| Part 1 Isolation,Identification and High-throughput Sequencing of EPCs-exo Stimulated by Extracorporeal Cardia Shock WavesObjective:To establish rat bone marrow derived endothelial progenitor cell model stimulated by extracorporeal cardiac shock waves(ECSW).The exosomes were extracted and the expression levels of related miRNA in exosomes were determined.Methods:The endothelial progenitor cells(EPCs)from rat bone marrow were isolated,cultured and identified.The EPCs were divided into two groups:Control group(CON group)and Shock wave group(SW group).SW group was intervened by 500 shots of ECSW with an energy of 0.09mJ/mm2.Simultaneously,the CON group was conducted experiments without ECSW treatment.Two groups of exosomes(CON-exo and SW-exo)were purified from culture medium and identified.The next generation sequencing was performed to screen the differentially expressed miRNAs in exosomes of the two groups.The target genes of differentially expressed miRNAs were predicted and the functional significance enrichment analysis was performed.The total miRNAs in the two groups of cells and exosomes were extracted,and the screened miRNAs were verified by qRT-PCR to detect the expression level of the target miRNA.Results:1.The EPCs were isolated from rat femoral bone marrow using density-gradient centrifugation.The adherent cells began to adhere to the wall on 3-4 days,showing oval and polygonal changes.On the 7th day,the number of cells obviously increased and showed a colony-like growth.The typical "paving stone"-like cell colonies appeared within 14 days,and the cells exhibited a long fusiform shape on the 21st day.Flow cytometry assay showed that the surface markers of EPCs such as CD31,CD34,CD 133 and VEGFR2 were all positive,while hematopoietic markers CD 14 and CD45 were negative.Functional identification showed that these cells have the ability of Dil-AcLDL uptake and FITC-UEAI binding and form the formation capillary-like structures on the surface of Matrigel.2.Exosomes were purified from the culture medium of EPCs.Transmission electron microscopy showed that the exosomes had double-layer membranes in spherical or cupshaped shape with complete morphology and uniform size.Nanoparticle tracking analysis showed that the size distribution of exosomes was 50-120nm,and the particle concentration is about 7×10^8 particles/ml.Western blot analysis showed that the exosomes expressed CD9,CD63 and CD81 as widely recognized exosome-specific markers,while did not express Calnexin protein.3.The results of next generation sequencing showed that the miRNAs expression profiles of SW-exo and CON-exo were basically overlapped,but significant analysis(Fold change>2,p<0.05)indicated that 18 miRNAs with relative high amounts differentially expressed between SW-exo and CON-exo.Compared with CON-exo,the expression of rno-miR-140-3p in SW-exo was significantly up-regulated(p<0.01).GO and KEGG enrichment analysis showed that target genes predicted of differential miRNAs may be involved in the regulation of multiple myocardial ischemiareperfusion injury-related pathways,such as PI3K/AKT,MAPK,Apoptosis,NF-κB,HIF-1α,Ras,etc.It is closely related to biological processes such as cell proliferation,growth,survival and apoptosis.The result of qRT-PCR showed that the relative expression levels of miR-140-3p in both EPCs and exosomes after ECSW stimulation were significantly higher than those in the control group(p<0.05),consistent with the results of sequencing.Conclusions:1.The rat bone marrow-derived EPCs were successfully obtained and stimulated by ECSW.2.The stimulation of ECSW has a regulatory effect on miRNA in exosomes of EPCs,and the expression level of miR-140-3p in EPCs-exo is significantly increased after ECSW.Part 2 Extracorporeal Cardiac Shock Waves Alleviate H9c2 Cardiomyocytes Hypoxia/reoxygenation Injury via EPCsexo/miR-140-3p/PTEN pathwayObjective:To establish hypoxia/reoxygenation(H/R)injury model in H9c2 cell and to explore the protective effect and mechanism of EPCs-exo induced by extracorporeal cardiac shock waves on cardiomyocytes hypoxia/reoxygenation injury.Methods:H9c2 cardiomyocytes were incubated with EPCs-exo labeled with PKH26 to verify their ability of exosome uptake.To establish H/R injury model in H9c2 cell,and then the different concentrations of EPCs-exo were used to determine the optimal concentration of intervention.The cardiomyocytes after H/R injury were incubated with CON-exo and SW-exo for 24 hours,respectively.The divided experimental groups were as follows:NC group,H/R group,H/R+PBS group,H/R+CON-exo group and H/R+SW-exo group.After treatment,CCK-8 and LDH assay were used to detect cell viability.The apoptosis rate of H9c2 cells was analyzed by flow cytometry analyses.The production of reactive oxygen species(ROS)was detected to evaluate intracellular oxidative stress level.The expression level of Bax,Bcl-2,Cleaved caspase-3,NF-κB were detected by western blot.To further verify the role of miR-140-3p in SW-exo.The H9c2 cells were divided into following groups:H/R group,H/R+miR-140-3p mimic NC group,H/R+miR140-3p mimic group,H/R+miR-140-3p inhibitor NC+SW-exo group,H/R+miR140-3p inhibitor+SW-exo group.After treatment,CCK-8 and LDH assay were used to detect cell viability.The apoptosis rate of H9c2 cells was analyzed by flow cytometry analyses.The production of reactive oxygen species(ROS)was detected to evaluate intracellular oxidative stress level.The expression level of Bax,Bcl-2,Cleaved caspase-3,NF-κB were detected by western blot.In addition,the expression of PTEN/PI3K/AKT protein in H9c2 cells after treatment were detected in all groups by western blot.The relative expression levels of miR-140-3p and PTEN mRNA in cells were detected by qRT-PCR.The dual luciferase reporter gene vectors and cell transfection were used to study the regulation of miR140-3p on the predicted target gene PTEN.Results:1.Exosome tracing experiment showed that after co-incubation of PKH26-labeled EPCs-exo with H9c2 cardiomyocytes,red fluorescent signals appeared in cardiomyocytes,which indicated that cardiomyocytes could uptake EPCs-exo.2.The exosome concentration screening showed that SW-exo and CON-exo can improve cell viability,and present a dose-dependent manner.When the concentration reached to 20μg/ml,the effects of SW-exo and CON-exo on cell viability were both significantly enhanced(p<0.01).Based on that,20μg/ml was determined as the optimal exosome concentration used for further experiments.3.Compare with NC group,The cell viability of the H/R group was significantly decreased,and the LDH activity,the rate of apoptosis and the level of intracellular ROS formation were significantly increased,while the expressions of Bax,Cleaved caspase3 and NF-κB were increased,and the expression of Bcl-2 was decreased(p<0.05).Compared with H/R+PBS group and H/R+CON-exo group,administration of SW-exo to H9c2 cells after H/R injury could significantly improve cell viability,inhibit cell apoptosis,and down-regulate oxidative stress level,with an increase of Bcl-2 protein and a decrease of Bax,Cleaved caspase-3 and NF-κB protein(p<0.05).4.Compared with the H/R+miR-140-3p mimic NC group,the H/R+miR-140-3p mimic group improved cell viability,inhibited LDH activity,cell apoptosis rate,and intracellular ROS formation,while the expressions of Bax,Cleaved caspase-3 and NFκB were decreased,and the expression of Bcl-2 was increased(p<0.05).The results of H/R+miR-140-3p inhibitor NC+SW-exo group and H/R+miR-140-3p mimic group were similar with no statistical difference(p>0.05),but miR-140-3p inhibitor could reverse the above protective effects of SW-exo resulting in decrease of cell viability and increase of LDH activity,cell apoptosis rate and cellular ROS formation.At the same time,the expressions of Bax,Cleaved caspase-3 and NF-κB were increased,and the expression of Bcl-2 was decreased(p<0.05).5.Western blot and qRT-PCR results showed that compared with the NC group,the expression of miR-140-3p in the H/R group decreased,following an increase of PTEN expression and a decrease of phosphorylation levels of p-PI3K/PI3K and p-AKT/AKT(p<0.01).Compared with H/R+PBS group and H/R+CON-exo group,the expression of miR-140-3p in H/R+SW-exo group increased,following a decrease of PTEN expression and an increase of phosphorylation levels of p-PI3K/PI3K and p-AKT/AKT(p<0.05).After H/R,transfection of miR-140-3p mimic in H9c2 cells could significantly up-regulate the level of miR-140-3p,inhibit the expression of PTEN,and promote the phosphorylation of p-PI3K/PI3K and p-AKT/AKT(p<0.01).miR-140-3p inhibitor could significantly inhibit the up-regulation effect of SW-exo on miR-140-3p,resulting in a decrease of miR-140-3p expression,an increase of PTEN expression,and a decrease of phosphorylation of p-PI3K/PI3K,p-AKT/AKT level(p<0.05).6.Dual luciferase reporter assay showed that luciferase activity significantly declined when PTEN 3’UTR-WT combined with miR-140-3p mimic(p<0.01).When PTEN 3’UTR-MUT combined with miR-140-3p mimic,the fluorescence intensity of the reporter gene did not change significantly(p>0.05).Conclusions:1.EPCs-exo could be taken up and utilized by H9c2 cardiomyocytes.SW-exo could significantly improve cell viability,inhibit cell apoptosis,and down-regulate inflammatory factors and oxidative stress levels triggered by hypoxia/reoxygenation injury in H9c2 cells.2.The expression level of miR-140-3p in H9c2 cells was down-regulated after H/R injury.SW-exo may deliver miR-140-3p to cardiomyocytes,playing a therapeutic role in cardiomyocytes H/R injury by inhibiting the expression of the target gene PTEN and activating PI3K/AKT pathway.Part 3 The Protective Effect of Extracorporeal Cardia Shock Waves on Myocardial Ischemia-Reperfusion Injury in Rats via EPCs-exo/miR-140-3p/PTEN pathwayObjective:To establish myocardial ischemia-reperfusion(I/R)model in ApoE knockout rats and to explore the protective effect and mechanism of extracorporeal cardiac shock waves induced-EPCs-exo on myocardial ischemia-reperfusion injury in vivo.Methods:The ApoE gene knockout rat model was constructed by using the gene editing technology CRISPR/Cas9 for modeling rat myocardial ischemia/reperfusion injury.The left anterior descending coronary artery was ligated briefly,after 45 minutes of ischemia,the ligature was released to start reperfusion.After reperfusion,the myocardium was injected with exosomes or PBS.PKH26-labeled EPCs-exo were used for myocardial injection to verify the exosome uptake ability of myocardial tissue in vivo.Rats were randomly divided into Sham group,I/R+PBS group,I/R+CON-exo group and I/R+SW-exo group.In order to further verify the effect of miR-140-3p in SW-exo on myocardial I/R injury,we prepared SW-exoinhibitor and SW-exoinhibitor NC based on original exosomes.The EPCs were transfected with miR-140-3p inhibitor or miR-140-3p inhibitor NC,and then subjected to extracorporeal cardiac shock waves.The two groups of exosomes were extracted respectively.qRT-PCR was used to detect the level of miR-140-3p in the two groups of exosomes.The rats were then randomly divided into I/R+PBS group,I/R+SW-exoinhibitor NC group and I/R+SW-exoinhibitor group.Echocardiography was used to detect the cardiac function of rats.HE staining of cardiac tissue sections was used to detect myocardial damage and Masson staining was used to evaluate the myocardial fibrosis after myocardial I/R injury.Cardiomyocyte apoptosis was assessed by TUNEL staining,and the expression levels of Bax,Bcl-2,Cleaved caspase-3,NF-κB,NOX2,and GAPDH protein were detected by Western blotting.In addition,the expression of PTEN/PI3K/AKT protein in myocardium after treatment were detected in all groups by western blotting.The relative expression levels of miR140-3p and PTEN expression were detected by qRT-PCR.Results:1.The ApoE gene knockout rat model was successfully constructed.After breeding,PCR and sequencing were carried out to identify the ApoE gene double knockout(ApoE-/-)rats,which were used for animal experiment modeling.2.Rat model of myocardial ischemia-reperfusion injury was successfully established by briefly ligating the left anterior descending coronary artery and restoring coronary blood flow.After the ligature,the ST-T was significantly elevated and the color of the heart became white below the ligation site.After unfastened,the myocardial tissue turned dark red,and the ST segment gradually decreased.3.In exosomes uptake assessments in vivo,the punctate PKH26 red fluorescence signal was observed in the myocardial tissue using fluorescence microscope.The fluorescence signal distribution was coincident with cardiomyocyte fibers,suggesting that the EPCs-exo can be taken up and utilized by myocardial tissue.4.The results of qRT-PCR showed that compared with SW-exoinhibitor NC,the level of miR-140-3p in SW-exoinhibitor was significantly reduced(p<0.01),indicating that the knockdown of miR-140-3p in SW-exo was successfully performed.which can be used for follow-up experiments.5.Compared with the Sham group,the left ventricle internal diastolic diameters(LVIDd),left ventricle internal systolic diameters(LVIDs),left ventricle end-diastolic volume(EDV),and left ventricle end-systolic volume(ESV)increased after I/R,while left ventricle ejection fraction(EF%)and left ventricle fractional shortening(FS%)were decreased(p<0.05).Compared with I/R+CON-exo,SW-exo improved EF%and FS%in rats more significant(p<0.05).The pathological results showed that myocardial tissue damage was alleviated in CON-exo and SW-exo groups with the reduction of surrounding inflammatory cells infiltration and coloration of collagen fibers in myocardial tissue.The effect of SW-exo group was more obvious than CONexo.SW-exo can significantly down-regulate the level of TUNEL-positive cells in myocardial tissue and inhibit cardiomyocyte apoptosis(p<0.01).Compared with I/R+PBS group and I/R+CON group,SW-exo significantly reduced the expressions of Bax,Cleaved caspase-3,NF-κB and NOX2,and promoted the expressions of Bcl-2(p<0.05).6.Compared with I/R+SW-exoinhibitor NC group,I/R+SW-exoinhibitor group increased LVIDd,LVIDs,EDV and ESV,decreased EF%and FS%,indicating worse cardiac function(p<0.05).However,compared with IR+PBS group,EF%and FS%in I/R+SW-exoinhibitor group still increased(p<0.05).The pathological results of myocardial tissue in I/R+SW-exoinhibitor NC group were consistent with those in SW-exo group,but the injury of myocardial tissue in I/R+SW-exoinhibitor group was aggravated with increased collagen fibers,which was slightly better than that in I/R+PBS group.Compared with I/R+S W-exoinhibitorNC group,the rate of TUNEL positive cells increased in I/R+SW-exoinhibitor group(p<0.01).Compared with I/R+SW-exoinhibitor NC group,I/R+SW-exoinhibitor group significantly increased the expressions of Bax,Cleaved caspase-3,NF-κB and NOX2,and reduced the expressions of Bcl-2(p<0.01).7.The results of Western blot and qRT-PCR showed that compared with the Sham group,the relative expression of miR-140-3p in myocardium of I/R+PBS group decreased,and the expression of PTEN increased(p<0.01).Compared with the I/R+PBS and I/R+CON-exo groups,the expression of miR-140-3p in the I/R+SW-exo group was up-regulated,following a decrease of the expression of PTEN and an increase of phosphorylation of PI3K/AKT(p<0.05).Compared with I/R+SWexoinhibitor NC group,the expression of miR-140-3p in I/R+SW-exoinhibitor group was significantly decreased with an increase of PTEN,which hindered the phosphorylation of p-PI3K/PI3K and p-AKT/AKT(p<0.05).Conclusions:1.EPCs-exo can be taken up and utilized by myocardial tissue.SW-exo could significantly improve cardiac function and inhibit ventricular remodeling in I/R rats.It could inhibit cardiomyocyte apoptosis and alleviate inflammatory response and oxidative stress to improve myocardial ischemia-reperfusion injury in rats.2.SW-exo may activate PI3K/AKT pathway through miR-140-3p/PTEN axis to exert cardioprotective effects.The miR-140-3p inhibitor can antagonize the most of the protective effects of SW-exo by inhibiting PI3K/AKT phosphorylation. |
PDF Full Text Request