The Role And Mechanism Of IL-17A In The Formation Of Deep Vein Thrombosis

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The study on the role and mechanism of IL-17 A in platelet of patients with deep vein thrombosisObjective: To explore the role and mechanism of IL-17 A in regulating human platelet activation by investigating the difference of IL-17 A level and platelet aggregation between healthy volunteer and patients with deep vein thrombosis(DVT),as well as the effects of IL-17 A intervention on human platelet activation.Methods: The expression levels of IL-17 A in serum of healthy volunteer and patients with DVT were observed by ELISA.Platelet aggregation of DVT patients was detected by platelet aggregation apparatus.Venous blood of healthy volunteers was extracted and sorted.The platelets from healthy volunteers were incubated with r IL-17 A and r IL-17+Cs A(an inhibitor of mitochondrial permeability transition pore,MPTP)respectively,and then activated by ADP.Thus,the treated platelets were divided into 3 groups: 1)ADP group;2)ADP+IL-17 A group;3)ADP+IL-17A+Cs A group.Next,the change of platelet mitochondrial membrane potential and the expression of human platelet activation markers CD62 p and PAC-1 were detected by flow cytometry.Moreover,platelet aggregation was also detected by platelet aggregation apparatus.Finally,the change of the ratio of P-ERK2/ERK2 protein and the expression of p53 protein were detected by Western-blot.Results: Both IL-17 A level and platelet aggregation were significantly higher in patients with DVT than in healthy volunteer.In human platelets in vitro,compared with ADP group,platelet mitochondrial membrane potential decreased and the expression of activation marker CD62 p and PAC-1 was significantly increased in ADP+IL-17 A group.In contrast,after applying inhibitor Cs A in vitro,the platelet mitochondrial membrane potential was increased,CD62 p and PAC-1 was decreased in ADP+IL-17A+Cs A group compared with ADP+IL-17 A group,suggesting that Cs A inhibited the effect of IL-17 A.Additionally,platelet aggregation in ADP+IL-17 A group was significantly increased compared with ADP group,and platelet aggregation in ADP+IL-17A+Cs A group was decreased compared to ADP+IL-17 A group,implying the pro-aggregation role of IL-17 A and the anti-aggregation role of Cs A.Molecular protein detection showed that the ratio of P-ERK2/ERK2 protein and the expression of p53 in ADP+IL-17 A group were higher than those in ADP group,indicating that IL-17 A can stimulate protein expression of platelet activating signal pathway.Conclusions: In DVT patients,the expression level of IL-17 A in peripheral blood and platelet aggregation are up-regulated.IL-17 A can significantly activate ERK2/p53 signaling pathway in platelets and induce platelet activation through promoting MPTP opening to facilitate the formation of DVT.Part ? The study on the role and mechanism of IL-17 A in platelet of DVT mouseObjective: To investigate the the expression of IL-17 A after DVT and the effects of IL-17 A or Anti-IL-17 A on thrombus of DVT as well as the molecular mechanisms of IL-17 A in regulating platelet activation in a mouse model of DVT.Methods: To mimic DVT injury,we established a DVT model in mouse by constricting IVC.The expression level of IL-17 A in DVT mice was detected by ELISA.Next,we seek to evaluate whether IL-17 A has an effect on DVT at the early phase(2,3,6,12,24,48h)after operation,and to determine the time point of maximum difference.Then,according to the maximum difference time point,we randomly divided Balb/c mice into 5 groups: 1)Control group;2)Sham group;3)DVT group(100 ?l PBS was injected into mice through caudal vein 10 min before operation);4)r IL-17 A group(r IL-17 A diluted in 100 ?l PBS was injected into mice through caudal vein 10 min before operation);5)Anti-IL-17 A group(IL-17 A monoclonal antibody diluted in 100 ?l PBS was injected into mice through caudal vein 10 min before operation).The thrombus size postoperative 24 h was evaluated by echocardiograph.After that peripheral blood,thrombus and inferior vena cava were obtained by sacrificing the mice.The thrombus was taken out to measure its weight and flow cytometry was used to detect the expression of CD61 and CD49? in peripheral blood platelets.Then,platelet aggregation was detected by platelet aggregation apparatus.Finally,the expression of ERK2 and p53 protein was detected by Western Blot.Results: The expression of IL-17 A protein in plasma was increased than that in Control group after DVT operation.In dynamic thrombus detection,the effect of r IL-17 A on DVT began to vary from postoperative 6h,and the difference reached the peak at 24 h.Further detection in 24 h time points showed that compared with DVT group,the length and weight of thrombus in r IL-17 A group increased,while the length and weight of thrombus decreased in Anti-IL-17 A group.Compared with the DVT group,r IL-17 A promoted the expression of platelet activation markers CD61 and CD49? and promoted platelet aggregation,which showed adhesion and aggregation enhancement,while neutralization of IL-17 A,the expression of CD61 and CD49? in platelet activation was reduced and platelet aggregation atteneuated.However,the above three groups were still higher than the Control and Sham groups.In signal pathway,the expression of P-ERK2 and p53 in r IL-17 A group was higher than that in group DVT.In contrast,after applying IL-17 A antibody,the expression level of P-ERK2 and p53 was lower than that in DVT group.Conclusions: In mouse DVT models,the expression level of IL-17 A in peripheral blood is up-regulated.After operation,IL-17 A can activate ERK2/p53 signaling pathway in platelets and facilitate the activation and aggregation of platelets,promoting the formation of DVT.However,after neutralization of IL-17 A,the expression of P-ERK2 and p53 is inhibited and the activation and aggregation of platelets decreased,which inhibits the formation of DVT.Part ? The study on the role and mechanism of IL-17 A in neutrophil infiltration and endothelial cell activation of DVT mouseObjective:To observe the effects of IL-17 A on neutrophil infiltration and endothelial cell activation,and explore the molecular mechanisms of IL-17 A in regulating neutrophil infiltration and endothelial cell activation by establishing a mouse model of DVT.Methods: We randomly divided Balb/c mice into 5 groups: 1)Control group;2)Sham group;3)DVT group(100 ?l PBS was injected into mice through caudal vein 10 min before operation);4)r IL-17 A group(r IL-17 A diluted in 100 ?l PBS was injected into mice through caudal vein 10 min before operation);5)Anti-IL-17 A group(IL-17 A monoclonal antibody diluted in 100 ?l PBS was injected into mice through caudal vein 10 min before operation).After 24 h,peripheral blood,thrombus and inferior vena cava were obtained from mice.Firstly,the infiltration of neutrophils in thrombus was detected by immunohistochemical,and then the expression of macrophage inflammatory protein-2(MIP-2)in plasma was detected by ELISA.Secondly,the secretion of citrulline histone H3(Cit H3)in thrombus were detected by immunofluorescence and the content of DNA in plasma was detected by extracellular DNA fluorescence.Thirdly,the expression of P-P38 protein in neutrophil was detected by Western Blot.Additionlly,for the inferior vena cava,we first detect the expression of intercellular adhesion molecule-1(VCAM-1)and intercellular adhesion molecule-1(ICAM-1),which are the activation markers of endothelial cells,by immunohistochemical staining and real-time quantitative PCR.Finally,we detected the expression of P-P38 protein in signaling pathway of endothelial cell(EC)activation by Western Blot.Results: In thrombosis,both neutrophil infiltration and secretion of Cit H3 were more prominent in r IL-17 A group than that in DVT group,while in Anti-IL-17 A group these elements were inhibited compared to DVT group.In plasma,MIP-2 expression and the DNA in r IL-17 A group were significantly higher than those in DVT group.In contrast,MIP-2 and DNA expression were decreased after appiying the IL-17 A antibody,suggesting that IL-17 A plays an important role in promoting neutrophil infiltration.In venous vessels,the expression of endothelial cell activation markers VCAM-1 and ICAM-1 in r IL-17 A group was higher than that in DVT group,and the expression in Anti-IL-17 A group was lower than that in DVT group,implying the activation level of ECs was promoted by IL-17 A.There was almost no VCAM-1 and ICAM-1 expression in the Control and Sham groups.Lastly,both the ratio of P-P38/P38 in neutrophil and EC were increased in r IL-17 A group than that in DVT group and the expression of P-P38 was inhibited by IL-17 A antibody compared to DVT group.Conclusions: IL-17 A can significantly promote the neutrophil infiltration by increasing the expression of MIP-2,which activates neutrophil by P38 protein to boost the NETs formation,and facilitate ECs activation through activating P38 signaling pathway,thereby promoting the formation of DVT.