Mechanism Of Endothelial Microparticles Transporting MicroRNA-126 To Regulate HMGB1 Expression In Airway Inflammation Of Chronic Obstructive Pulmonary Disease

Objective:1.To clarify the intervention effect of endothelial microparticles（EMPs）derived from normal primary pulmonary microvascular endothelial cells on airway inflammation induced by cigarette smoke extract in human bronchial epithelial cells（HBECs）and chronic obstructive pulmonary disease（COPD）mice;2.To explore whether micro RNA-126（miR-126）transferred by EMPs can regulate the expression of high mobility group box 1（HMGB1）in airway,and then affect the downstream nuclear factor of kappa B（NF-κB）p65inflammatory pathway activation.Methods:1.Isolation and identification of EMPs:EMPs were collected by gradient centrifugation;EMPs were identified by transmission electron microscopy,flow cytometry and western blotting.2.Intervention effect of EMPs on inflammatory response of HBECs induced by CSE:The colocalization of EMPs and HBECs was observed by fluorescence microscope;The effect of EMPs on releasing protein levels of TNF-αand IL-1βin cell supernatant of HBECs induced by CSE was detected by enzyme linked immunosorbent assay（ELISA）;The effect of EMPs on m RNA changes of TNF-α,IL-1βand IL-6 in HBECs induced by CSE was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction（q RT-PCR）;The effect of EMPs on protein level changes of p-p65 to NF-κB p65 in HBECs induced by CSE was detected by western blotting.3.Intervention effect of EMPs on airway inflammation of COPD mice:The COPD mouse model was established by smoking for 28 days and intraperitoneal injection of CSE,and EMPs were treated to mice by intratracheal instillation;Hematoxylin-eosin（HE）staining was used to detect the effects of EMPs on alveolar mean linear interval（MLI）and destructive index（DI）in COPD mice,and the airway inflammation was scored semi quantitatively;Wright Giemsa staining was used to evaluate the effect of EMPs on the classification and count of cells in bronchoalveolar lavage fluid（BALF）of COPD mice;The effect of EMPs on releasing protein levels of TNF-αand IL-1βin COPD mouse BALF was detected by ELISA;The effect of EMPs on m RNA changes of TNF-α,IL-1βand IL-6 in COPD mouse lung tissue was detected by q RT-PCR;The effect of EMPs on protein level changes of p-p65 to NF-κB p65,HMGB1 in COPD mouse lung tissue was detected by western blotting;The effect of EMPs on the expression of HMGB1 protein in the lung tissue of COPD mice was detected by immunohistochemistry.4.Intervention effect of EMPs on inflammatory response of HBECs induced by CSE and LPS:The effect of EMPs on releasing protein levels of TNF-αand IL-1βin cell supernatant of HBECs induced by CSE and LPS was detected by ELISA;The effect of EMPs on m RNA changes of TNF-α,IL-1βand IL-6 in HBECs induced by CSE and LPS was detected by q RT-PCR;The effect of EMPs on protein level changes of p-p65 to NF-κB p65 in HBECs induced by CSE and LPS was detected by western blotting.5.Intervention effect of EMPs on airway inflammation of AECOPD mice:On the basis of COPD mouse model,the acute exacerbation mouse model of COPD was established by intranasal drip of LPS,and EMPs were treated to mice by intratracheal instillation;HE staining was used to detect the effects of EMPs on alveolar MLI and DI in AECOPD mice,and the airway inflammation was scored semi quantitatively;Wright Giemsa staining was used to evaluate the effect of EMPs on the classification and count of cells in BALF of AECOPD mice;The effect of EMPs on releasing protein levels of TNF-αand IL-1βin AECOPD mouse BALF was detected by ELISA;The effect of EMPs on m RNA changes of TNF-α,IL-1βand IL-6 in AECOPD mouse lung tissue was detected by q RT-PCR;The effect of EMPs on protein level changes of p-p65 to NF-κB p65,HMGB1,NLRP3,Cleaved caspase-1 in AECOPD mouse lung tissue was detected by western blotting;The effect of EMPs on the expression of HMGB1 protein in the lung tissue of AECOPD mice was detected by immunohistochemistry.6.Intervention effect of EMPs transferring miR-126 on inflammatory response of HBECs induced by CSE:The colocalization of EMPs and miR-126 was observed by fluorescence microscope;The miR-126 levels in HBECs after intervention of EMPs transferring miR-126mimic/inhibitor were detected by q RT-PCR;The effect of EMPs transferring miR-126 mimic/inhibitor on m RNA changes of TNF-α,IL-1βand IL-6 in HBECs induced by CSE was detected by q RT-PCR;The effect of EMPs transferring miR-126 mimic/inhibitor on protein level changes of p-p65 to NF-κB p65,HMGB1 in HBECs induced by CSE was detected by western blotting;The effect of EMPs transferring miR-126mimic/inhibitor combined with si-HMGB1 on protein level changes of p-p65 to NF-κB p65,HMGB1 in HBECs induced by CSE was detected by western blotting.Results:1.Isolation and identification of EMPs:transmission electron microscopy showed that EMPs were round or oval;Flow cytometry analysis shows that the diameter of EMPs is distributed in 0.1-1μm;Western blotting showed that EMPs expressed endothelial marker protein（PECAM/CD31,E-Selectin/CD62E）and apoptosis specific protein（Annexin-V）.2.Intervention effect of EMPs on inflammatory response of HBECs induced by CSE:Fluorescence microscopy showed that CFSE green fluorescence carried by EMPs was clearly displayed in or around the nucleus of HBECs after 24 hours of co-incubation;Elevated TNF-αand IL-1βprotein levels in cell supernatant of HBECs induced by CSE were significantly reversed by EMPs;EMPs treatment markedly reversed increased pro-inflammatory gene（TNF-α,IL-6,and IL-1β）m RNA levels induced by CSE;Treatment of EMPs could significantly diminish the increased protein level of p-p65 to p65 in HBECs induced by CSE.3.Intervention effect of EMPs on airway inflammation of COPD mice:HE staining showed that EMPs significantly decreased alveolar MLI,DI and airway inflammation score in COPD mice;After intervention of EMPs,the total number of cells,neutrophils and macrophages in BALF of COPD mice decreased significantly;Increased TNF-αand IL-1βprotein levels in COPD mouse BALF were significantly reversed by EMPs;EMPs treatment markedly decreased pro-inflammatory gene（TNF-α,IL-6,and IL-1β）m RNA levels in COPD mouse lung tissue;Treatment of EMPs could significantly decreased protein levels of p-p65 to p65,HMGB1 in COPD mouse lung tissue.4.Intervention effect of EMPs on inflammatory response of HBECs induced by CSE and LPS:Elevated TNF-αand IL-1βprotein levels in cell supernatant of HBECs induced by CSE and LPS were significantly reversed by EMPs treatment;EMPs treatment markedly reversed increased pro-inflammatory gene（TNF-α,IL-6,and IL-1β）m RNA levels induced by CSE and LPS;Intervention of EMPs could significantly diminish the increased protein level of p-p65 to p65 in HBECs induced by CSE and LPS.5.Intervention effect of EMPs on airway inflammation of AECOPD mice:HE staining showed that EMPs significantly decreased airway inflammation score in AECOPD mice,while EMPs had no effects on alveolar MLI and DI;After intervention of EMPs,the total number of cells,neutrophils and macrophages in BALF of AECOPD mice significantly decreased;Increased TNF-αand IL-1βprotein levels in AECOPD mouse BALF were significantly reversed by EMPs;EMPs treatment markedly decreased pro-inflammatory gene（TNF-α,IL-6,and IL-1β）m RNA levels in lung tissue of AECOPD mouse;Treatment of EMPs could significantly decreased protein levels of p-p65 to p65,HMGB1,NLRP3 and Cleaved caspase-1 in COPD mouse lung tissue.6.Intervention effect of EMPs transferring miR-126 on inflammatory response of HBECs induced by CSE:Fluorescence microscopy showed that after Ca Cl₂transfection,the CFSE labeled EMPs showed Cy3 red fluorescence carried by miR-126 mimic;TNF-αand IL-1βprotein levels in cell supernatant of HBECs were significantly decreased after the intervention of EMPs transferring miR-126 mimic,while EMPs transferring miR-126 inhibitor could significantly increase TNF-αand IL-1βprotein levels in cell supernatant of HBECs;EMPs transferring miR-126 mimic significantly decreased TNF-α,IL-6,and IL-1βm RNA levels in HBECs;Protein levels of p-p65 to p65,HMGB1 in HBECs were significantly decreased after the treatment of EMPs transferring miR-126mimic,while EMPs transferring miR-126 inhibitor significantly increased protein levels of p-p65 to p65,HMGB1 in HBECs;The results of western blotting showed that the intervention of miR-126 mimic loaded EMPs combined with si-HMGB1 could further inhibit the expression level of HMGB1 protein and the relative protein expression level of p-p65 to p65compared with miR-126 mimic loaded EMPs group and si-HMGB1group;Compared with si-HMGB1 group,EMPs loaded with miR-126inhibitor combined with si-HMGB1 could partially reverse the decrease of HMGB1 protein expression level and the relative protein expression level of p-p65 and p65.Conclusions:EMPs derived from normal primary pulmonary microvascular endothelial cells can regulate HMGB1 expression by transporting miR-126,and then inhibit airway inflammation in COPD model.