Studies On The Role And Mechanism Of TNF-α In The Formation Of Deep Vein Thrombosis

Deep vein thrombosis(DVT)is a common clinical vascular disease with potential mortality risk due to the subsequent blockage of pulmonary artery after thrombus shedding.The pathogenesis of deep vein thrombosis is complicated,more and more studies have suggested that inflammation plays a key role in the pathogenesis of venous thrombosis.The role of inflammatory factors in the pathogenesis of deep vein thrombosis has not been fully explained.As an important inflammatory factor,tumor necrosis factor alpha(TNF-α)has a series of physiological functions such as regulating vascular permeability,stimulating the release of procoagulant factors and inducing the formation of hypercoagulant state.Previous studies have shown that TNF-α may be involved in the process of arterial thrombosis.So far,there are very few studies focusing on the potential relationship between TNF-α and venous thrombosis.This study explored the role of TNF-α in the formation process of deep vein thrombosis through combining a variety of experimental methods.We first detected the expression level of plasma TNF-α in DVT patients and healthy controls.The expression of TNF-α m RNA and the expression of tissue factor(TF),plasminogen activator inhibitor-1(PAI-1),vascular endothelial cell adhesion molecule-1(VCAM-1)and intercellular adhesion molecule-1(ICAM-1)were observed by isolating and culture peripheral blood mononuclear cells(PBMCs).After TNF-α inhibitor was added,the changes of the above factors were observed to explore the correlation between TNF-α and the expression of TF,PAI-1,ICAM-1 and VCAM-1.In the second part of the study,we used the PCR-LDR method to detect the polymorphism of TNF-α gene rs1800629(-308G/A).The association between TNF-α rs1800629(-308G/A)polymorphism and venous thrombosis susceptibility was systematically and comprehensively evaluated by meta-analysis.In the third part,a mouse model of deep vein thrombosis was established to observe whether TNF-α can promote venous thrombosis in in vivo experiments.TNF receptor gene knockout mice were adopted to verified which type of TNF receptor produced the effect of TNF-α on thrombosis.In addition,ATL(15-Epi-Lipoxin A4)was used in mouse thrombosis model to verify whether ATL can inhibit the inflammatory response in the process of acute venous thrombosis,and whether ATL can inhibit the pro-thrombotic effect induced by TNF-α.The expression of pPI3 K,PI3K,p-AKT1,AKT1 and NF-κB in tissues of the inferior vena cava of mice in each group was detected to verify the specific signaling pathway mechanism of TNF-α involved in the process of venous thrombosis.Results from the first part of the study showed a significant increase in TNF-α expression in patients with deep vein thrombosis and a decrease in TNF-α expression after anticoagulant and thrombolytic therapy.TNF-α related genes may be activated during the pathogenesis of DVT.The expressions of TF,PAI-1,ICAM-1 and VCAM-1 in PMBCs were significantly increased in DVT group.The high expression of TNF-α was correlated with the high expression of TF,PAI-1,ICAM-1 and VCAM-1,and the addition of TNF-α inhibitor could decrease the expression of these factors.Results from the second part of the study showed no significant association between TNF-α rs1800629 polymorphism and DVT susceptibility.The third part of the study showed that TNF-α has a promoting effect on venous thrombosis.The pro-thrombotic effect of TNF-α may be activated by the PI3K/AKT/NF-κB signaling pathway mediated by TNFR2 receptor,TNF-α may increase of the expressions of various procoagulants,fibrinolytic inhibitors and adhesion factors such as TF,PAI-1,ICAM-1 and VCAM-1.ATL can inhibit the inflammatory response during the occurrence of venous thrombosis,reduce the expression of TF,PAI-1,ICAM-1 and VCAM-1,and also show a certain inhibitory effect on the pro-thrombogenesis induced by TNF-α.The specific mechanism of ATL inhibition may be through the inhibition of PI3K/ Akt /NF-κB signaling pathway and down-regulation of TNF-α-mediated expressions of a series of pro-coagulation factors and adhesion factors.The research of this subject has conducted a more in-depth exploration of the specific role of TNF-α in the process of deep vein thrombosis and related mechanisms,which may provide novel insights and methods for the diagnosis and treatment of deep vein thrombosis in the future.The research of this subject was divided into three parts.The purpose,methods,results and conclusions of each part are described below.Part I: Expression of TNF-α in patients with deep venous thrombosis and its possible mechanism in the formation of deep venous thrombosisObjective: To observe the expression of TNF-α in patients with deep venous thrombosis(DVT)and the change of TNF-α expression after anticoagulation and thrombolytic therapy;To observe the expression of TNF-α and the expression of tissue factor(TF),plasminogen activator inhibitor-1(PAI-1)and cell adhesion factor(ICAM-1 / VCAM-1)by isolating and culturating peripheral blood mononucle cells(PBMCs)from healthy controls and DVT patients.To preliminarily investigate the possible mechanism of TNF-α in the formation of deep venous thrombosis.Methods: A total of 50 patients with isolated acute deep venous thrombosis without obvious causes were included,and the control group consisted of 50 healthy people who underwent physical examination at the same time.The expression levels of TNF-α in the two groups were detected by enzyme-linked immunosorbent assay(ELISA)method.Patients in the case group were treated with enoxaparin and urokinase.The levels of TNF-α in case group were detected by ELISA after one week of treatment,and the changes of TNF-α levels before and after treatment were compared.Peripheral blood mononucocyte(PBMCs)of healthy control population and DVT patients were isolated and cultured.The expression of TNF-α m RNA was detected by real-time quantitative PCR(RT-q PCR),and the expression of TF,PAI-1,ICAM-1 and VCAM-1 were detected by Western Blot method.Finally,TNF-α inhibitor was added to detect the expression changes of TF,PAI-1,ICAM-1 and VCAM-1.Results: The expression level of plasma TNF-α in the patients with DVT was significantly increased,the expression level of TNF-α decreased after anticoagulant and thrombolytic therapy.The expression of TNF-α m RNA in DVT group was significantly higher.Compared with the healthy control group,the expressions of TF,PAI-1,ICAM-1,and VCAM-1 were also significantly higher than those of the healthy control group.After adding TNF-α inhibitor in PBMCs of case group,the expression of TF,PAI-1,ICAM-1 and VCAM-1 were significantly decreased.Conclusion: TNF-α is significantly overexpressed in patients with deep venous thrombosis,and its expression level is reduced after anticoagulant and thrombolytic therapy.Cell experiments confirmed that TNF-α is related to the activation of pro-coagulation factor and fibrinolytic system inhibitor such as TF and PAI-1,as well as the high expression of adhesion factors such as ICAM-1 and VCAM-1.Part II Association study of TNF-α gene rs1800629 polymorphism and susceptibility to deep venous thrombosisObjective: To explore the relationship between rs1800629(-308G> A)polymorphism of TNF-α gene and deep venous thrombosis in Han population.To comprehensively analyze the correlation between the polymorphism of rs1800629 and the susceptibility to venous thrombosis through the meta-analysis method.Methods: Fifty patients with deep venous thrombosis who were hospitalized in Lianyungang First People’s Hospital from November 2017 to January 2019 were collected as the case group.Fifty healthy people were recruited as the control group.The diagnostic criteria for deep venous thrombosis were adopted by the third edition of the 2017 Chinese Deep Vein Thrombosis Diagnosis and Treatment Guidelines.All patients were diagnosed by vascular ultrasound or venography.The basic clinical data and peripheral blood samples were collected of the case group and the control group.PCR-LDR method was used to detect the polymorphism of rs1800629 in the case group and the control group.Subsequently,through systematic searching of pubmed,embase,web of science,CNKI and Wanfang databases,the previous research literatures on the association between the polymorphism of rs1800629 and venous thromboembolism were collected.After extracting the research data,the data on the association between the rs1800629 polymorphism and the susceptibility to venous thrombosis were integrated,and a meta-analysis was conducted using Revman 5.3 software.Results: In the case group,the distribution frequency of each genotype of the rs1800629 was 42 cases of GG type(84%),6 cases of GA type(12%),and 2 cases of AA type(4%).The genotype distribution frequencies in the control group was 44 cases of GG type(88%),5 cases of GA type(10%),and 1 case of AA type(2%).The control group complies with Hardy-Weinberg equilibrium.There was no statistically significant difference in genotype distribution between case group and control group.Allelic model(G vs.A),heterozygous model(GA vs.GG),homozygous model(AA vs.GG),dominant model(GA + AA vs.GG)and recessive model(AA vs.GG + GA)gene comparison models were used for statistical analysis,no significant correlation was found between the rs1800629 polymorphism and the susceptibility to deep vein thrombosis.In the meta-analysis section,through systematic retrieval of relevant databases and combining the data from the current study,a total of 5 related studies were included.The total sample size of the case group included in the meta-analysis was 1,396,and the total sample size of the control group was 1,311.After calculation,the statistical results of the five gene comparison models were as follows: allele model(OR: 1.05 95% CI 0.91-1.23 p = 0.49),dominant model(OR: 1.06 95% CI 0.89-1.25 p = 0.52),heterozygous model(OR : 1.05 95% CI 0.88-1.25 p = 0.59),homozygous model(OR: 1.14 95% CI 0.66-1.97 p = 0.65)and recessive model(OR: 1.14 95% CI 0.66-1.96 p = 0.65),no significant difference was found in the comparison models.Additionally,no significant heterogeneity was found in all gene comparison models(I2 <50%).The results of the subgroup analysis according to different ethnic groups also found no significant difference in five genetic comparison models.Conclusion: There is no significant correlation between the polymorphism of rs1800629 in TNF-α gene and the susceptibility to deep venous thrombosis.Part III Study on the effect and mechanism of TNF-α in mouse model of deep vein thrombosisObjective: To establish a mouse deep vein thrombosis model,observe whether TNF-α can promote venous thrombosis,and explore possible mechanisms;To study the specific receptor pathways and signal pathway mechanisms of TNF-α on the formation of venous thrombosis;Further experiments verify whether ATL(15-epi-lipoxin A4)can inhibit the inflammatory response during venous thrombosis and whether it can inhibit the TNF-α-induced prothrombotic effect;and investigate the possible signal pathway mechanism of ATL’s effect.Methods: 1.Establish a mouse deep vein thrombosis model by ligating the inferior vena cava;the model mice are injected intravenously with PBS or TNF-α in PBS,and observe the effect of TNF-α on venous thrombosis in mice at different time points(6h,12 h,24h,48 h after surgery).Determine the time point with the largest difference in thrombus by measuring the length and weight of the thrombus;2.Mice were randomly divided into 4 groups: ○1 control group ○2 sham group ○3 DVT operation group(PBS injection with tail vein 10 minutes before operation and then thrombus modeling)○4 TNF-α + surgery group(Through vein injection of 0.4 μg/kg TNF-α in PBS 10 minutes before surgery for thrombus modeling);At the time point when the difference in thrombosis was greatest,the mice were sacrificed and the thrombosis of each group of mice was measured;ELISA method was used to detect the levels of TNF-α in the thrombus group and the control group and sham operation group;Flow cytometry was used to detect the expression of platelet surface activation markers CD61 and CD49β,platelet aggregation test was used to detect the platelet aggregation of mice in each group;Western Blot method was used to measure the expression of TF,PAI-1,ICAM-1 and VCAM-1 in the inferior vena cava vascular tissue of each group of mice.3.The DVT model was established in TNFR1-/-and TNFR2-/-mice(10 minutes before the operation,0.4 μg / kg PBS with TNF-α dissolved in the tail vein was injected for thrombus modeling).The venous thrombosis of TNFR1-/-and TNFR2-/-mice was measured;The expression of TF,PAI-1,ICAM-1 and VCAM-1 in the inferior vena cava vascular tissue of each group of mice were measured.4.The mice were then divided into two groups,one is the ATL + DVT surgery group(100 μg / kg 15-epi-lipoxin A4 in PBS),another group was ALT + TNF-α + surgery group(100 μg / kg 15-epi-lipoxin A4 in PBS dissolved 1 hour before surgery,0.4 μg / kg TNF-α in PBS10 minutes before surgery),the expression of TF,PAI-1,ICAM-1 and VCAM-1 were measured in the inferior vena cava vascular tissue of each group of mice;HE staining and immunohistochemistry were used to analyze the expression of inflammatory cells in thrombus tissues and the expression of TF,PAI-1 and ICAM-1 in the inferior vena cava vascular tissue of each group;RT-q PCR method was used to detect the effects of TNF-α and ATL on the expression of PI3 K,AKT1 and NF-κB m RNA in the inferior vena cava vascular tissue of mice;Western Blot method was used to detect the expression of p-PI3 K,PI3K,p-AKT1,AKT1 and NF-κB in the inferior vena cava endothelial tissue.Results: 1.Plasma TNF-α levels of mice in the DVT surgery group were significantly increased.TNF-α has been shown to promote venous thrombosis,and the effect of TNF-α on thrombosis was greatest at 24 hours after surgery.Compared with the control group,the sham operation group and the DVT operation group,the thrombus length and weight of the mice in the TNF-α + operation group were increased,and the difference was statistically significant.2.There was no significant difference in the platelet aggregation test between the TNF-α + surgery group mice and the DVT surgery group,and the flow cytometry method also showed no significant difference in the activated platelets in the plasma of the two groups.3.Compared with the control group,sham operation group,and DVT operation group,the expression of adhesion factors such as TF,PAI-1,ICAM-1,and VCAM-1 in the TNF-α + surgery group were increased.4.TNFR1-/-mice showed no significant difference in the length and weight of thrombus formation,while TNFR2-/-mice showed significant reduction in the length and weight of thrombus formation;The expression of TF,PAI-1,ICAM-1 and VCAM-1 decreased,and the difference was statistically significant.5.HE staining results showed that the inflammatory cell infiltration in the thrombus tissue of the TNF-α + surgery group was significantly higher than that in the surgery group,while the inflammatory cell infiltration in the thrombus tissue of the ATL + surgery group was significantly lower than that in the DVT surgery group.The results of immunohistochemistry and Western Blot showed that the expressions of TF,PAI-1 and ICAM-1 in the ATL + surgery group were inhibited compared with those in the DVT surgery group,while the TF and PAI-1 in the ATL + TNF-α + surgery group were suppressed.The expression of ICAM-1 and VCAM-1 was significantly lower than that of TNF-α + surgery group mice.6.The results of RT-q PCR and Western Blot showed that the expressions of PI3 K,ATK1,and NF-κB m RNA and protein in the inferior vena cava of the TNF-α + surgery group were significantly higher than DVT group.ATL can inhibit the expression of PI3 K,ATK1 and NF-κB m RNA and protein.Conclusion: In the mouse deep vein thrombosis model,the expression of TNF-α was elevated.TNF-α has a stimulating effect on venous thrombosis,TNF-α had no effect on platelet aggregation and activation.Its prothrombotic effect may be mainly achieved through the activation of the TNFR2 receptor-mediated PI3 K / AKT / NF-κB signaling pathway and the increase of factors such as TF,PAI-1,ICAM-1,and VCAM-1.ATL can inhibit the inflammatory response and the expression of TF,PAI-1,ICAM-1,and VCAM-1 during the occurrence of venous thrombosis.It also shows that it has a certain inhibitory effect on the TNF-α-induced prothrombotic effect.The inhibitory effect of ATL may be achieved by inhibiting the PI3 K / AKT / NF-κB signaling pathway and then down-regulating the expression of a series of procoagulant and adhesion factors mediated by TNF-α.