Neuroprotective Effects And Mechanisms Of Natrium Benzoate On Traumatic Spinal Cord Injury By Regulating DJ-1

BackgroundsTraumatic spinal cord injury(t-SCI)is one of the most serious injuries worldwide,which has a high mortality and disability rate and seriously threatens human life and health.The pathogenesis of t-SCI is complex,involving a series of pathological,physiological and biochemical changes.Generally speaking,it can be divided into primary injury and secondary injury.Primary injury refers to the acute mechanical damage of spinal cord nerve cells,processes and blood vessels caused by external forces at the time of injury,resulting in spinal cord compression,contusion and even severance,resulting in the destruction of blood-spinal cord barrier(BSCB),spinal cord hemorrhage,ischemia and cell necrosis.Secondary injury is a series of molecular,cellular and biochemical reactions caused by primary injury,which continues to progress throughout the t-SCI process.These include hemorrhagic injury,ischemic injury,oxidative stress injury,excitatory toxicity of glutamate,inflammation,apoptosis,axonal demyelination and degeneration,extracellular matrix(ECM)remodeling,glial hyperplasia,and so forth.These mechanisms are synthetical and mutually reinforcing,and cause and effect each other,further aggravating spinal cord injury.Among all these factors,oxidative stress is one of the key pathogenesis of t-SCI.During oxidative stress,excessive production of reactive oxygen species(ROS)leads to a series of harmful effects,including lipid peroxidation,protein oxidation and DNA damage.It also activates several signaling pathways to induce cell apoptosis.Neuronal apoptosis is an important pathological change after t-SCI,which leads to neurological dysfunction.Inhibition of neuronal apoptosis can significantly improve neurological function after t-SCI.DJ-1 is a highly conserved and widely expressed protein,which is expressed in many tissues of the whole body.Its roles involve many aspects of cell life activities,including oxidative stress,mitochondrial function regulation,transcriptional regulation,protein-RNA interaction,molecular chaperone activity and so forth.The most important one is to resist oxidative stress injury.Studies have shown that DJ-1 plays an important neuroprotective role in Parkinson's disease(PD),Alzheimer's disease(AD)and other neurodegenerative diseases and ischemic stroke.Its neuroprotective effects may depend on its anti-oxidative stress and anti-apoptotic ability.Natrium benzoate(NaB)is a sodium salt of aromatic carboxylic acid,which has a wide range of pharmacological effects.Many studies have shown that NaB plays a neuroprotective role by increasing the expression of DJ-1.However,the underlying mechanism remains unclear.Whether NaB can play a neuroprotective role by increasing DJ-1 in t-SCI and its corresponding mechanisms have not been studied yet.Therefore,the aim of this study was to investigate the neuroprotective effects and mechanisms of NaB on t-SCI by regulating DJ-1,and to provide potential drug and possible target for the treatment of t-SCI.MethodsPart 1Experiment 1:SD rats were divided into 7 groups:sham group,t-SCI 3h group,t-SCI 6h group,t-SCI 12h group,t-SCI 24h group,t-SCI 48h group,t-SCI 72h group.The expression of DJ-1 was detected by western blot(WB),the co-staining of DJ-1 and NeuN was detected by immunofluorescence(IF),and the microstructure of spinal cord were observed by hematoxylin and eosin(HE)staining.Experiment 2:SD rats were divided into six groups:sham group,t-SCI+vehicle group,t-SCI+scramble siRNA group,t-SCI+DJ-1 siRNA group,t-SCI+NaB group.t-SCI+NaB+DJ-1 siRNA group.The expressions of DJ-1,Nrf-2,HO-1,SOD2,p-p38 MAPK,Bcl-2,Bax,CC-3,MMP-9,Claudin-5,Occludin and ZO-1 were detected by WB.Meanwhile,ROS levels were detected.Spinal cord water content(SCWC)was measured by dry-wet weight method,and BSCB destruction was detected by evans blue(EB)extravasation method.Experiment 3:SD rats were divided into three groups:sham group,t-SCI+vehicle group,t-SCI+NaB group.The rats in t-SCI+vehicle group and t-SCI+NaB group received treatment for 7 consecutive days after operation.Basso,Beattie,and Bresnahan(BBB)and inclined plate test(IPT)scores were used to evaluate the locomotor function on day 1,3,7,14,21 and 28 after operation.Part 2Experiment 1:The expression of p-Akt was detected by WB using the samples and grouping of the experiment 1 in the first part.Experiment 2:The expression of p-Akt was detected by WB using the samples and grouping of the experiment 2 in the first part.Experiment 3:SD rats were divided into 5 groups:sham group,t-SCI+vehicle group,t-SCI+NaB group,t-SCI+MK2206 group,t-SCI+NaB+MK2206 group.The expressions of DJ-1,p-Akt,Nrf-2,HO-1,SOD2,p-p38 MAPK,Bcl-2,Bax and CC-3 were detected by WB.Meanwhile,ROS levels were detected.The co-staining of CC-3,TUNEL and NeuN was detected by IF.The ultrastructure of neurons was observed by transmission electron microscopy(TEM).Experiment 4:Human neuroblastoma cells(SH-SY5Y)were divided into five groups:control group,injury+vehicle group,injury+NaB group,injury+MK2206 group,injury+NaB+MK2206 group.Cell viability was detected by MTT assay and TB staining.The expressions of DJ-1,p-Akt,Nrf-2,HO-1,SOD2,p-p38 MAPK,Bcl-2,Bax and CC-3 were detected by WB.Meanwhile,ROS levels were detected.ResultsPart 1After t-SCI,the level of DJ-1 increased significantly and decreased gradually after 24 hours reaching the peak.At 24 hours after t-SCI,the proportion of DJ-1 positive neurons increased significantly,the levels of DJ-1,Nrf-2,HO-1,SOD2,ROS,p-p38 MAPK and CC-3 increased significantly,and Bcl-2/Bax ratio decreased significantly.DJ-1 siRNA significantly decreased the levels of DJ-1,Nrf-2,HO-1 and SOD2,increased levels of ROS,p-p38 MAPK and CC-3,and decreased Bcl-2/Bax ratio.NaB treatment significantly increased the levels of DJ-1,Nrf-2,HO-1 and SOD2,decreased the levels of ROS,p-p38 MAPK and CC-3,and increased Bcl-2/Bax ratio,and these effects can be reversed by DJ-1 siRNA.At 24 hours after t-SCI,the SCWC and EB extravasation increased significantly,the level of MMP-9 increased significantly,and the levels of Claudin-5,Occludin and ZO-1 decreased significantly.NaB treatment significantly improved the above situations.The BBB and IPT scores decreased significantly after t-SCI.NaB treatment significantly increased the above scores on day 21 and 28 after t-SCI.Part 2After t-SCI,the level of p-Akt increased significantly and decreased gradually after 24 hours reaching the peak.At 24 hours after t-SCI,DJ-1 siRNA significantly decreased the level of p-Akt,while NaB treatment significantly increased the level of p-Akt,and this effect can be reversed by DJ-1 siRNA.At 24 hours after t-SCI,the proportion of CC-3 and TUNEL positive neurons increased significantly,TEM showed apoptotic changes in the ultrastructure of neurons,and the proportion of mitochondrial vacuolation increased significantly.NaB treatment significantly improved the above situations,and these effects can be reversed by MK2206.In vitro experiments showed that the cell viability decreased significantly after injury.NaB treatment significantly improved the above situations,significently increased the levels of DJ-1,p-Akt,Nrf-2,HO-1 and SOD2,decreased the levels of ROS,p-p38 MAPK and CC-3,and increased Bcl-2/Bax ratio,and these effects can be reversed by MK2206.ConclusionsPart 1NaB treatment can increase the levels of DJ-1 and antioxidant proteins,reduce ROS and ROS-induced neuronal apoptosis,BSCB damage,spinal cord edema,and improve long-term locomotor function.Part 2DJ-1 mediates the downstream antioxidant stress effects by promoting Akt phosphorylation,thereby reducing oxidative stress-induced neuronal apoptosis.