| Background and purpose:About 80% of the thrombus in acute pulmonary embolism（APE）came from detachment of deep venous thrombosis（DVT）in the lower extremities,a small part came from the right ventricular system,and a very small part was the formation of pulmonary artery thrombosis in situ.Vascular endothelial injury,blood flow stasis and hypercoagulability are the basic conditions for thrombosis.Pathophysiological changes and related clinical signs caused by thrombus of any origin are resulted from partial or total disruption of blood flow due to partial or total occlusion of the pulmonary artery.Vascular occlusion not only leads to the body’s ischemia and hypoxia,neurohumoral activation and the release of circulating endocrine factors,but also leads to the injury and dysfunction of pulmonary microvascular endothelial cells（PMVECs）.Mitochondria plays an important role in maintaining the stability of endothelial cells and the degree of mitochondrial injury is closely related to cell injury and apoptosis.Over the years,anticoagulant therapy has always been the first choice for pulmonary circulation revascularization and achieved a good effect.However,few studies have been conducted on what happened to the mitochondria of PMVECs after occurrence of APE and how anticoagulants repair mitochondrial function other than revascularization is not known very well.Since APE has become a disease with high morbidity and mortality in cardiovascular system,new oral anticoauglants（NOACs）have been applied in clinic continuously in recent years.As a single-target inhibitor in the coagulation pathway,dabigatran binds specifically to thrombin and has many advantages including quick absorption and stable blood drug concentration of its oral dosage form,little impact with food and drugs,no need to monitor routine blood clotting and low risk of bleeding,good inhibition of thrombosis and assisting fibrinolytic system to dissolve thrombus.Since dabigatran has a good effect on restoring pulmonary blood flow,many domestic and foreign guidelines have listed it’s oral dosage form-darbigatron etexilate as the first-line recommendation for treating APE.At the same time,some scholars have begun to pay attention to whether dabigatran can repair occluded PMVECs and mitochondrial injury.And relevant research results have not been reported yet.By establishing a mouse model of oxygen and glucose deprivation（OGD）in PMVECs,this research was to observe the injury of PMVECs and mitochondria in ischemia/low oxygen at the cellular level and the effect of different concentrations of dabigatran on PMVECs and mitochondrial injury to investigate the protective effect on pulmonary vessels other than anticoagulation.Methods:Pulmonary microvascular endothelial cells（PMVECs）were isolated by enzyme digestion method and cultured.The morphology of PMVECs was observed by inverted phase contrast microscope,and then PMVECs were identified by flow cytometry and immunofluorescence.Subsequently,PMVECs were used to construct the oxygen and glucose deprivation（OGD）model and the following studies were conducted:（1）Dabigatran effects on proliferation,cycle and apoptosis of PMVECsAfter OGD models were successfully constructed,PMVECs were cultured with 10 nM,100nM,300 nM dabigatran for 6h,24 h and 72 h,respectively and control groups were set up.The proliferation activity of PMVECs was evaluated by CCK-8 method,the apoptosis morphology of PMVECs was evaluated by immunofluorescence staining and the cell cycle and the apoptosis rate of PMVECs was evaluated by flow cytometry.（2）Dabigatran effects on inflammatory cytokines released by PMVECs and mitochondrial function① Elisa and RT-PCR were used to detect the changes in the concentration and mRNA expression levels of inflammatory cytokines IL-1,IL-6,IL-8,PAF,TGF-β and TXA2in control group,OGD group and dabigatran group after treatment for 24 h.② The effect of dabigatran on mitochondrial reactive oxygen species（mtROS）labeled by DCFH-DA fluorescent probe and the change of JC-1 labeled mitochondrial membrane potential（MMP）were detected by flow cytometry.ATP content kit,lactic acid test kit and glucose uptake test kit were used to detect the changes of ATP synthesis,lactic acid level and glucose uptake level of dabigatran on PMVECs mitochondria.③ Mito-tracker Green staining was used to observe the mitochondrial morphological changes of PMVECs in three groups.Transmission electron microscope was used to observe the ultrastructure changes of PMVECs in three groups.The mitochondrial kinetic changes of PMVECs in three groups were evaluated by western blot.④ Western blot was applied to detect the expression of mitochondrial apoptosis-related proteins Bax,Bcl-2,Cleaved Caspase 3,Cleaved Caspase 9 and Cyt-c protein in three groups.（3）Dabigatran effects on PI3K/AKT/mTOR signaling pathway and its role in mitochondrial repair of PMVECsWestern blot was used to detect the expression of key proteins of p-PI3 K,p-AKT,p-mTOR in the PI3K/AKT/mTOR signaling pathway in three groups.Results:（1）Dabigatran effects on proliferation,cycle and apoptosis of PMVECs①Dabigatran could significantly increase the proliferation rate of PMVECs induced by OGDCompared with the control group,the proliferation rate of PMVECs decreased significantly in the OGD group and three different concentrations of dabigatran groups at 6h,24 h and 72h（P<0.05）.Compared with the OGD group,the proliferation rate of PMVECs increased significantly in three different concentrations of dabigatran groups at 6h,24 h and 72h（P<0.05）.And 24 h cell was selected to conduct follow-up experiments.②Dabigatran accelerates the DNA synthesis of PMVECs by promoting the transformation of PMVECs from G0/G1 phase to S and G2/M phaseCompared with the control group,the proportion of G0/G1 phase increased significantly（P<0.05）,the proportion of S phase decreased significantly（P<0.05）and the proportion of G2/M phase showed a decreasing trend in the OGD group and dabigatran groups.Compared with the OGD group,the proportion of G0/G1 phase decreased significantly（P<0.05）,the proportion of S phase increased significantly（P<0.05）and the proportion of G2/M phase showed an increasing trend and was dose-dependent in dabigatran groups.③ Dabigatran could inhibit OGD-induced apoptosis of PMVECsThe cells in the control group were lightly and uniformly stained,while the cells in the OGD group were relatively dense in color,with obvious nuclear chromatin condensation and irregular morphology.With the increase of dabigatran dose,the cell color changed from dark to light,the degree of chromatin agglutination decreased gradually,and the nucleus morphology tended to be regular.Compared with the control group,the apoptosis rate of the OGD group and dabigatran groups increased significantly（P<0.05）.The apoptosis rate of dabigatran groups was significantly lower than that of OGD group（P<0.05）,and the apoptosis rate reduced more significantly with the increase of dabigatran concentration（P<0.05）.（2）Dabigatran effects on inflammatory cytokines released by PMVECs and mitochondrial function① Dabigatran could inhibit the levels of inflammatory cytokines released by PMVECs induced by OGDThe levels of IL-1,IL-6,IL-8,PAF,TXA2 released by PMVECs in the OGD group and dabigatran groups were significantly higher than those in the control group（P<0.05）.The levels of above inflammatory cytokines released by PMVECs in dabigatran groups were lower than those in the OGD group and were concentration-dependent,but there was no statistical difference in IL-1 mRNA expression levels between 300 nM and 100 nM dabigatran group.mRNA expression levels of above inflammatory cytokines released by PMVECs in the OGD group and dabigatran groups were higher compared with the control group.However,there was no significant difference in IL-8 when 100 nM dabigatran group and above inflammatory cytokines when 300 nM dabigatran group compared with the control group.mRNA expression levels of above inflammatory cytokines released by PMVECs in dabigatran groups were lower than that in the OGD group and were concentration-dependent.However,there was no statistical difference in IL-8 between 10 nM dabigatran and the OGD group.And there was no significant difference in IL-8 and TXA2between 300 nM dabigatran and 100 nM dabigatran group.② By inhibiting the mtROS production of PMVECs induced by OGD,dabigatran could improve the MMP level of PMVECs,promote the recovery of mitochondrial ATP synthesis function,decrease the level of lactic acid and increase the glucose uptake concentration of PMVECsCompared with the control group,the mean fluorescence intensity of DCF in the OGD group and dabigatran groups were significantly enhanced（P<0.05）.The mean fluorescence intensity of DCF in dabigatran groups were significantly lower than that in the OGD group（P<0.05）.In the control group,JC-1 aggregates in PMVECs mitochondrial matrix in the form of polymer,presenting red fluorescence which is the normal MMP expression.The red fluorescence PMVECs proportion in the OGD group and dabigatran groups decreased significantly（<0.05）,that means MMP deceased significantly.Red fluorescence was the least in PMVECs and green fluorescence was the most in the OGD group,which means the lowest PMVECs MMP.Compared with the OGD group,the proportion of PMVECs with red fluorescence in dabigatran groups increased significantly（<0.05）and showed a concentration-independence,that means MMP increased gradually.Compared with the control groups,the concentration of mitochondrial ATP synthesis in PMVECs in the OGD group and dabigatran groups significantly decreased（all P<0.05）,the lactic acid production level significantly increased,and glucose uptake level significantly decreased（all P<0.05）.However,compared with the OGD group,the concentration of mitochondrial ATP synthesis in PMVECs in dabigatran groups significantly increased（all P<0.05）,the lactic acid production level significantly decreased,and glucose uptake level significantly increased（all P<0.05）,in a concentration dependent manner（all P<0.05）.③ Dabigatran could significantly improve the mophology and ultrastructure of PMVECs mitochondria,promote the mormalization of mitochondrial mophology and ultrastructure,inhibit OGD-induced mitochondrial fission and promotes mitochondrial fusionUnder an inverted fluorescence microscope,the PMVECs nuclei were dyed blue by DAPI,the mitochondria were dyed green by mito-tracker,and the blue nucleus were surrounded by green mitochondria in the Merge staining.The nuclear volume was the largest and the number of mitochondria was the most in the control group.The nuclear volume was the smallest,the number of mitochondria was the least and the morphology was fuzzy and irregular.With the increase of dabigatran dose,the nuclear volume increased,the number of mitochondria increased and mitochondrial morphology tended to be clear and regular.Under transmission electron micorscopy,the PMVECs mitochondria in the control group were elliptic or rod-shaped bilayer structures with neat inner cristae and no expansion of the inner cristae.In the OGD group,PMVECs mitochondria were swollen and deformed into globar structures,and the inner cristae were reduced and some of them were vacuolized.With the increase of dabigatran dose,dabigatran groups showed a small degree of mitochondrial swelling and deformation of PMVECs,increased elliptic or rod-like structures,decreased globar structures,increased mitochondrial cristae and obvious decrease in vacuolization.Compared with the control group,the expression levels of p-DRP1（s637）and OPA1 in the OGD group and dabigatran groups were significantly decreased（P<0.05）,while the expression level of DRP1 and p-DRP1（s616）were significantly increased（<0.05）.Compared with the OGD group,the expression levels of p-DRP1（s637）and OPA1 in dabigatran groups were significantly increased（P<0.05）,while the expression level of DRP1 and p-DRP1（s616）were significantly decreased（<0.05）with concentration dependence（P <0.05）.④ Dabigatran effects on expression of PMVECs mitochondrial apoptosis-related proteinsCompared with the control group,the expression levels of pro-apoptotic protein Bax,Cleaved Caspase 3,Cleaved Caspase 9 and Cyt-c proteins in the OGD and dabigatran groups were increased significantly（P<0.05）and expression level of anti-apoptotic protein Bcl-2 was decreased significantly（P <0.05）.Compared with the OGD group,the expression levels of pro-apoptotic protein Bax,Cleaved Caspase 3,Cleaved Caspase 9 and in dabigatran groups were decreased significantly（P <0.05）while the expression of anti-apoptotic protein Bcl-2 was increased significantly（P <0.05）with concentration dependence（P <0.05）.（3）Dabigatran effects on PI3K/AKT/mTOR signaling pathway and its role in mitochondrial repair of PMVECs① Dabigatran activates the PI3K/AKT/mTOR signaling pathway to promote the repair of PMVECs mitochondrial injuryCompared with the control group,the expression levels of p-PI3 K,p-AKT,p-mTOR protein in the OGD group and dabigatran groups were significantly decreased（P<0.05）.Compared with the OGD group,the expression levels of p-PI3 K,p-AKT,p-mTOR protein in dabigatran groups were significantly increased（P<0.05）and showed a concentration dependence（P<0.05）.Conclusions:① Dabigatran could play a protective role on PMVECs by enhancing cell proliferatioin activity,promoting cell DNA and inhibiting cell apoptosis in OGD models of APE.② Dabigatran could promote the recovery of mitochondrial function,morphology and structure injury of PMVECs and reduce mitochondrial apoptosis of PMVECs by inhibiting the mtROS pathway mediated by inflammatory response of PMVECs in OGD models of APE.③ Dabigatran could further inhibit the production of mtROS by activating the PI3K/ AKT/mTOR signaling pathway in OGD models of APE,thus promoting the repair of mitochondrial injury of PMVECs.④ Dabigatran could also protect pulmonary small vascular endothelial cells in addition to good anticoagulant effect through inhibiting blood coagulation factor Ⅱ in the treatment of APE. |
