Exosome Derived From Bone Mesenchymal Stem Cells Protects The Injured Spinal Cord Via Inhibition Of Pericyte Pyroptosis

Spinal cord injury often results in permanent functional impairment.The clinical manifestations are mainly motor sensory dysfunction,urinary incontinence,neuralgia,sexual dysfunction,etc.Chronic complications usually include the respiratory system,cardiovascular system,urinary reproductive and gastrointestinal dysfunction.The incidence of spinal cord injury has the characteristics of low incidence,high disability,high mortality and heavy economic burden.Therefore,exploring the pathological mechanism and treatment of SCI is particularly important for the rehabilitation of patients with spinal cord injury.A large number of studies have shown that bone marrow mesenchymal stem cells can play a neuroprotective role in spinal cord injury by secreting multiple neurotrophic factors and inhibiting neuronal apoptosis,etc.However,the therapeutic benefits of MSC are limited by many disadvantages,including low survival rates,possible tumor formation,immune rejection,and genetic variation.There are increasing evidences that exosomes derived from mesenchymal stem cells can replace MSC transplantation and show similar neuroprotective effects in different disease models including SCI.However,the therapeutic role and efficacy of exosomes derived from bone mesenchymal stem cells in spinal cord injury need further research and elaboration.Pericytes,also called mural cells,cover the most of the blood vessel walls throughout the body and are inserted into the microvascular basement membrane.As the central part of the neurovascular unit,pericytes play key roles in the CNS including:(1)regulating BSCB/BBB permeability;(2)regulating capillary blood flow and maintenance of the vascular system;(3)removing toxic molecules;(4))Regulate the entry of immune cells into the central nervous system;(5)Show stem cell characteristics in some cases.Several studies have found that the loss of pericytes after spinal cord injury increases the permeability of BSCB,directly leading to the destruction of the microcirculation system and the leakage of harmful blood components.Pyroptosis is a new form of programmed cell death that relies on the activation of caspases 1,4,5,and 11 and the release of pro-inflammatory cytokines.A recent study found that in animal models of sepsis induced by cecal ligation and puncture,pericyte pyroptosis,rather than apoptosis,leads to vascular leakage.Therefore,the existence of pericytes pyroptosis in the SCI model and its effect on the permeability of BSCB are worth further exploration.This study is based on a summary analysis of previous research to further explore the pathological changes after spinal cord injury and the therapeutic effects and underlying mechanisms of bone mesenchymal stem cell-derived exosomes on spinal cord injury.Methods:1.Spinal cord injury model were conducted by with a spinal cord impactor.The rats are randomly divided into 4 groups:Sham group,EXO group,SCI group,SCI+EXO group.The exosome suspension was injected intravenously in EXO group and SCI+EXO group after SCI,and other groups were given the same volume of PBS.2.Exosomes derived from BMSC was extracted by ultracentrifugation and observed by Transmission electron microscopy.Specifically,specimens on coppers were made by two methods:dripping and floating.Nano sight technology(Particle Metrix,Meerbusch,Germany)was performed to analyze the size distribution of exosomes.Several exosome markers such as CD9,CD63,and CD81 were verified by western blot.3.Nissl staining and FJB staining was used to assess neuron survival.Luxol Fast Blue(LFB)staining and NF-200 Immunohistochemistry analysis was used to verify fiber degeneration.BBB scores was used to examine the motor function of spinal cord injury rats.4.Evans blue assessment was conducted to assess the integrity of BSCB and water content was performed to assess spinal cord edema.5.Elisa was used to determine the expression of Il-1β和 Il-18.6.Immunofluorescence analysis to analyze pericyte pyroptosis and the pericyte coverage on endothelia cells in injury spinal cord.Results:1.Typical structures of exosome were observed by TEM,which showed numerous saucer-shaped vesicles.Besides,specimens prepared by the floating method were more stereoscopic and clearly in morphology than those prepared by the dripping method.Nanoparticle Tracking Analysis further revealed the particle size,with a diameter of approximately 100nm.In addition,western blot analysis confirmed distinctive exosome markers including CD9,CD63,and CD81.2.Nissl bodies.and FJB positive neurons in the SCI+EXO group is dramatically ameliorated when compared with the SCI group.Fiber degeration was greatly attenuated by BMSC-EXOs in SCI rats.Basso Beattie Bresnahan(BBB)scores shows that BMSC-EXOs treatment improved locomotor function from 2 weeks post-SCI when compared with the SCI group(P<0.05).3.The infiltration of EB was significantly reduced in the SCI+EXO group compared with the SCI group(P<0.05).The water content of spinal cord tissues was similarly decreased in SCI rats because of the injection of BMSC-EXOs.These data suggest protective effects of BMSC-EXOs on BSCB integrity.4.Compared with rats in the Sham group,the expression of caspase1、Il-1βand Il-18 were increased in SCI rats,which means the pyroptosis in SCI.Compared with rats in the Sham group,the co-localization of Pdgfr-β with caspase-1 positive cells,was increased in spinal cord tissue of SCI rats.A marked loss of pericyte coverage on the vascular wall and a decreased PC/EC coverage was observed in the spinal cord of SCI rats at 72 hours post-surgery.Compared to the SCI group,BMSC-EXOs treatment reverses these pathological process in SCI-EXO group(P<0.05).Conclusion:BMSC-EXOs can effectively suppress inflammatory factor release and pericytes pyroptosis,maintain BSCB integrity,and thereby ameliorate related pathogenesis after SCI.