| Objective: Tissue injury after acute myocardial infarction(AMI)triggers a strong inflammatory response and necrosis,which involves extensive infiltration of immune cells such as monocytes and soluble mediators.Our previous study found that microparticles(MPs)are generated during AMI,and the levels of micro RNA(miRNA)contained in these MPs are significantly different from those in circulating MPs under normal physiological conditions.However,the mechanism of inflammation and tissue damage induced by these MPs remains unclear.The main research contents include:(1)To clarify the correlation between EMPs and miR-126,mitochondrial components,adhesion molecules,and myocardial injury at the clinical level.(2)To investigate the mechanism of miR-126 in mediating MK2 and ROS dependent cell injury under inflammatory stimulation at the cellular level.(3)To explore the mechanism of MK2 signaling pathway and miR-126 expression regulation in inducing myocardial tissue injury at the animal level.Methods: The study was conducted at the clinical,cellular,and animal levels in three parts:(1)A case-control study design was adopted to include 50 patients with AMI,50 patients with stable coronary artery disease(SCAD)and 50 healthy subjects(Control)who were admitted to the People’s Hospital of Xinjiang Uygur Autonomous Region from September 2021 to September 2022.The basic information and clinical data of patients were collected through the electronic medical record system,and peripheral blood of the Control group,SCAD group and AMI group was collected under resting state.The morphology of MPs was observed by transmission electron microscopy(TEM),the number of MPs was detected by nanoparticle tracking analysis(NTA),MPs were identified by flow cytometry(FCM)and EMPs were analyzed,miR-126 expression in EMPs was detected by PCR,ROS content in EMPs and the expression of EMPs surface adhesion molecules were detected by ELISA.Logistic multivariate regression analysis was used to analyze the influencing factors of AMI patients,and the receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of each factor for AMI.(2)Human umbilical vein endothelial cells were cultured and treated with TNF-α(10ng/ml)for 6h,12 h and 24 h,respectively.The MK2 pathway inhibition model and ROS inhibition model were constructed by si RNA transfection and targeting the mitochondrial antioxidant mito TEMPO,respectively.EMPs were obtained by centrifugation and analyzed by FCM.WB was used to detect the expression of MK2 pathway proteins in endothelial cells.PCR was used to detect the expression of miR-126 in EMPs.The expressions of ATP,m PTP,ΔΨm,ROS and adhesion molecules in EMPs were detected by ELISA.(3)Thirty male C57BL/6 mice were divided into six groups,with 5 mice in each group.The mice were divided into Sham group,AMI group,MK2 knockdown(sh MK2)group,miR-126 overexpression(miR-126 mimic)group,AMI+sh MK2knockdown(AMI+sh MK2)group and AMI+miR-126 overexpression(AMI+miR-126mimic)group.HE staining was used to observe the infiltration of inflammatory cells in the myocardium of mice,and Masson staining was used to evaluate the collagen volume fraction of mice.EMPs were analyzed by FCM,the expression levels of MK2 and miR-126 in EMPs were detected by PCR,the content of mitochondrial ROS in EMPs was detected by FCM,the expression of adhesion molecules in EMPs was detected by ELISA,and the monocyte levels in bone marrow,blood,spleen and heart were detected by FCM.WB and PCR were used to detect the expression of HMGB2,integrin α4,integrin β2 and MCP-1 in bone marrow,blood,spleen and heart.Results:(1)(1)TEM: The MPs membrane was intact,the outline was clear,and the diameter was about 100nm-400 nm.(2)NTA: Compared with the Control group,the number of MPs in the plasma of AMI group and SCAD group was significantly increased(P<0.01,P<0.001).(3)Identification of MPs by FCM: TSG101 and HSP70 proteins were positive on the surface of MPs isolated from plasma,indicating that the isolated samples were MPs.(4)FCM analysis of EMPs: Compared with the Control group and the SCAD group,the plasma level of EMPs in the AMI group was significantly increased(P<0.001).(5)PCR detection of miR-126 in EMPs: Compared with the Control group,the expression of miR-126 in plasma EMPs in AMI group was significantly down-regulated(P<0.001).(6)ELISA detection of ROS in EMPs: Compared with the Control group,the level of ROS in plasma EMPs in AMI group and SCAD group was significantly increased(P<0.001).(7)Adhesion molecules in EMPs detected by ELISA: Compared with the Control group,the expression of adhesion molecules in plasma EMPs was up-regulated in SCAD and AMI patients(P<0.05,P<0.01,P<0.001).(8)Multivariate regression analysis: miR-126,ROS and P-selectin were independently associated with the occurrence of AMI.miR-126(OR=0.026,95%CI=0.003-0.210,P=0.001)was a protective factor,ROS(OR=1.009,95%CI=1.005-1.013,P<0.001)and P-selectin(OR=1.063,95%CI=1.022-1.105,P=0.002)were risk factors.(9)ROC: The area under the curve of combined diagnosis of miR-126,ROS and P-selectin was 0.950.(2)(1)EMPs were measured by FCM: Compared with Control group,EMPs in TNF-α group were significantly increased(P<0.01,P<0.001).Compared with TNF-α group,EMPs in TNF-α+si MK2 and TNF-α+mito TEMPO groups were significantly decreased(P<0.01,P<0.001).(2)The MK2 pathway factors of endothelial cells were detected by WB: Compared with Control group,MK2 pathway protein expression in TNF-α group was increased(P<0.05,P<0.01,P<0.001).Compared with TNF-α group,the expression of MK2 pathway protein in TNF-α+si MK2 and TNF-α+mito TEMPO groups decreased(P<0.05,P<0.01,P<0.001).(3)miR-126 in EMPs was detected by PCR: Compared with Control group,the expression of miR-126 in EMPs of TNF-α group was significantly decreased(P<0.01,P<0.001).Compared with TNF-αgroup,the expression of miR-126 in EMPs in TNF-α+si MK2 and TNF-α+mito TEMPO groups was increased(P<0.05).(4)ELISA was used to detect ATP,m PTP,ΔΨm and ROS in EMPs: Compared with the Control group,the contents of ATP and m PTP in EMPs in the TNF-α group were significantly decreased(P<0.001),while the contents of ΔΨm and ROS were significantly increased(P<0.001).Compared with the TNF-α group,the TNF-α+si MK2 and TNF-α+mito TEMPO groups had significant increases in the contents of ATP and m PTP(P<0.01,P<0.001)and significant reductions in the contents of ΔΨm and ROS(P<0.01,P<0.001).(5)ELISA detection of adhesion molecules in EMPs:Compared with the Control group,the expression of adhesion molecules in EMPs was significantly increased in the TNF-α group(P<0.001).Compared with the TNF-α group,the TNF-α+si MK2 group had a significant reduction in the expression of adhesion molecules in EMPs(P<0.05,P<0.001),and the TNF-α+mito TEMPO group had a significant reduction in the expression of VCAM-1 and ICAM-1(P<0.001).The expression of E-selectin and P-selectin was increased(P<0.05).(3)(1)HE staining:Compared with AMI group,AMI+sh MK2 group and AMI+miR-126 mimic group had reduced myocardial infarction area and inflammatory cell infiltration.(2)Masson staining:Compared with AMI group,the collagen volume fraction in AMI+sh MK2 group and AMI+miR-126 mimic group was significantly reduced.(3)EMPs detected by FCM:Compared with Sham group,the level of EMPs in plasma of AMI group was significantly increased(P<0.001).Compared with AMI group,the plasma levels of EMPs in AMI+sh MK2 group and AMI+miR-126 mimic group were significantly decreased(P<0.01,P<0.001).(4)PCR detection of MK2 and miR-126 in EMPs: Compared with the Sham group,the m RNA level of MK2 in plasma EMPs of AMI group was significantly increased,and the level of miR-126 was significantly decreased(P<0.01,P<0.001).Compared with AMI group,the MK2 m RNA levels in plasma EMPs of AMI+sh MK2 group and AMI+miR-126 mimic group were significantly decreased(P<0.001).(5)ELISA detection of adhesion molecules: Compared with the Sham group,the levels of adhesion molecules in the plasma of the AMI group were significantly increased(P<0.01,P<0.001);Compared with AMI group,the levels of adhesion molecules contained in EMPs in the plasma of AMI+sh MK2 group and AMI+miR-126 mimic group were decreased(P<0.05,P<0.01,P<0.001).(6)FCM detection of monocytes: Compared with the Sham group,the levels of monocytes in the bone marrow and spleen of the AMI group were significantly decreased(P<0.001),and the levels of monocytes in the blood and heart were significantly increased(P<0.001).Compared with AMI group,the levels of monocytes in the bone marrow and spleen of AMI+sh MK2 group and AMI+miR-126 mimic group were significantly increased(P<0.001),and the levels of monocytes in the blood and heart were significantly decreased(P<0.01,P<0.001).(7)WB and PCR detection of HMGB2,integrinα4,integrin β2,MCP-1 protein and m RNA expression: Compared with Sham group,the levels of HMGB2,integrin α4,integrin β2 and MCP-1 in bone marrow,blood,spleen and heart of AMI group were significantly increased(P<0.001).Compared with AMI group,the levels of HMGB2,integrin α4,integrin β2 and MCP-1 in bone marrow,blood,spleen and heart of AMI+sh MK2 group and AMI+miR-126 mimic group were decreased(P<0.05,P<0.01,P<0.01).Conclusions:(1)At the clinical level,miR-126,ROS,P-selectin and the combined indicators of the three in EMPs have diagnostic value for AMI,and the combined indicators of the three have the highest diagnostic value,indicating that they may be potential diagnostic indicators for AMI patients.(2)At the cellular level,inhibition of MK2 by si RNA transfection or ROS production by targeting mitochondrial antioxidant mito TEMPO was found to increase miR-126 and decrease the expression of VCAM-1 and ICAM-1 in EMPs.It was confirmed that inhibition of MK2 pathway or ROS production could reduce EMPs-induced inflammation in endothelial cells.(3)At the animal level,inhibition of MK2 gene or overexpression of miR-126 can reduce the expression of adhesion molecules in EMPs in plasma of mice after AMI,reduce the migration of monocytes to the infarct site,and thus reduce myocardial inflammation and infarct size. |
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