The Study Of Improving The Permeability Of Pulmonary Vascular Endothelial Cells By Regulating Mitochondrial Autophagy Of Pulmonary Endothelial Cells In Acute Lung Injury

Objective To investigate the effects of human placental mesenchymal stem cells(hpMSCs)and human placental mesenchymal stem cell exosomes(hpMSCs-ex)on lipopolysaccharide(LPS)induced injury of pulmonary vascular endothelial monolayer and acute lung injury(ALI),Objective to study the effect and mechanism of ALI on pulmonary vascular endothelial permeability.Methods1.Isolation and identification of hpMSCs and hpMSCs-ex.(1)The morphological characteristics of hpMSCs were observed under light microscope.Hp MSCs were cultured in specific differentiation medium to observe their transformation into adipose,osteogenic and chondrogenic cells.The expression of stem cell specific antigen on the surface of hpMSCs was detected by flow cytometry.(2)The culture supernatant of hpMSCs was collected,and hpMSCs-ex were extracted with Exo Quick exosome extraction kit.The morphological characteristics of hpMSCs-ex were observed under transmission electron microscope,and the expression of surface specific antigen was detected by Western blot(WB).2.In vitro experiment:To observe the effect of hpMSCs and hpMSCs-ex on LPS induced injury of human pulmonary microvascular endothelial cells(HPMECs).(1)Establishment of cell injury model:(1)HPMECs were treated with 200 ng/ml LPS for 6 h to induce cell injury model.(2)Methods and groups:groups:(1)They were divided into normal group(N Group),LPS group,hpMSC group and hpMSC-ex group.(2)Methods:hpMSCs or hpMSCs were co cultured with LPS damaged HPMECs in transwelltm co culture system for 48 hours.(3)Detection indexes:(1)Morphological characteristics of HPMECs were observed under light microscope;(2)observe the effect of hpMSC and hpMSCs-ex on HPMECs injured by LPS:observe the transfer of DIL labeled hpMSCs-ex into HPMECs by fluorescence microscope;CCK-8 method was used to detect the proliferation rate of HPMECs in each group;(3)the mitochondrial function and autophagy of HPMECs in each group were observed;JC-1 staining was used to detect the mitochondrial membrane potential of HPMECs in each group;WB method was used to detect the expression level of apoptosis inducing factor(AIF)released by mitochondria in HPMECs;The autophagy of HPMECs in each group was observed by transmission electron microscope(TEM),and the protein of HPMECs in each group was collected and extracted.The expression level of autophagy related protein in HPMECs in each group was detected by WB method.(4)Detection of monolayer pulmonary vascular endothelial permeability:FITC dextran was used to detect the monolayer pulmonary vascular endothelial permeability;the expression of E-cadherin in HPMECs was detected.3.In vivo experiment:to detect the effect of hpMSCs and hpMSCs ex on LPS induced ALI mice.(1)Preparation of ALI mouse model:LPS(15 mg/kg body weight)was injected intraperitoneally to establish ALI mouse model.(2)Methods and groups:(1)groups:The experimental groups were divided into N group(N Group),ALI mouse model group(LPS group),hpMSC group and hpMSC-ex group.(2)Methods:hpmsc group:Ali mice were intratracheal instillation of hpmscs for 24 hours;hpmsc ex group:Ali mice were intratracheal instillation of hpmscs ex for 24 hours.(3)Detection indexes:the pathological injury scores of lung tissue was calculated,Changes in lung tissues of each group of mice under transmission electron microscope(TEM),the level of inflammatory factor TNF-αin bronchoalveolar lavage fluid(BALF)was measured,Evans blue exudation in lung tissue was detected and the expressions of E-cadherin,AIF,autophagy related protein beclin-1,LC3Ⅱ/I and PINK1/parkin in lung tissue were detected.Results:1.(1)Under the inverted microscope,hpMSC were willow shaped,whirlpool like adherent rowth.Specific culture medium could induce them to transform into osteoblasts,chondroblasts and adipocytes.The surface antigen of hpMSCs conformed to the typical MSCs antigen phenotype.Flow cytometry showed that the antigen of hpMSC highly expressed CD105,CD90 and CD73,but did not express CD34,CD45 and HLA-DR.(2)Under TEM,hpMSC-ex bilayer membrane structure was observed,which was cup-shaped,with a diameter of 30-120nm.The surface antigen CD9 and CD63 were positive.2.In vitro results:(1)HPMECs was found to be in good shape and similar in size under light microscope,and the shape was oval,circular or short shuttle.(2)The effects of hpMSCs-ex on HPMECs were as follows:(1)Under fluorescence microscope,DIL labeled hpMSCs-ex could be transferred into LPS induced HPMECs,showing bright granules;(2)CCK-8 method was used to detect the proliferation rate of HPMECs:LPS group was significantly lower than normal group(P<0.01);The proliferation rate of hpmecs in hpMSC group and hpMSC-ex group was significantly higher than that in LPS group(P<0.01).(3)The mitochondrial function and autophagy related protein expression of HPMECs in each group were as follows:(1)JC-1 fluorescence staining showed that the mitochondrial membrane potential of HPMECs in LPS group was significantly lower than that in N group(P<0.01);the mitochondrial membrane potential of HPMECs in hpMSC group and hpv ex group was significantly higher than that in LPS group(P<0.01);(2)the expression level of apoptosis inducing factor released by mitochondria in HPMECs:compared with N group,the expression level of AIF protein released by mitochondria in pulmonary vascular endothelial cells was significantly higher Compared with LPS group,the expression of AIF protein in HPMECs of hpMSC group and hpMSC-ex group was significantly decreased(P<0.01);(3)The results of TEM were as follows:No autophagosome or autophagy lysosome was found in HPMECs in N group;In LPS group,the number of mitochondria in pulmonary vascular endothelial cells decreased significantly,and some mitochondria cristae disappeared to form vacuoles,and no autophagosome or autophagy lysosome was observed;In MSC group,the mitochondria showed dense changes,but the structure was basically normal.There were double membrane structure of mitochondrial autophagosomes,and the cristae structure was not completely dissolved;In hpMSC-ex group,the structure of mitochondria in HPMECs was normal,and the autophagosome and autophagy lysosome were clearly observed.(4)Autophagy and mitochondrial autophagy related protein detection results:compared with the N group,the expression of autophagy related protein becline-1 and LC3Ⅱ/I in LPS group were increased(P<0.05);compared with LPS group,the expression of autophagy related protein becline-1 and LC3Ⅱ/I in hpMSC group and hpMSC-ex group were increased(P<0.05)The expression of LC3Ⅱ/I increased(P<0.01),suggesting that the autophagy level of hpMSC group and hpMSC-ex group was higher than that of LPS group;compared with the N group,the expression of PINK1 and parkin in LPS group was increased(P<0.01);compared with LPS group,the expression of PINK1 and parkin in hpMSC group and hpMSC-ex group were significantly increased(P<0.01),The results showed that the expression of mitochondrial autophagy in hpMSC group and hpMSC-ex group was higher than that in LPS group.(4)Single layer pulmonary vascular endothelial permeability was measured:(1)The permeability of pulmonary endothelial monolayer in LPS group was significantly lower than that in N group(P<0.01);compared with the N group,Hp MSC group and hpMSC-ex group had a significant decrease in monolayer pulmonary vascular endothelial permeability(P<0.01);(2)HPMEC cadherin expression:compared with the N group,the expression of cadherin in LPS group was significantly decreased(P<0.01);and the expression of E-cadherin in hpMSC group and hpMSC-ex group was significantly higher than that in LPS group(P<0.01).3.In vivo results:(1)The results of lung pathological injury scores showed that:compared with the N group,the lung pathological injury scores of LPS group increased significantly;compared with the LPS group,the lung pathological injury scores of hpMSC group and hpMSC-ex group decreased significantly;(2)The results of transmission electron microscopy and electron microscopy showed that the intracellular organelles of PMECs and type II cells in the normal group were normal,and the basement membrane was intact.The mitochondrial cristae of PMECs in the LPS group disappeared,the basement membrane was obviously thickened and the double-layer membrane structure was destroyed;the type II cell nucleus was changed like a concentration,the normal lamellar structure of the osmophilic corpuscle plate disappeared,a large number of corpuscles were emptied,and the alveolar surface of the cells was micro The villi disappeared,and polynuclear leukocytes exuding to the alveoli were seen.In the hpMSC group,the nucleus of PMECs was normal,the mitochondria were swollen but the mitochondrial cristae was still intact,and the basement membrane was thicker than the normal group.The integrity of the thickened part of the basement membrane was destroyed,and a clear double-layer membrane structure was visible.The structure exists,the microvilli on the alveolar surface of the cells exist,and some of them are broken and disappeared.In the hpMSC-ex group,the nucleus of PMECs was normal,the mitochondria were swollen but the mitochondrial cristae space was widened,the basement membrane was thickened compared with the normal group,and the integrity of the thickened part was destroyed,and a clear double-layer membrane structure was visible(D1-c).TypeⅡcells have normal nuclei,the lamellar structure of osmophilic bodies,and the presence of microvilli on the alveolar surface of the cells,and some of them are broken and disappeared.(3)TNF-αlevel in BALF:compared with N group,TNF-αlevel in BALF of mice with lung injury was significantly increased(P<0.01),and TNF-αlevel in BALF of hpMSC group and hpMSC-ex group was significantly decreased(P<0.01)compared with LPS group;(4)Evans blue penetration test results:compared with N group,Evans blue content in lung tissue of mice in LPS group increased significantly(~&P<0.01);compared with LPS group,Evans blue content in lung tissue of mice in hpMSC group and hpMSC-ex group decreased significantly.(*p<0.01);(5)The results of E-cadherin expression showed that compared with the N group,the expression of E-cadherin in LPS group was significantly decreased(P<0.01);compared with LPS group,the expression of E-cadherin in hpMSC group and hpMSC-ex group was significantly increased(P<0.01);(6)The expression of mitochondrial apoptosis inducing factor(AIF):compared with the N group,the expression of AIF increased in LPS group,and decreased significantly in hpMSC group and hpMSC-ex group(P<0.01).(7)Autophagy related protein expression:compared with the N group,the protein expression of beclin-1,LC3Ⅱ/I and PINK1/parkin in LPS group increased(P<0.01);compared with the LPS group,the protein expression of beclin-1,LC3Ⅱ/I and PINK1/parkin in hpMSC and hpMSC-ex groups increased significantly(P<0.01).Conclusion:1.human placental mesenchymal stem cell exosomes can promote the repair of pulmonary vascular endothelial cells and improve the permeability of pulmonary vascular endothelial cells in acute lung injury.2.Exosomes of human placental mesenchymal stem cells can promotemitochondrial autophagy of pulmonary vascular endothelial cells in acute lung injury.