Mechanism By Which MiR-96/VAMP8 Regulate Lipopolysaccharide Induced Platelet Activation

Background:Platelet activation is the initiating factor and the core of acute myocardial infarction(AMI).Thus,dual antiplatelet therapy(DAPT)is the cornerstone of AMI treatment.Studies have shown that current antiplatelet drugs may fail to inhibit other platelet activation pathways,leading to a significantly higher risk of ischemic cardiovascular and cerebrovascular events recurrence.Meanwhile,coronary heart disease is a chronic inflammatory disease and the inflammatory effect runs through the whole process of atherosclerosis development.Platelets are not only effector cells of thrombosis,but also play a role in regulating immune and inflammatory responses.Platelets may therefore induce platelet activation through an inflammatory signaling pathway in which "platelet escape" occurs,resulting in high platelet reactivity(HPR).However,the specific mechanism of platelet activation induced by inflammation remains unclear.Therefore,this study will explore the role and mechanism of inflammation induced platelet activation from clinical cases,animal models and platelet culture in vitro at multiple levels,so as to provide a theoretical basis for exploring the pathogenesis of HPR after antiplatelet therapy and searching for new therapeutics targets.Part Ⅰ.The effect of high platelet reactivity on platelet function after antiplatelet therapy Objective:To observe the effect of high platelet reactivity(HPR)on platelet activation and aggregation after antiplatelet therapy.Methods:1.Twenty patients with initial acute myocardial infarction(AMI)and 20 patients with HPR admitted to the department of cardiovascular medicine of the Second Affiliated Hospital of Nanchang University from July 1 to August 31,2022 were randomly included.Twenty patients with non-coronary artery disease confirmed by coronary angiography were randomly selected as control.2.General clinical baseline data of patients were collected,including age,gender,systolic blood pressure,diastolic blood pressure,past medical history,smoking history,blood routine,biochemical test and drug use.3.The maximum platelet aggregation rate(MAR)and average platelet aggregation rate(AAR)of each group were detected by platelet function analyzer.4.Platelet miRNAs and proteins were extracted from the patients,and the expression levels of miR-96 were detected by qRT-PCR,while the expression levels of VAMP8,CD62 P and PAC-1 were detected by Western Blot(WB).Results:1.Compared with the control group,patients in the HPR and AMI groups were older and had a higher proportion of previous hypertension and diabetes(P<0.05);White blood cell count,neutrophil absolute value,serum glucose and glycosylated hemoglobin were significantly increased,while HDL cholesterol was significantly decreased(P<0.05);The HPR group was more likely to use clopidogrel,while the AMI group was more likely to use ticagrelor(P<0.05).2.Platelet function analyzer results showed that the platelet MAR and AAR in HPR and AMI groups were significantly higher than those in control group(P<0.05).3.The results of qRT-PCR showed that the expression level of platelet miR-96 in HPR and AMI groups were significantly lower than that in control group,and the decrease was more significant in AMI group(P<0.05).4.WB results showed that the expression levels of platelet VAMP8,CD62 P and PAC-1 in HPR and AMI groups were significantly higher than those in control group,especially in AMI group(P<0.05).Conclusions:1.Platelet activation and aggregation were enhanced in HPR patients after antiplatelet therapy.2.The expression of platelet miR-96 decreased and VAMP8 protein increased in HPR patients after antiplatelet therapy.Part Ⅱ.The effect of lipopolysaccharide induced inflammation on platelet function Objective:To observe the effect of lipopolysaccharide(LPS)induced inflammation on platelet activation and aggregation.Methods:1.In this part,about 250 g male SD rats and C57B6 J mice aged 6-8 weeks were used as research objects.2.After 1 week of adaptive feeding,the rats and mices were divided into control group(continuous gavage of normal saline for 5 days and intraperitoneal injection of normal saline on the 6th day),LPS group(continuous gavage of normal saline for 5days and intraperitoneal injection of LPS on the 6th day),DAPT group(continuous gavage of double antiplatele drugs for 5 days and intraperitoneal injection of normal saline on the 6th day)and DAPT+LPS group(continuous gavage of double antiplatele drugs for 5 days and intraperitoneal injection of LPS on the 6th day),6rats per group and 10 mice per group.3.The intragastric doses of the double antiplatele drugs were aspirin 10mg/Kg/day and ticagrelor 9 mg/Kg/day,and the intragastric dose of normal saline was 0.9% sodium chloride solution of the same volume for continuous intragastric administration for 5 days.On the 6th day,LPS 10 mg/Kg was injected intraperitoneally to induce acute inflammation,and the dose of normal saline was0.9% sodium chloride solution of the same volume.Cardiac blood was collected and platelets were isolated after LPS treatment for 1 h.4.The expression level of platelet miR-96 was detected by qRT-PCR,and the expression level of platelet VAMP8 and platelet activating moleculars protein were detected by WB.Platelet function analyzer was used to measure platelet aggregation rate.The time of thrombus induced vascular occlusion after carotid artery injury induced by ferric chloride was evaluated.The time of bleeding was evaluated by tail amputation test.The morphology and structure of platelet mitochondria were observed by transmission electron microscopy(TEM).Mitochondrial membrane potential and reactive oxygen species(ROS)levels were evaluated by fluorescence microscope.Results:1.The activity of rats and mices in the group of aspirin and ticagrelor intragastric administration were lower than before.After intraperitoneal injection of LPS,the rats and mices showed diarrhea of different degrees,with unformed yellow loose stools,and their mental state were worse than before injection,but there was no diarrhea and no change in mental state after intraperitoneal injection of normal saline.2.The results of qRT-PCR showed that the expression level of platelet miR-96 in LPS group and DAPT+LPS group were significantly lower than that in control group and DAPT group,and the expression level of platelet miR-96 in LPS group was lower than that in DAPT+LPS group(P<0.05).3.The results of WB showed that the expression levels of platelet VAMP8,CD62 P and PAC-1 in LPS group and DAPT+LPS group were significantly higher than those in control group and DAPT group(P<0.05),but there was no significant difference in the expression levels of VAMP8,CD62 P and PAC1 in control group and DAPT group,LPS group and DAPT+LPS group(P>0.05).4.Platelet function analyzer results showed that platelet MAR in LPS and DAPT+LPS groups were significantly higher than that in control group and DAPT group,and the highest platelet MAR in LPS group(P<0.05).Compared with the control group,AAR of platelets was significantly increased in LPS group(P<0.05),the AAR of platelets in DAPT+LPS group was significantly higher than that in DAPT group(P<0.05),but there was no significant difference between the DAPT+LPS group and the control group(P>0.05).5.Compared with the control group,the time of thrombus induced vascular occlusion after carotid artery injury and tail bleeding were significantly shortened in LPS group,and significantly prolonged in DAPT group(P<0.05).The time of thrombus induced vascular occlusion after carotid artery injury and tail bleeding in DAPT+LPS group was longer than that in LPS group and shorter than that in DAPT group(P<0.05).6.The results of TEM showed that the platelet mitochondria of rats in the control group and DAPT group were oval and short rod-like,the bilayer membrane structure was intact,no rupture or fusion occurred,and the inner mitochondrial ridge structure was normal.The outer membrane of platelet mitochondria was ruptured and the inner ridge fusion disappeared in LPS group.Platelet mitochondria in DAPT+LPS group were significantly enlarged,and mitochondrial inner ridge structure was fused and enlarged.7.The results of fluorescence microscopy showed that the platelet mitochondrial membrane potential was significantly decreased and ROS level was significantly increased in LPS and DAPT+LPS groups(P<0.05).The mitochondrial membrane potential of platelet in DAPT+LPS group was higher than that in LPS group,but lower than that in DAPT group.ROS levels were lower than LPS group and higher than DAPT group(P<0.05).Conclusions:1.LPS-induced inflammation can enhance platelet activation and aggregation,thus promoting thrombosis,in which miR-96 and VAMP8 may be involved in LPS-induced platelet activation.2.LPS-induced inflammation can damage the structure and function of platelet mitochondria.Part Ⅲ: The role and mechanism of miR-96/VAMP8 in regulating platelet activation induced by lipopolysaccharide Objective:To investigate the role and mechanism of miR-96/VAMP8 pathway in lipopolysaccharide(LPS)induced platelet activation.Methods:1.Human Megakaryoblastic leukemia cells(Meg-01 cells)were used as the research object.They were divided into control group,LPS group,DAPT group and DAPT+LPS group.The control group was not treated with any drug,and the DAPT group and DAPT+LPS group were treated with aspirin with a final concentration of 2mmol/L and ticagrelor with a final concentration of 10 μmol/L for 1 hour.After 1hour,LPS group and DAPT+LPS group were added with a final concentration of 5μg/m L for 4 hours.2.The expression level of miR-96 was detected by qRT-PCR,and the expression level of VAMP8 and platelet activating moleculars protein was detected by WB.The VAMP8 expression level was observed by immunofluorescence.The morphology and structure of mitochondria were observed by TEM.The changes of mitochondrial membrane potential and ROS were detected by fluorescence microscopy and flow cytometry.3.The level of miR-96 was regulated by transfection of miR-96 mimic and inhibitor,and the expressions of VAMP8 and platelet activating moleculars protein were detected by WB.The VAMP8 expression level was observed by immunofluorescence.The morphology and structure of mitochondria were observed by TEM.The changes of mitochondrial membrane potential and ROS were detected by fluorescence microscopy and flow cytometry.4.VAMP8 expression was decreased by transfection of VAMP8 interference fragment,and the expression of platelet activating moleculars protein were detected by WB.The VAMP8 expression level was observed by immunofluorescence.The morphology and structure of mitochondria were observed by TEM.The changes of mitochondrial membrane potential and ROS were detected by fluorescence microscopy and flow cytometry.Results:1.The results of qRT-PCR showed that the expression level of miR-96 in LPS group and DAPT+LPS group was significantly lower than that in control group and DAPT group(P<0.05).WB results showed that the expression levels of VAMP8,CD62 P and PAC-1 protein were significantly higher than those in control group and DAPT group,and VAMP8 protein expression level in LPS group was significantly higher than that in DAPT+LPS group(P<0.05).Immunofluorescence results showed that the VAMP8 fluorescence intensity in LPS group and DAPT+LPS group were significantly stronger than that in control group and DAPT group,and the VAMP8 fluorescence intensity in LPS group was stronger than that in DAPT+LPS group(P<0.05).The results of TEM showed that the mitochondria of the control group and DAPT group were normal,with the presence of bilayer membrane structure and the inner ridge structure.The mitochondria of the LPS group were ruptured and the inner ridge fusion disappeared.The mitochondria of the DAPT+LPS group were ruptured and the inner ridge structure was obviously widened or even disappeared.Fluorescence microscopy and flow cytometry showed that mitochondrial membrane potential was significantly decreased and ROS production was significantly increased in LPS and DAPT+LPS groups(P<0.05).2.After transfecting miR-96 mimic and inhibitor into cells,qRT-PCR results showed that miR-96 expression was significantly increased in miR-96 mimic group,while significantly decreased in miR-96 inhibitor group(P<0.05).WB results showed that the expression levels of VAMP8,CD62 P and PAC-1 protein in DAPT+LPS group,DAPT+LPS+miR-96 mimic group and DAPT+LPS+miR-96 inhibitor group were significantly higher than that in control group(P<0.05).Compared with DAPT+LPS group,the expression levels of VAMP8,CD62 P and PAC-1 protein were significantly decreased in DAPT+LPS+miR-96 mimic group,while significantly increased in DAPT+LPS+miR-96 inhibitor group(P<0.05).The results of immunofluorescence showed that the trend of VAMP8 fluorescence intensity was the same as that of VAMP8 protein expression.The results of TEM showed that the membrane structure of mitochondria in DAPT+LPS group,DAPT+LPS+miR-96 mimic group and DAPT+LPS+miR-96 inhibitor group were damaged to varying degrees,and the inner ridge structure was widened or merged.The damage of mitochondria was heaviest in DAPT+LPS+miR-96 inhibitor group.Flow cytometry results showed that mitochondrial membrane potential and ROS production levels were significantly decreased in DAPT+LPS group,DAPT+LPS+miR-96 mimic group and DAPT+LPS+miR-96 inhibitor group.The change of DAPT+LPS+miR-96 inhibitor group was most obvious(P<0.05).3.After transfection of Si-VAMP8 into cells,the immunofluorescence results showed that the fluorescence intensity of VAMP8 in DAPT+LPS and DAPT+LPS+Si-VAMP8 groups was significantly higher than that in control group,while the fluorescence intensity of VAMP8 in DAPT+LPS+Si-VAMP8 group was lower than that in DAPT+LPS group(P<0.05).WB results showed that the expression levels of CD62 P and PAC-1 protein in DAPT+LPS and DAPT+LPS+Si-VAMP8 groups were significantly higher than those in control group,while the expression levels of CD62 P and PAC-1 protein in DAPT+LPS+Si-VAMP8 group were significantly lower than those in DAPT+LPS group(P<0.05).The results of TEM showed that the membrane structure was damaged in DAPT+LPS group and DAPT+LPS+Si-VAMP8 group,and the inner ridge structure was widened or fusing.The damage was most obvious in DAPT+LPS group.Flow cytometry showed that the mitochondrial membrane potential in DAPT+LPS group and DAPT+LPS+Si-VAMP8 group were significantly lower than control group,while the mitochondrial membrane potential in DAPT+LPS+Si-VAMP8 group was slightly higher than that in DAPT+LPS group(P<0.05);ROS production in DAPT+LPS group and DAPT+LPS+Si-VAMP8 group were significantly increased,while DAPT+LPS+Si-VAMP8 group was slightly decreased compared with DAPT+LPS group(P<0.05).Conclusion:LPS induced inflammation can cause damage to platelet mitochondria and promote platelet activation.miR-96/VAMP8 may be an important pathway for LPS induced platelet activation.