Efficacy And Mechanism Of Hypoxia-preconditioned Adipose-derived Mesenchymal Stem Cell-derived Exosomes In Spinal Cord Injury

Objective:Spinal cord injury is a central nervous system injury with very high morbidity and mortality.With the development of industries such as construction and road transportation,the incidence of spinal cord injury is increasing substantially.However,because the pathophysiological mechanism of spinal cord injury is extremely complex,it is still a major problem in the medical field.Therefore,it is very important to develop new methods that can effectively treat spinal cord injury.This study aimed to explore the effect of hypoxia-preconditioned adipose-derived mesenchymal stem cell-derived exosomes on neurological recovery after spinal cord injury in rats.At the same time,the mi RNA expression profile of adipose-derived mesenchymal stem cells derived from hypoxia preconditioning was revealed.The role and mechanism of highly expressed mi RNAs in exosomes in regulating neuronal apoptosis after spinal cord injury were explored.This study may provide new treatment strategy for clinical treatment of spinal cord injury.Method:1.Isolate and culture primary adipose-derived mesenchymal stem cells(ADSCs).Ultracentrifugation was used to extract exosomes from hypoxia-preconditioned ADSCs;Exosomes are verified and identified by scanning electron microscopy,nanoparticle tracking analysis,and Western Blot.The uptaking of exosome by neuronal cells was visualized by immunofluorescence.2.PC12 and SHS-Y5 Y cells were used to establish the Oxygenglucose Deprivation model in vitro.After treated with different exosomes,CCK8 was used for cell proliferation toxicity experiments,flow cytometry and scratch experiments were used to verify and evaluate the effect of exosomes on regulating of neuronal apoptosis.3.The rat spinal cord contusion model was established by the modified Allen’s method.The BBB score and Catwalk gait analysis of the rats were performed after spinal cord injury in the hypoxic exosome group,the normal exosome group and the PBS group and the sham group,respectively to assess the recovery of neurological function after spinal cord injury in rats.4.Fluorescently labeling exosomes were used to track the distribution of exosomes in the spinal cord of rats with spinal cord injury;HE staining was used to detect the formation of syringomyelia after spinal cord injury in rats;TUNEL staining was used to detect the neuronal apoptosis in the spinal cord of each group;Western Blot was used to detect the expression of apoptosis-related proteins in the injured spinal cord;Aiming to verify the pathological changes in the spinal cord of rats after hypoxic exosome treatment,and to evaluate the therapeutic effect of exosomes.5.Exosomal mi RNA sequencing and q RT-PCR were used to explore the effect of hypoxia treatment on the expression profile of exosomal mi RNA,and search for potential apoptosis-inhibiting active substances;Function gain and loss experiments were performed to explore and verify mi RNA from exosomes that inhibit apoptosis in exosomes by using mi RNA mimics.6.The 45-Cignal Pathway Array was used to explore the downstream signaling pathway of highly expressed mi RNA in exosmes.7.The activation of MAP/JNK signaling pathway in vivo and in vitro were verified by western blot.The expression level of target gene JNK3 were verified by western blot.The expression level of JNK3 in rats was verified by immunofluorescence.8.Bioinformatics method was Predict the binding site of mi R-499-5p and JNK3 3’-UTR by.The targeted inhibition of mi R-499-5p on JNK3 was validated in vitro by Western Blot and q RT-PCR in both PC12 and SHSY5 Y cells.9.Construct a reporter gene containing the binding site of mi R-499-5p on JNK3 3’-UTR,and verify and determine whether mi R-499-5p directly acts on JNK3 by luciferase reporter gene assay.Results:1.Successfully extracted and identified exosomes secreted by adipose-derived mesenchymal stem cells under both hypoxia and normoxia condition,and verified that neurons can effectively take up exosomes;2.After treated with hypoxic exosomes,PC12 and SHS-Y5 Y cells presented more resistance to apoptosis and more proliferative in an oxygen-glucose deprivation model;3.Compared with the normoxic exosome group and the PBS group,the hypoxic exosome group obtained better BBB score after spinal cord injury than the normoxic exosome group and the PBS group;Twenty-eight days after spinal cord injury,the Catwalk results showed that the gait performance of the hypoxic exosome group was significantly better than that of the other groups;4.The PKH-26 fluorescently labeled exosomes showed the local recruitment of exosome group;HE staining showed that hypoxic exosomes could effectively reduce the formation of syringomyelia after spinal cord injury in rats;TUNEL staining showed the proportion of neuronal apoptosis in the spinal cord of the exosome group was less than that of the other groups,and the hypoxic exosomes group were even less;WB detection showed that the expression levels of pro-apoptosis-related molecules BAD and cleaved caspase-3 in the hypoxic exosome group were significantly reduced,and the expression level of Bcl2 was significantly increased.5.MiRNA sequencing found that a series of mi RNAs such as mi R-499-5p were highly expressed in hypoxic exosomes.After verification by q RT-PCR,it was finally determined that mi R-499-5p was the most important differential component of different exosomes;The mi R-499-5p mimics were validated in vitro with anti-apoptotic effects similar to those of hypoxic exosomes.6.The results of 45-Cignal Pathway Array showed that after mi R-499-5p mimic treatment,most signaling pathways in PC12 cells were inhibited,and many signaling pathways including MAP/JNK signaling pathway were significantly inhibited.7.The results of Western Blot and immunofluorescence showed that after the treatment of hypoxic exosomes or mi R-499-5p mimics,the expression level of JNK3 was significantly decreased,the phosphorylation level of the downstream protein c-jun was significantly decreased,and the apoptosis-related protein cleaved caspase-3 expression decreased.8.Through bioinformatics methods,it was found that mi R-499-5p has a JNK3 3’-UTR binding site;transient transfection with mi R-499-5p mimics can significantly induce JNK3 protein in PC12 and SHS-Y5 Y cells and down-regulation of m RNA levels.9.By luciferase reporter gene assay,it was verified that mi R-499-5p has a JNK3 3’-UTR binding site,and it was verified and confirmed that mi R-499-5p directly targets JNK3.Conclusion:1.The exosomes secreted by ADSCs after hypoxia preconditioning can effectively reduce the apoptosis of neurons s after oxygen and glucose deprivation.2.The exosomes secreted by ADSCs after hypoxia preconditioning can recruit locally to the injury area in vivo,inhibit the hyperplasia of scar tissue after injury,and be beneficial to the recovery of nerve function after spinal cord injury in rats.3.MiRNA-499-5p enriched exosomes from hypoxia-preconditioning ADSCs inhibits JNK3,negatively regulates MAP/JNK signaling pathways,and promotes the recovery of neurological function.