| Objective:Spinal cord ischemia-reperfusion injury is a serious complication of thoracic aortic disease and abdominal aortic disease after endovascular isolation or surgical reconstruction.It’s important mechanism of action and the exploration of more effective protective strategies are the top priorities for the spinal cord to resist ischemiareperfusion injury.miRNAs are involved in the regulation of neuronal apoptosis,proliferation,differentiation,and autophagy.According to literature reports,mesenchymal stem cell-derived exosomes overexpressing certain miRNAs have a protective effect on ischemia-reperfusion injury.The study found that miR-455-5p could inhibit hypoxia-induced injury of human primary cardiomyocytes,promote cardiomyocyte proliferation,and inhibit apoptosis.Therefore,we speculated that BMSCs overexpressing miR-455-5p exosomes could play a protective role against spinal cord ischemia-reperfusion injury.In order to verify this speculation,the first part of this experiment intends to establish a model of spinal cord ischemia-reperfusion injury in rats,combined with the knowledge of cell biology,experimental zoology,molecular biology,pathology,and other related disciplines,to verify the overexpression of miR-455-Effects of 5p-derived bone marrow mesenchymal stem cell exosomes on spinal cord ischemiareperfusion injury.However,the mechanism of action of BMSC exosomes overexpressing miR-455-5p in spinal cord ischemia-reperfusion injury still needs to be further explored.In order to study the downstream mechanism of miR-455-5p,we predicted the target of miR-455-5p by bioinformatics tool(RNAhybrid).Since Nogo-A plays an important role in the regeneration and reconnection of axons after CNS injury,the relationship between NogoA and miR-455-5p was further investigated.It is known that down-regulation of Nogo-A protein can promote autophagy,inhibit cell apoptosis,and increase cell proliferation in ischemia-reperfusion injury.Therefore,the second part of this experiment intends to build a rat spinal cord ischemia-reperfusion injury model,combined with experimental Knowledge of zoology,cell biology,molecular biology,immunohistochemistry and other related disciplines,verify the targeted binding and regulatory relationship between miR455-5p and Nogo-A protein,and explore the bone marrow mesenchymal cells overexpressing miR-455-5p The effect of mesenchymal stem cell(BMSC)exosomes on the expression of Nogo-A protein in spinal cord ischemia-reperfusion tissue,further clarifying the effect of miR-455-5p overexpressing bone marrow mesenchymal stem cell exosomes on spinal cord ischemia-reperfusion injury mechanism.Methods:1 Part Ⅰ Bone marrow mesenchymal stem cell derived exosomal miR-455-5p protects against spinal cord ischemia reperfusion injury1.1 Isolation and identification of BMSCs:Male Sprague Dawley rats aged 10-14 days,weighing 30-40 g,were used for this experiment.All animal experimental procedures were permitted by Ethics Review Board of The First Hospital of China Medical University and were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals BMSCs were isolated from the femur and tibia of rats.Bone marrow was flushed from femurs and tibias with DMEM/F12 medium and dissociated into cell suspension.Red blood cells were removed with lysis buffer.After centrifuging and washing with phosphate-buffered saline(PBS),BMSCs were obtained and cultured in DMEM/F12 containing 10%fetal bovine serum.When BMSCs at the third passage reaching 80-90%confluence,the cells were collected and resuspended in PBS.BMSCs were incubated with antibodies against CD29,CD90,CD34,and CD45 respectively.NovoCyte flow cytometer was used to acquire data.1.2 Isolation and identification of BMSCs-exosomes:The P3-BMSCs medium was collected and subjected to ultracentrifugation to separate the exosomes.The initial centrifugations were as follows:2000 g for 30 min,and 10000 g for 45 min at 4℃.The supernatant was filtered by a 0.45 μm filter membrane.Then,the filtering liquid was centrifuged at 100000 g for 70 min at 4℃,the pellet was resuspended with PBS,followed by another centrifugation at 100000 g for 70min at 4℃.The pellet was resuspended with PBS and BMSCs-exosomes were obtained.Transmission electron microscopy was used to observe the morphology of exosomes.Briefly,10 μL exosome sample was dropped onto a copper grid,and the grid was dried by filter paper after 1 min.Then,the grid was stained with uranyl acetate for 1 min.After drying at room temperature for several minutes,the images were visualized by transmission electron microscopy.The exosome size was measured by nanoparticle tracking analysis(NTA)with NanoFCM.The exosome markers(CD63,CD9,CD81)were detected by Western blot analysis.1.3 Cell infection:Rat miR-455-5p lentivirus gene transfer vector was constructed by Addgene,and the lentivirus vector without miR-455-5p were used as control(miR-NC).Lentivirus was added to BMSCs at the multiplicity of infection(MOI)of 50.1.4 Animal experiments:All animal experimental procedures were permitted by Ethics Review Board of The First Hospital of China Medical University.The use of laboratory animals was carried out in accordance with the National Institutes of Health guide for care and use of Laboratory animals.Male Sprague Dawley rats(6 weeks,180-200 g)were divided into the following groups:Sham,SCIR,SCIR+BMSC-Exo,SCIR+BMSC-LVNC-Exo,and SCIR+BMSC-LV-miR-455-5p-Exo.Exosomes(2 μg/μL in PBS,10 μL)were administered by intrathecal injection,and SCIR rats were injected with 10 μL PBS.Animals were included in the establishment of SCIR model only if they had normal hindlimb motor function.Three days after intrathecal injection,rats were anesthetized by intraperitoneal injection of 50mg/kg pentobarbital sodium.The rats were placed in a supine position.The aorta was exposed by a transperitoneal approach from the beginning of the left renal artery to the aortic bifurcation.During surgery,the body temperature of rats was maintained at 36.5 ± 0.5℃.Heparin(130 U/kg)was administered intravenously 5 min before occlusion.The aorta was cross clamped using 2 bulldog clamps from directly below the left renal artery to the aortic bifurcation.Aortic occlusion was lasted for 30 min,then bulldog clamps were removed.After blood flow restored,the wound was sutured.The rats needed to assist in urination after surgery,until autonomic urination was restored.The same procedure was performed on Sham group without ischemia reperfusion.Neurological evaluation was performed at the indicated time after reperfusion.Twenty-four hours after reperfusion,the rats were euthanized,and lumbar spinal cord segments between L2 and L5 were collected for subsequent experiments.1.5 Neurological evaluation:The Locomotor function of hind-limb after SCIR was assessed using the Basso,Beattie,and Bresnahan(BBB)grading scale,which ranges from scores of 0(complete paralysis)to 21(normal locomotion).The BBB score was evaluated 6,12,24,and 48h after reperfusion.1.6 Histopathological evaluation:The fixed lumbar spinal cord segments were dehydrated and embedded in paraffin.The paraffin blocks were sliced into 5 μm-thick coronal sections.The sections were dewaxed,and then stained with hematoxylin and eosin.After dehydrated and sealed,the sections were observed with a microscope at 200×magnification.Semiquantitative scoring was performed to evaluate the pathological changes of spinal cord.For Nissl staining,the sections were stained with 0.5%cresyl violet acetate.After washing,the sections were differentiated with 0.25%glacial acetic acid ethanol.Finally,the slices were visualized using an Olympus microscope(at 200×magnification).1.7 For TUNEL/NeuN double immunofluorescence staining:the sections were permeabilized in 0.1%Triton X-100 for 8 min at room temperature.Subsequently,the sections were subjected to citric acid-sodium citrate solution for epitope retrieval.The enzyme solution(5 μL)was mixed with the labeling solution(45 μL)to obtain TUNEL reaction solution.Each section was incubated with 50 μL of TUNEL reaction solution at 37℃ for 60 min.After blocked with goat serum,the sections were incubated with the antibody against NeuN.The sections were incubated with the FITC-labeled secondary antibody.The nuclei were stained with DAPI.Finally,the samples were photographed under a microscope at 400× magnification.The proportion of TUNEL-positive neurons was calculated.1.8 For LC3/NeuN double immunofluorescence staining:the sections were blocked with goat serum after epitope retrieval.The antibodies against LC3 and NeuN were added to each section and incubated overnight at 4℃.The sections were incubated with the FITC-labeled and Cy3-labeled secondary antibody.After stained with DAPI,the samples were photographed,and the proportion of LC3-positive neurons was calculated.1.9 Western blot:Western blot was used to detect the expression levels of apoptotic proteins Bax,caspase 3 and cleaved caspase-3 in spinal cord tissue,and to detect autophagy proteins Beclin-1 and LC3 in spinal cord tissue-II,expression level of LC3-I.2 Part Ⅱ Mechanism study on the Bone marrow mesenchymal stem cell derived exosomal miR-455-5p protects against spinal cord ischemia reperfusion injury2.1 Preparation and identification of Bone marrow mesenchymal stem cell derived exosomal miR-455-5p:Take the bone marrow of male Sprague-Dawley(SD,10-14 d,60-100 g)rat tibia and femur,isolate and culture BMSC cells,The third-generation cells(P3)were identified by flow cytometry,miR-455-5p/miR-NC lentiviral overexpression vector was constructed,BMSC cells were infected,exosomes were prepared 48 h later,and the exosome marker CD9 was detected by Western blot,the expression levels of CD63 and CD81;the level of miR-455-5p in exosomes was detected by Real-time PCR.2.2 Animal experiments:After adaptive feeding,male SPF SD rats(180-200 g)were randomly divided into 5 groups of 6 rats in each group.BMSC-Exo,BMSC-LV-NC-Exo,BMSC-LV-miR-455-5p-Exo were injected,and then the SCIR model was established by the same method.The lumbar(L2-L5 segment)spinal cord tissue and serum samples of rats were collected for subsequent experiments.2.3 Immunohistochemistry:The sections were incubated in 3%H2O2 for 15 min,followed by epitope retrieval.The slices were blocked with goat serum at room temperature for 15 min.Afterward,the sections were incubated with primary antibody rabbit anti-Nogo-A overnight at 4℃,and then incubated with secondary antibody of HRPlabeled goat anti-rat IgG at 37℃ for 60 min.Then,the sections were colored with diaminobenzidine,followed by counterstaining with hematoxylin for 3 min.Then sections were immersed in water and rinsed by water.After dehydrated and sealed,the sections were observed under a microscope.2.4 Western blot:L2-L5 segments of spinal cords were homogenized in Cell lysis buffer and Western blot was used to detect the expression levels of Nogo-A.2.5 Dual-luciferase assay:The sequence of wild-type Nogo-A3’UTR(WT)was cloned to the downstream of luciferase reporter gene pmirGLO.Then the Nogo-A binding site was subjected to site-directed mutagenesis and Nogo-A-mutant type(MUT)vector was constructed.Afterwards,the two plasmids and miR-455-5p mimics or NC mimics were co-transfected into PC12 cells.After 48 h,cells luciferase activity was measured by a dual luciferase reporter assay kit.Results:1 Part Ⅰ Bone marrow mesenchymal stem cell derived exosomal miR-455-5p protects against spinal cord ischemia reperfusion injury1.1 Characterization of exosomes derived from bone marrow mesenchymal stem cells(BMSCs)and Identification of miR-455-5p-overexpressed exosomes:BMSCs at firstpassage(P1)with spindle-shaped,fusiform,and polygonal morphology were observed under light microscope.BMSCs at third passage(P3)were uniform spindle-shaped in appearance.Based on the spindle-shaped morphology of P3-BMSCs and their adhesion to culture dish,the cells were identified as BMSCs.Flow cytometry analysis showed that the cells were positive for CD29 and CD90,and negative for CD34 and CD45.The results suggested that the cells we isolated were in line with the characteristics of BMSCs.Next,exosomes we extracted from the cultured supernatant of BMSCs.To verify the characteristics of exosomes,NTA,TEM and Western blot analysis were employed.NTA showed the particles with diameters ranging from 30 to 150 nm,with an average diameter of 73.22 nm.The exosomes were round or oval shape under TEM.The expression levels of specific exosome markers(CD63,CD9,CD81)were remarkably higher in the BMSCExos compared with the BMSCs.These results indicated that the particles we extracted were indeed exosomes.BMSCs were infected with Lv-miR-455-5p or Lv-NC for 48 h.Real-time PCR showed that the expression of miR-455-5p was higher in miR-455-5pinfected BMSCs than that in miR-NC-infected BMSCs.After extracted from the supernatant of these above BMSCs,exosomes were identified by the positive expression of exosome marker.Meanwhile,the expression of miR-455-5p was up-regulated in BMSC-LV-miR-455-5p-Exo compared with BMSC-LV-NC-Exo.1.2 Exosomes containing miR-455-5p promoted recovery of locomotor function after SCIR:The expression of miR-455-5p was determined at 24 h of after SCIR modeling.It was downregulated in spinal cords of SCIR rats compared with Sham group.miR-455-5p enriched exosomes significantly enhanced the level of miR-455-5p in spinal cords.At 6,12,24,and 48 h after SCIR modeling of rats,BBB scores were used to evaluate the effect of BMSC-LV-miR-455-5p-Exo on locomotor functional recovery.The limb motor function scores were lower at each time point after reperfusion compared with the Sham group.The scores of the SCIR+BMSC-LV-miR-455-5p-Exo group were significantly higher than the SCIR+BMSC-LV-NC-Exo group from 12 to 48 h after SCIR.These results suggested that BMSC-LV-miR-455-5p-Exo promoted recovery of locomotor function after SCIR.1.3 Effects of miR-455-5p-enriched exosomes on histopathological changes after SCIR:To assess the effect of miR-455-5p-enriched exosomes on spinal cord damage caused by SCIR,HE staining and Nissl staining were performed.The histologic findings in spinal cord tissues of Sham rats were normal.In the SCIR group,we observed the disordered nerve fibers,degenerated neurons in the gray matter,and vacuolation in the white matter.However,neatly arranged nerve fibers,less degenerated neurons and reduced vacuolation were presented in spinal cord tissues of SCIR+BMSC-LV-miR-455-5p-Exo group.The pathological score was higher in the SCIR group than that in the Sham-operated group.The increased pathological score SCIR rats was markedly reduced by BMSC-LVmiR-455-5p-Exo administration.Nissl staining showed the number of intact motor neurons was reduced in the SCIR group compared with the Sham group.However,the number of intact motor neurons in the SCIR+BMSC-LV-miR-455-5p-Exo group was increased.These results suggested that BMSC-LV-miR-455-5p-Exo effectively mitigated spinal cord damage after SCIR.1.4 Exosomes containing miR-455-5p reduced apoptosis of neurons after SCIR:To further investigate the neuroprotective effects of BMSC-LV-miR-455-5p-Exo on SCIR injury,we evaluated neuron apoptosis by double immunofluorescence staining of TUNEL and NeuN(a neuron marker).The number of TUNEL-positive neurons after SCIR was higher than that in the Sham group,indicating that SCIR triggered neuron apoptosis in the spinal cord.BMSC-LV-miR-455-5p-Exo administration reduced the number of TUNELpositive neurons in SCIR rats.Western blot showed that the levels of cleaved-caspase 3 and Bax in spinal cords were increased significantly in SCIR rats.However,the increased levels of cleaved-caspase 3 and Bax in SCIR rats were reduced by BMSC-LV-miR-4555p-Exo.These results indicated that exosomes containing miR-455-5p protected neuron from apoptosis in SCIR rats.1.5 Exosomes containing miR-455-5p promoted autophagy activation in neurons after SCIR:In order to examine autophagy activation after SCIR,we performed LC3/NeuN double immunofluorescence staining and Western blot analysis.The results showed a decreased number of LC3-positive neurons in the spinal cord section of the SCIR group,while there were more LC3-positive neurons in SCIR rats administrated with exosomes containing miR-455-5p.The levels of Beclin-1 and LC3-II in spinal cords were reduced in the SCIR group.The administration of BMSC-LV-miR-455-5p-Exo resulted in significant upregulation of Beclin-1 and LC3-II in SCIR rats.These results indicated that exosomes containing miR-455-5p activated autophagy in neurons after SCIR.2 Part Ⅱ Mechanism study on the Bone marrow mesenchymal stem cell derived exosomal miR-455-5p protects against spinal cord ischemia reperfusion injury2.1 Characterization of exosomes derived from bone marrow mesenchymal stem cells(BMSCs)and Identification of miR-455-5p-overexpressed exosomes:P3 generation cells were arranged in an orderly manner,with plump cell bodies,mainly spindle-shaped.The results of flow cytometry of P3 generation cells indicated that the extracted cells were BMSC cells.The diameter of the extracted BMSC exosomes was about 30-200 nm;the exosomes were complete,spherical,and uniform in size by transmission electron microscopy;CD9,CD63 and CD81-specific proteins were all expressed in the BMSC exosomes.The real time PCR results showed that the expression level of miR-455-5p was significantly increased in the exosomes of BMSC-LV-miR-4555p.2.2 BMSC exosomes overexpressing miR-455-5p can reduce the expression of NogoA protein in spinal cord tissue:To study the downstream mechanism of miR-455-5p,we predicted the target of miR-455-5p by bioinformatic tool.Since Nogo-A plays an important role in the regeneration and reconnection of axons after CNS injury,the relationship between Nogo-A and miR-455-5p was further investigated.Both immunohistochemical staining and Western blot analysis showed that the expression of Nogo-A in the spinal cords of SCIR group was higher than that in the Sham group.The administration of exosomes containing miR-455-5p significantly decreased the expression of Nogo-A in the spinal cords of SCIR rats.2.3 Exosomal miR-455-5p targeted Nogo-A in the injured spinal cords:We further verified the binding of miR-455-5p and 3 ’-UTR of Nogo-Ausing a dual-luciferase reporter assay.The sequence of miR-455-5p,wild-type and mutated sequences of Nogo-A 3 ’-UTR were presented,the luciferase activity was significantly inhibited by miR-455-5p mimics in the group with the Nogo-A-WT vector.However,the luciferase activity failed to be inhibited by miR-455-5p in the Nogo-A-MUT vector group.Therefore,we concluded that miR-455-5p targets Nogo-A to exert its neuroprotective role in SCIR injury.Conclusion:1.Exosomes derived from BMSCs overexpressing miR-455-5p alleviate spinal cord injury,increase autophagy to inhibit neuron apoptosis,take a protective effect on spinal cord ischemia.2.There is a targeted binding relationship between miR-455-5p and Nogo-A protein.miR-455-5p can inhibit the expression of Nogo-A protein in spinal cord tissue and promote recovery of locomotor function after SCIR through downregulating Nogo-A.This is the first time to report the role of BMSC-exosome-miR-455-5p in SCIR injury,which might open a new avenue for SCIR treatments. |
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