| Background and objective:Spinal cord injury(SCI)is a serious traumatic disease of the central nervous system,which results in motor and sensory impairment and poor prognosis.Currently,researches for treating SCI mainly include two areas:neuroprotection and regeneration.However,the complicated pathologic changes following SCI,especially caused by secondary injury,have hindered the development of effective and reliable treatments.Mesenchymal stem cells(MSCs)play an important role in the treatment of SCI,which had been studied widely.Nevertheless,most studies have shown that the direct transplantation of MSCs exists the potential risks.Although the transplantation of MSCs have neuron-like characteristics,it is difficult to treat them as real neurons.Based on the current evidence,the efficacy of MSCs seems to be related to their paracrine activity,rather than the mechanism of cell replacement.Exosomes have been shown to act as messengers of intercellular communication and participate in the pathological processes of SCI.Given the characteristics of cell-free therapy and the ability to cross the blood brain barrier or blood spinal cord barrier freely,the therapeutic potential of exosomes in SCI has attracted more attention.Exosomes are recognized as ideal vectors to deliver therapeutic molecules such as miRNAs,which play crucial roles in the physiopathology of SCI.Therefore,in the current study,a model of contusion SCI in Sprague-Dawley(SD)rats was established,and we investigated the therapeutic potential of exosomes derived from bone marrow mesenchymal stem cells(BMSCs)transfected with miR-338-5p mimics,as well as the underlying molecular mechanisms.Methods:Part 1:The bilateral femurs of SD rats(150-200g)were extracted and BMSCs were isolated by whole bone marrow adherent method.The cell-surface antigens including CD44,CD90,CD29,CD45,CD24 on BMSCs at passage 3 were identified using flow cytometry.BMSCs were transfected with miR-338-5p mimics or negative control,and the cell supernatants were collected.Exosomes were isolated from the cell supernatants and identified by using transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and western blot analysis.q RT-PCR was conducted to measure the expression level of miR-338-5p. Part 2:The normal male SD rats(200-280g)were randomly divided into two groups: sham group and SCI groups.Rats in the sham group were subjected to the laminectomy without contusion injury.In the SCI groups,a contusion injury was carried out using a modified Allen's weight drop apparatus on the exposed dura of the spinal cord.Several rats in the SCI groups were randomly divided into three groups and sacrificed at 1,4,and7 days.The spinal cord containing the injured site was harvested for q RT-PCR analysis of miR-338-5p level.Several rats in the SCI groups were randomly divided into four groups:(1)control group(the rats were only subjected to SCI),(2)PBS group(the rats were treated with PBS),(3)control exosomes group(the rats were treated with control exosomes packaged with miR-338-5p mimics-NC),and(4)miR-338-5p exosomes group(the rats were treated with exosomes packaged with miR-338-5p mimics),and sacrificed at 4 days.The expression levels of neurofilament protein-M(NF-M),growth-associated protein-43(GAP43),myelin-associated glycoprotein(MAG)and glial fibrillary acidic protein(GFAP)were detected by western blot.Exosomes(100?g)or an equal volume of PBS were directly microinjected into the injured site of the spinal cord and combined with injection via the tail vein after SCI.The pathologic changes of spinal cord were observed by using HE staining and Nissl staining.The recovery of hindlimb locomotor function was assessed by using Basso-Beattie-Bresnahan(BBB)scores on SCI rats at 1,3,7,14,and 21 days.Part 3:Oxidative stress cell model was established in PC12 cells induced by H2O2.To determine the concentration and action time of H2O2,expression of miR-338-5p was assessed by q RT-PCR.Cell viability was determined using a CCK-8 assay and cell apoptosis was measured by flow cytometric assays in H2O2-induced PC12 cells.The intracellular ROS and SOD levels were measured.The expression levels of NF-M,GAP43,MAG,GFAP proteins and apoptosis-associated proteins were measured in H2O2-induced PC12 cells by western blot.The possible targets of rno-miR-338-5p were searched by retrieving the Target Scan,miRDB,miRWalk,miRMap and micro T-CDS databases.After a search of the KEGG pathway database and a literature review,cannabinoid receptor 1(Cnr1)was selected for further study.The negative regulatory relationship between miR-338-5p and Cnr1 was determined by dual-luciferase reporter assays and Rap1 was the downstream gene by KEGG pathway analysis.The possible regulatory pathways were searched and western blot was applied to analysis the related proteins whether these proteins regulate the cell proliferation and survival after treated with miR-338-5p.Results:Part 1:Flow cytometric analysis showed that the cell-surface antigens were positive for CD44,CD90,and CD29 but negative for CD45 and CD34,suggesting that the isolated BMSCs had mesenchymal specific phenotypes rather than hematopoietic phenotype.Exosomes were extracted from the cell supernatants of BMSCs by multistep ultracentrifugation.q RT-PCR results showed that the miR-338-5p expression levels in the BMSC-exosomes transfected with miR-338-5p mimics were significantly higher than those of the exosomes transfected with miR-338-5p mimic-NC.Meanwhile,the results of TEM and NTA analysis demonstrated that exosomes were lipid bilayer structures with the diameters of ranged from50 to 200 nm and the average diameter of exosomes were 100 nm.Western blot analysis showed the positive expression of exosome-specific protein markers,including CD63,CD9,TSG101 and HSP70.Part 2:q RT-PCR showed that the expression levels of miR-338-5p were downregulated at 1,4 and 7 days after acute spinal cord injury in rats.The expression level of miR-338-5p at day 4 was significantly downregulated after SCI(p<0.001).The results of western blot and immunofluorescence staining showed that miR-338-5p exosomes increased the expression levels of NF-M and GAP43,while decreased the expression levels of MAG and GFAP;moreover,we found that exosomes derived from BMSCs resulted in similar effects,which were lower than those of miR-338-5p exosomes.HE staining and Nissl staining showed that exosomes overexpressing miR-338-5p improved the pathological changes of SCI,such as reduction of tissue hemorrhage,inhibition of neuron degeneration and promotion of microglial proliferation as well as reduction of Nissl bodies lost.The BBB scores results as follows: changes of hindlimb locomotor function were not obvious between five groups after SCI at 1 and 3 days(p>0.05);the hindlimb locomotor function in the miR-338-5p exosomes group and control exosomes group were significantly improved than that of in the control group following SCI at 7,14 and 21 days(p<0.001);the mean scores of the miR-338-5p exosomes group were 5.8,11 and 14.2 at 7,14 and 21 days respectively;the mean scores of the control exosomes group were 5,8.2 and 10.6 at 7,14 and 21 days respectively;the mean score of the control group and PBS group were 7.6 and 7.4 at 21 days following SCI respectively.Part 3:Oxidative stress cell model was established in PC12 cells induced by H2O2.we found that 300?M H2O2 for 12 h significantly reduced the expression of miR-338-5p(p<0.01).In addition,300?M H2O2 for 12 h H2O2 treatment significantly decreased cell viability and increased the percentage of apoptotic cells(p<0.05).Hence,300?M for 12 h was selected as an H2O2-stimulating condition in the following experiments.The analysis of ROS and SOD levels suggested that miR-338-5p and its overexpression exosomes notably promoted the antioxidative ability in PC12 cells stimulated by oxidative stress.The western blot results showed miR-338-5p exosomes upregulated the NF-M and GAP43 protein levels while downregulating the MAG and GFAP protein levels,which was consistent with the results of animal experiments.Furthermore,miR-338-5p and miR-338-5p-overexpressing exosomes repressed H2O2-induced apoptosis,as evidenced by upregulated Bcl-2 and downregulated Bax and cleaved caspase-3 expression.Cnr1 was the target gene of miR-338-5p by dual-luciferase reporter assays.Cnr1 was one of the G protein-coupled receptors,and Cnr1 inhibited adenylyl cyclase activity and reduces cyclic AMP(c AMP)accumulation by activating heterotrimeric Gi/o type G proteins.Overexpression of miR-338-5p increased c AMP accumulation as a consequence of downregulated Cnr1 expression and that Rap1 was activated by c AMP.Finally,the upregulation of the p-PI3 K and p-Akt proteins inhibited cell apoptosis and promoted neuronal proliferation and survival.Conclusion:miR-338-5p-overexpressing exosomes derived from BMSCs promoted the recovery of neurological function and motor function in rats.Moreover,miR-338-5p-overexpressing exosomes also provided neuroprotective effects and improved the living environment of nerve cells.The underlying mechanism of miR-338-5p was increased c AMP accumulation as a consequence of downregulated Cnr1 expression and that Rap1 was activated by c AMP.Eventually,the activation of the PI3K/Akt pathway activation attenuated cell apoptosis and promoted neuronal proliferation and survival by c AMP-mediated Rap1 activation. |
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