0.05), and given 30μmol/L concentration of SIRT1 agonist pretreatment for 2h, H2O2 on the above gene and protein upregulation was reversed (P<0.05). ③Added the SIRT1 specific inhibitor EX527, regulation effects of SIRT1 on P-selectin, PSGL-1, vWF and TM mRNA and protein expression disappeared (P<0.05).Conclusion:1. Oxidative stress plays an important role in the occurrence and development of DVT. PI3-K/Akt/GSK3p, MAPKs (c-Jun, ERK1/2, p38), NF-κB, Nfr2/ARE signaling pathway are involved in venous endothelial cells apoptosis induced by oxidative stress.2. SIRT1 could inhibit pro-apoptotic pathway (MAPKs), inhibit inflammatory response pathway (NF kappaB), activate anti-apoptotic pathway (PI3-K/Akt/GSK3β), proteting endothelial cells from oxidative stress induced apoptosis and injury.3. Overexpression of SIRT1 could inhibit up-regulating of P-Selectin, PSGL-1, vWF and TM mRNA and protein in oxidative induced endothelial cells, suggesting a new target for prevention and treatment of DVT.