Effects Of Macrophages-derived Exosomes On The Apoptosis Of Pulmonary Vascular Endothelial Cells In COPD And Its Mechanism

Chronic obstructive pulmonary disease(COPD)is a chronic disease caused by airway structural remodeling and abnormal alveolar function after exposure to toxic particles or gases.Cigarette smoke exposure is the most common risk factor.Previous studies showed that the apoptosis of pulmonary microvascular endothelial cells caused by smoking is an important pathological mechanism of COPD.Exosomes are carriers of information transmission between cells,which regulate the biological functions of other cells by transmitting their contents.Macrophages are the primary resident cells in the lung,but it is unclear whether exosomes derived from macrophages mediate the apoptosis of pulmonary microvascular endothelial cells in COPD.In recent years,the role of arachidonic acid metabolic disorder in lung diseases has attracted much attention.The metabolic disorder of cyclooxygenase-2/soluble epoxide hydrolase can lead to uncontrolled macrophage inflammation,but whether it is involved in regulating the effect of Macrophage Secretion on the apoptosis of pulmonary microvascular endothelial cells and then regulating the occurrence and development of COPD has not been reported.Objective: To clarify the mechanism of macrophage-derived exosomes mediated apoptosis of pulmonary microvascular endothelial cells in COPD,we intend to investigate:(1)the effects of exosomes derived from bronchoalveolar lavage fluid(BALF)on apoptosis of pulmonary microvascular endothelial cells in COPD mice;(2)the effects of macrophage-derived exosomes stimulated by cigarette smoke extract(CSE)on apoptosis of pulmonary microvascular endothelial cells and its mechanism;(3)the effects of the dual COX-2/s EH inhibitor PTUPB on apoptosis of pulmonary microvascular endothelial cells induced by macrophage-derived exosomes stimulated by CSEMethods:1.The effects of exosomes derived from BALF on apoptosis of pulmonary microvascular endothelial cells in COPD miceAdult male C57BL/6J mice were used in this study.COPD was established by intratracheal injection of LPS and cigarette smoke exposure.HE staining was used to observe the pathological changes in lung tissue of COPD mice,and Western Blot was used to detect the expression of apoptosis and inflammatory proteins in lung tissue.BALF of normal mice or COPD mice were further collected.Exosomes were extracted from BALF by exosome separation kit.The morphology of exosomes was observed by transmission electron microscope,the diameter of exosomes was detected by NTA particle size analysis,and the surface marker protein of exosomes was detected by Western Blot.The exosomes are administered intratracheally into mouse airways.After the model intervention,HE staining was used to observe the pathological changes in mouse lung tissue;q PCR and Western Blot were used to detect the expression of apoptosis and inflammatory genes or proteins in mouse lung tissue.The apoptosis of pulmonary microvascular endothelial cells was detected by immunofluorescence CD31 and TUNEL.Mouse pulmonary microvascular endothelial cells were isolated by immunomagnetic bead sorting technology.Mouse exosomes derived from BALF were used to intervene pulmonary microvascular endothelial cells.The expression of apoptosisrelated genes was detected by q PCR.2.The effects of macrophage-derived exosomes stimulated by CSE on apoptosis of pulmonary microvascular endothelial cells and its mechanismMouse primary macrophages were stimulated by CSE,and macrophage supernatant was collected.The pulmonary microvascular endothelial cells were intervened with macrophage supernatant.The apoptosis of pulmonary microvascular endothelial cells was observed by Hoechst 33342/PI staining.After further pretreatment with exosome inhibitor GW4869,the apoptosis of pulmonary microvascular endothelial cells was detected by q PCR and Western Blot.The exosomes were isolated by differential centrifuge and ultracentrifuge method.Exosomes were used to intervene pulmonary microvascular endothelial cells.The apoptosis of pulmonary microvascular endothelial cells was detected by Hoechst33342/PI staining,q PCR and flow cytometry.The macrophage-derived exosomes of the control group and CSE treatment group were sequenced for lnc RNA to screen the differentially expressed lnc RNA.The target mi RNA differentially expressing lnc RNA was predicted by bioinformatics software,and the expression amount of mi RNA was further verified by q PCR.mi RNA was silenced or overexpressed in pulmonary microvascular endothelial cells.The apoptosis of pulmonary microvascular endothelial cells was detected by Western Blot and flow cytometry.3.The effects of inhibiting COX-2/s EH on apoptosis of pulmonary microvascular endothelial cells induced by macrophage-derived exosomes stimulated by CSEThe expressions of COX-2 and s EH in COPD patients,COPD mice and CSE stimulated macrophages were detected by immunofluorescence,immunohistochemistry or Western Blot.In vitro,macrophages were pretreated with PTUPB and stimulated with CSE for 24 h.The expressions of COX-2 and s EH in macrophages were detected by Western Blot;The activity of LDH in macrophage supernatant was determined;TNF secreted by macrophages into cell supernatant was detected by ELISA;The content of ROS was detected by flow cytometry;q PCR and Western Blot were used to detect the expression of inflammatory genes and proteins in macrophages.The effects of PTUPB on the apoptosis rate of pulmonary microvascular endothelial cells induced by CSE stimulated macrophagederived exosomes were further detected by flow cytometry.Results:1.The effects of exosomes derived from BALF on apoptosis of pulmonary microvascular endothelial cells in COPD miceThe lung tissue of COPD mice showed obvious emphysema destruction and inflammatory cell infiltration.The mean lining interval(MLI)and alveolar destruction index(DI)increased significantly,and the apoptosis and inflammatory proteins in the lung tissue increased,suggesting that the model was successfully replicated.BALF exosomes from COPD mice were dropped into the airway of normal mice.The alveolar wall of mice was damaged,and the alveolar cavity was expanded and fused.The infiltration of inflammatory cells,the increase of MLI and Di,the expression of apoptosis-related genes and proteins were significantly increased,and the apoptotic pulmonary microvascular endothelial cells in the lungs of mice were significantly increased by immunofluorescence CD31 and TUNEL double-labeling method.At the same time,the expression of inflammation related genes and proteins was also increased.Further,BALF exosomes from COPD mice were dropped into the airway of COPD mice.It was found that BALF exosomes could aggravate the degree of alveolar destruction,promote the expression of inflammatory and apoptotic proteins in lung tissue,and increase the apoptosis of pulmonary microvascular endothelial cells.In vitro experiments also confirmed that BALF exosomes of COPD mice can directly promote the apoptosis of pulmonary microvascular endothelial cells.2.The effects of macrophage-derived exosomes stimulated by CSE on apoptosis of pulmonary microvascular endothelial cells and its mechanismThe macrophage supernatant stimulated by CSE can promote the apoptosis of pulmonary microvascular endothelial cells.After inhibiting the secretion of macrophages by exosome inhibitor GW4869,the apoptosis of pulmonary microvascular endothelial cells induced by macrophage supernatant stimulated by CSE was significantly reduced.Further extraction of exosomes secreted by macrophages stimulated by CSE and direct intervention of pulmonary microvascular endothelial cells can promote the apoptosis of pulmonary microvascular endothelial cells.The exosomes derived from macrophages stimulated by CSE and the exosomes derived from macrophages without intervention were sequenced by lnc RNA,18 significantly up-regulated lnc RNAs and 39 significantly down regulated lnc RNAs were screened.The top 20 mi RNAs with the highest binding potential were predicted by differentially expressed lnc RNAs.Among them,mi R-15a-3p,mi R-149-3p,mi R-212-3p and mi R-125a-3p,which are closely related to apoptosis,were selected for q PCR verification.It was found that only mi R-149-3p had differential expression and was statistically significant.Overexpression of mi R-149-3p in pulmonary microvascular endothelial cells can promote the apoptosis of pulmonary microvascular endothelial cells.After inhibiting mi R-149-3p in pulmonary microvascular endothelial cells and intervening with CSE,macrophage exosomes acted on pulmonary microvascular endothelial cells,and it was found that the effect of promoting apoptosis was weakened.3.The effects of inhibiting COX-2/s EH on apoptosis of pulmonary microvascular endothelial cells induced by macrophage-derived exosomes stimulated by CSEThe expression of COX-2 and s EH increased significantly in COPD patients,COPD mice and CSE stimulated macrophages.The increased expression of COX-2 and s EH in macrophages induced by CSE can be inhibited by PTUPB in vitro.At the same time,PTUPB can significantly reduce the damage of CSE to macrophages and inhibit the production of ROS,the activation of the NLRP3 inflammasome and the secretion of inflammatory factors.It was further found that after the intervention with PTUPB,the apoptosis of pulmonary microvascular endothelial cells caused by macrophage-derived exosomes stimulated by CSE was alleviated.In addition,PTUPB can reduce the apoptosis caused by mi R-149-3p mimic.Conclusion:1.BALF exosomes derived from COPD mice can promote lung inflammation and apoptosis of pulmonary microvascular endothelial cells,and then induce and aggravate COPD in mice;2.CSE-stimulated macrophage regulate apoptosis of pulmonary microvascular endothelial cells through mir-149-3p in exosomes;3.Inhibition of COX-2 / s EH can improve the lung function of COPD mice,alleviate the macrophage inflammation and oxidative stress induced by CSE,and reduce the apoptosis of pulmonary microvascular endothelial cells induced by macrophage exosomes stimulated by CSE.