Neuroprotective Effects Of Sivelestat Sodium On Acute Spinal Cord Injury

Background: ASCI is trauma induced central nervous system injury with a high incidence of morbidity.It is one of the most devastating damage to the CNS.ASCI always cause disruption to the structure and function of the spinal cord,sometimes even paraplegia.ASCIs were often caused by accident,sports injury,natural disaster,falls and force trauma.And the incidence of ASCI has always been very high with no sign of reduction.Spinal cord injury is more common in young males aged 16-30,of which thoracolumbar injuries are most common.All kinds of initial physical impact,such as accidents,sports,natural disasters,drop and violence,which is called as a primary cause of ASCI and is often unpredictable.Following the primary injury,the secondary injury causes the death of additional neurons.Several pathways can activate the secondary damages,especially inflammation and apoptosis.So far,treatment for ASCI is mainly early impulsive therapyof methylprednisolone.However,impulsive methylprednisolone therapy can significantly increase the incidence of complications like gastrointestinal bleeding and hyperglycemia.More importantly,the scheme does not reduce the 2-week mortality of acute injury for ASCI.Therefore,it is urgent to find effective new drugs for the early treatment of spinal cord injury.After ASCI,a necrotic cavity was formed in the center of the injured tissue at first week after the primary spinal cord injury.At the same time,severe secondary injury was formed,such as the blood-brain barrier damage with peripheral inflammatory cell infiltration,a large amount of neutrophils migrate and infiltrate into the gray matter of the spinal cord,excessive activation of the neutrophils,which release a large amount of reactive oxidative species and elastase cytokines.These changes then cause progressive necrosis of the injured tissue and form the spinal cavity.Secondary injury is a major cause of neuron apoptosis,which makes secondary injury an important target for the treatment of ASCI.Recent research showed that GAPDH/Sial1 are very important proteins in the induction of cell apoptosis.The nucleus transportation of GAPDH/Sial1 can trigger the apoptotic process of neuron.GAPDH-Siah1 are key signal molecules for neuron apoptosis after CNS injury.When subjected to severe trauma,infection or outside adverse stimulation a small amount of neutrophil elastase(NE)negatively regulate the inflammatory process.However,excessive amount of NE not only degrades tissue elastic protein and breaks intercellular tight junctions,but also activates PMN and release more NE that forms a positive disastrous feedback.Recent research showed that application of neutrophil elastase inhibitor(NEI)is effective in reducing excessive inflammatory response in the acute lung injury(ALI).Sivelestat sodium is an NEI and it is the only drug currently available in clinical practice for neutrophil function inhibition.There are evidence showed that sivelestat sodium is effective in reducing neutrophil infiltration in ischemic reperfusion injuries in the liver,heart and spinal cord.It inhibits NE production,reduces inflammatory cytokines and improves dysfunction in metabolic disorders.The aim of the present study is to investigate the neuroprotective effects of Sivelestat sodium on secondary damage of traumatic SCI in a rat model,with focus on inflammatory responses after ASCI and its anti-apoptosis mechanism.Part 1.Neuroprotective Effect of Sivelestat Sodium on Acute Spinal Cord Injury Objective To investigate the effect of repeated Sivelestat sodium on acute spinal cord injury in rats.Methods The rat spinal cord model of blunt injury was used.A total of 50 male Sprague-Dawley rats(300-350g)were randomly divided into five groups :(1)Sham group: Vertebral lamina and spinous process from T7 to T9 were removed.(2)Control group: After anesthetized with chloral hydrate,vertebral lamina and spinous process from T7 to T9 were removed and the spinal cord injury was established with modified Allen?s bump equipment.(3)1.6mg/kg group: Sivelestat sodium was given to the rats intraperitoneally immediately after injury for 1.6mg/kg.(4)4.8mg/kg group: Sivelestat sodium was given to the rats immediately after injury for 4.8mg/kg.5.10mg/kg group: Sivelestat sodium was given to the rats immediately after injury for 10mg/kg.To evaluate function of the rear limbs,Basso Beattie & Bresnahan(BBB)rating scale were evaluated according to the criteria as reported.Neurological function was assessed at 1d,3d,7d,14 d,and 21 d after injury by an observer who was blind to the grouping.A histopathological evaluation for injured neurons was assessed over HE staining,and detection of apoptotic neurons the in rat spinal cords were calculated after TUNEL staining at 24 h after reperfusion.Results(1)Neurologic outcome: There were no significant difference in neurologic outcomes among groups on 1d and 3d after injury.But all Sivelestat sodium treatment groups showed better BBB scales compared to control group on 7d,14 d,and 21 d after injury(P < 0.05 vs.control,respectively).BBB score in 4.8mg/kg and 10mg/kg groups were higher than that in 1.6mg/kg group(P < 0.01)with no difference between 4.8mg/kg group and 10mg/kg group.(2)Histopathological evaluation: Lots of neuronal damage was observed in animals in control group.Cavities were formed in the grey matter spinal cord and nucleus condensed;only little normal neurons were observed.The injury was significanly decreased in all Sivelestat sodium treated groups compared to that in control group,of which 4.8mg/kg and 10mg/kg groups showed more survival neurons than that of 1.6mg/kg group.(3)TUNEL test show a lot of apoptotic motor neurons in the anterior horn of the spinal cord.Sivelestat sodium treatment significantly reduced TUNEL positive neurons in the injured spinal cord(P < 0.05),of which 4.8mg/kg and 10mg/kg showed more potent anti-apoptotic effect compared to 1.6mg/kg group.Conclusion Sivelestat sodium treatment could inhibit apoptosis after spinal cord injury improves motor function after acute spinal cord injury and there is an obvious dose-effect response of Sivelestat sodium treatment.Part 2.Mechanism underlying the neuroprotective effect of repeated Sivelestat sodium treatment on acute spinal cord injury Experiment 1.The effect of Sivelestat sodium on inflammation cytokines after acute spinal cord sinjury.Objective To investigate the effect of Sivelestat sodium repeated on acute inflammation after spinal cord injury.Methods Twenty-four male Sprague-Dawley rats were randomly divided into five groups:(1)Control group: ASCI model goup.(2)1.6mg/kg group: Sivelestat sodium was given to the rats intraperitoneally immediately after injury for 1.6mg/kg.(3)4.8mg/kg group: Sivelestat sodium was given to the rats intraperitoneally immediately after injury for 4.8mg/kg.Spinal cord of T7 to T9 was removed at 24 h after injury.IL-1?,TNF-?,NF-?B and myeloperoxidase(MPO)activity were tested using ELISA.Results IL-1?,TNF-?,NF-?B expressions and MPO activity were significantly lesser in sivelestat sodium treated groups(P < 0.05 vs.control group).And 4.8mg/kg sivelestat sodium is more effective in reducing these cytokines compared to 1.6mg/kg group..Conclusion Sivelestat sodium treatment reduced IL-1??TNF-?,NF-kb expression and MPO activity after acute spinal cord injury.Experiment 2.The expression of GAPDH and Siah1 in the injured spinal cord after acute spinal cord injury.Objective To investigate the Expression of GAPDH and Siah1 in the nucleus of cells in the injured spinal cord segments after acute spinal cord injury.Methods Fifty male Sprague-Dawley rats were randomly divided into 2 groups(1)Sham group.(2)Control group.Procedures were the same in Part 1,experiment 1.Spinal cord of T7 to T9 were removed at 2h,4h,12 h,24h and 72 h after injury(n=5 each).Expression of GAPDH and Siah1 in the cell nucleus of injured spinal cord was tested using WB.Results GAPDH and Siah1 expression were significantly higher in control group compared to that in sham group(P < 0.05)at each time point respectively.And the peak of GAPDH and Siah1 appeared at 24 h after injury.Conclusion Expression of GAPDH and Siah1 in the cell nucleus in the injured spinal cord was elevated after acute spinal cord injury.Experiment 3.The effect of Sivelestat sodium on the Expression of GAPDH and Siah1 in injured spinal cord segments.Objective To investigate the Effect of sivelestat sodium on the expression of GAPDH and Siah1 in the injured spinal cord segments after acute spinal cord injury.Methods Effect of Sivelestat sodium on GAPDH and Siah 1 nuclear protein expression.Twenty SD rats were randomly divided into 4 groups:(1)Sham group.(2)Control group.(3)1.6mg/kg sivelestat sodium treatment group.(4)4.8mg/kg sivelestat sodium treatment group.Procedures were the same in Part 1,experiment 1.At 24 h after acute spinal cord injury,spinal cords were removed for WB test of nuclear GAPDH and Siah 1 expression.Results(1)m RNA expression of GAPDH and Siah1 are elevated after spinal cord injury(P<0.05,control group vs.sham group).And 4.8mg/kg sivelestat sodium treatment significantly inhibited this elevation(P<0.05,4.8mg/kg group vs.control group).(2)Nuclear protein expression of GAPDH and Siah1 are elevated after spinal cord injury(P<0.05,control group vs.sham group).And sivelestat sodium treatment significantly inhibited this elevation(P<0.05,for both 1.68mg/kg group vs.control group and 4.8mg/kg group vs.control group).Between the two dosages,protein expression of GAPDH and Siah1 in cell nucleus was lower in 4.8mg/kg group compared to 1.6mg/kg group.Conclusions Sivelestat sodium treatment significantly reduced both m RNA and nuclear protein expression of GAPDH and Siah1 in the injured spinal cord after acute spinal cord injurSummary 1.Early treatment of acute spinal cord injury with sivelestat sodium improved the lower limb motor function,inhibited neuronal apoptosis,promoted cell survival after acute spinal cord injury in rats.The neuroprotective effect of sivelestat sodium is dose dependent,which proved the neuroprotective effect of sivelestat sodium against acute spinal cord injury.2.Expression o f Inflammatory cytokines like IL-1??TNF-?,NF-?B were elevated in the injured spinal cord after acute spinal cord injury.Sivelestat sodium treatment reduced the elevation of cytokines expression,mainly because of the inhibitory effect of sivelestat sodium against neutrophil function after acute spinal cord injury.Anti-apoptosis effect was the key mechanism of sivelestat sodium,and inhibiting apoptosis sequence of GAPDH/Siah1 play a key role during the treatment.3.After acute spinal cord injury,GAPDH and Siah1 combination was enhanced in the cell nucleus of the injured spinal cord tissue,which in turn induced cell apoptosis.Immediate application of sivelestat sodium significantly reduced expression of GAPDH and Siah1,indicated that sivelestat sodium provided neuroprotection by inhibiting cell apoptotic related protein-GAPDH and Siah1-expression,thus reduced cell apoptosis and improved neurological function.