| Background:The spinal cord is the lower central part of the nervous system,which is mainly responsible for the transmission of nerve impulses between the brain and the body.spinal cord injury（SCI）can cause the destruction or interruption of the connectivity of the nervous system,which often leads to motor,sensory,and autonomic nerve dysfunction or loss below the level of spinal cord injury.SCI not only causes serious physical and psychological burden to patients,but also brings heavy burden to their families and society.Therefore,SCI treatment is still one of the most challenging medical problems at present.The main clinical treatment strategies for SCI include surgical treatment and drug therapy.Among them,surgical treatment refers to early spinal cord decompression after injury,which is used to relieve the persistent damage caused by spinal cord compression and restore the stability of spinal structure,but it cannot be treated against the inflammatory reaction.Methylprednisolone is the only recommended drug in clinical SCI treatment guidelines.However,because of its need for early high-dose shock administration and continuous administration,accompanied with serious systemic side effects,its clinical application is controversial.Therefore,it is urgent to develop new strategies for the treatment of SCI in clinic.The difficulty in treatment of SCI may be related to its complex pathological changes.This complex pathological process mainly includes the following four aspects:（1）SCI causes microvascular injury in spinal cord tissue and destroys the blood circulation of the tissue,as well as micro vasospasm and thrombus,which leads to ischemia and microcirculatory disturbance in the injured site.（2）after SCI,the blood-spinal cord barrier could be destroyed to a certain extent,resulting in the recruitment and activation of inflammatory cells（such as macrophages and microglia）in the injured site,and the secretion of a large number of pro-inflammatory factors such as interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）.（3）in addition,reactive oxygen species（reactive oxygen species,ROS）accumulated excessively in the injured site.Excessive ROS destroys the oxidative balance in the spinal cord and eventually leads to DNA oxidative damage,protein oxidation and lipid peroxidation,and leads to cell death or induce apoptosis.（4）the death of necrotic and apoptotic cells can further promote the production of inflammatory factors and ROS,resulting in the loss of neurons and glial cells.The above pathophysiological changes are interrelated and influence each other,which makes the spinal cord tissue trapped in the circulation of multi-level secondary injury,resulting in the permanent loss of spinal cord nerve function.In recent years,a series of new therapeutic strategies for SCI have been proposed,including nano-drug therapy,cell transplantation,tissue engineering and so on.With the development of nanomaterials,more and more applications in the field of biomedicine,and show a variety of functions and advantages.Therefore,the use of nano-materials to treat SCI is the current research hotspot.The preparation of nano-drugs by physical or chemical combination of drugs and/or carriers can not only improve the bioavailability and plasma half-life of therapeutic drugs,but also reduce systemic side effects.Therefore,nano-drugs have become a promising strategy for the treatment of SCI.Objective:EGCG-Se NPs（epigallocatechin gallate-selenium nanoparticles）with ROS scavenging properties was designed and constructed.At the same time,the biocompatibility,antioxidation,anti-inflammatory effect and neuroprotective properties of EGCG-Se NPs in vivo and in vitro were discussed,and its mechanism was preliminarily explored to provide new strategies and ideas for the treatment of SCIMethods:EGCG-Se NPs was prepared by redox method.The size and morphology of the nanoparticles were characterized by dynamic light scattering（DLS）,transmission electron microscopy（TEM）and Malvern particle size analyzer.The structure and composition of EGCG-Se NPs were characterized by Fourier transform infrared（FTIR）,inductively coupled plasma emission spectrometer（ICP-MS）and X-ray surface photoelectron energy（XPS）.At the same time,the antioxidant activity of EGCG-Se NPs was detected by DPPH.In vitro experiment:firstly,the effects of different concentrations of EGCG-Se NPs(6.25,12.5,25,50,100μg m L-1)on the biocompatibility of PC12 cells were verified by MTT method.At the same time,the protective effect of EGCG-Se NPs on rat adrenal pheochromocytoma cells（rat pheochromocytoma cells,PC12）in 500μM H2O2 simulated oxidative stress environment was tested by MTT method.The protective effect of EGCG-Se NPs on oxidative damage of rat adrenal pheochromocytoma cells was further confirmed by living/dead cell staining.DCF fluorescence staining was used to understand that EGCG-Se NPs protects cells by reducing intracellular ROS.In addition,the effect of EGCG-Se NPs on the expression of proinflammatory factorα（IL-6,TNF-α）in mouse microglia（BV2 mouse microglia,BV2）stimulated by lipopolysaccharide（lipopolysaccharide,LPS）was investigated by Elisa method.In vivo experiment:the model of spinal cord contusion in rats was made by using standard spinal cord percussion device.After successful establishment of the model,normal saline,30mg kg-1 methylprednisolone sodium succinate（MP）,9.5 mg kg-1EGCG,10 mg kg-1EGCG-Se NPs and 5mg kg-1EGCG-Se NPs 1ml were injected through the tail vein of syringe to systematically study the therapeutic effect and mechanism of EGCG-Se NPs on rat SCI.One day after the establishment of the model,cardiac perfusion samples were taken,and the effects of EGCG-Se NPs on anti-inflammation and anti-apoptosis in vivo were evaluated by immunofluorescence staining（Iba1&CD68,SOD,caspase-3）.The motor function of the hindlimb of the remaining SD rats was continuously evaluated by BBB score at 1 day after modeling,and the recovery time of spontaneous micturition was recorded at the same time.Eight weeks after the establishment of the model,the samples were collected after cardiac perfusion,and the therapeutic effects of EGCG-Se NPs on acute SCI and related bladder muscle changes were systematically evaluated by gastrocnemius muscle,bladder Hemore and Masson staining,spinal cord image,spinal cord Hype and LFB staining and spinal cord immunofluorescence staining（GFAP&Iba1&CD68,Neu N&NF200,GPX1,SOD）.Results:EGCG-Se NPs with good solubility,uniform size and radius of 91.3±35.7nm was prepared,in which the content of Se was about 4.7%.The shape of the nanoparticles is round under electron microscope.The results of in vitro experiments showed that EGCG-Se NPs had no obvious toxicity to PC12 cells at the concentration of 100μg·m L-1,and the DPPH scavenging efficiency of EGCG-Se NPs at the concentrations of 1.25μg·m L-1,2.5μg·m L-1,5μg·m L-1,10μg·m L-1and 20μg·m L-1 were 11%,18%,36%,71%and 91%,respectively.Under 500μM H2O2 simulated oxidative stress,EGCG-Se NPs effectively increased the survival rate of PC12 cells by reducing intracellular ROS content,and inhibited the production of inflammatory factors TNF-αand IL-6 by BV2 cells stimulated by LPS.The in vivo experiment showed that compared with the normal saline group,in the spinal cord injury model of EGCG-Se group,syringomyelia was significantly reduced,nerve demyelination was inhibited,neurons were protected,glial scar formation and inflammatory cell infiltration were reduced,bladder intimal thickening,ectopic growth and bladder fibrosis were significantly alleviated.At the same time,the expression of pro-apoptotic protein caspase-3 decreased and the expression of antioxidant enzymes increased.BBB score showed that the motor function of hindlimb was significantly improved in EGCG-Se NPs group compared with Saline group and EGCG-Se NPs group.Similarly,by comparing the recovery time of spontaneous micturition,the bladder micturition function of EGCG-Se NPs group was better than that of Saline group and EGCG groupConclusion:A kind of nanoparticles（EGCG-Se NPs）with both scavenging ROS and anti-inflammation was designed and constructed to protect the nerve after SCI.EGCG-Se NPs can effectively promote the recovery of hindlimb motor function and micturition function in rats with spinal cord injury.Moreover,it can effectively remove ROS from the injured spinal cord tissue at the lesion site of SCI,and play a significant anti-inflammatory and neuroprotective function.In summary,the prepared EGCG-Se NPs can effectively improve the neurological function after SCI,which is expected to provide a new idea for the treatment of SCI. |
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