The Therapy Of Targeting IKK/NF-κB Pathway On Spinal Cord Injury And Its Related Mechanisms

Objective：Spinal cord injury (SCI) is a highly debilitating pathology, and can victimize activeindividuals at any age. It is difficult for finding an effective therapy because of itscomplex pathophysiology. Inflammatory responses are a major component of secondaryinjury and play a central role in mediating the pathogenesis of acute spinal cord injury.The nuclear factor-κB (NF-κB) family of transcription factors is a principal player in theregulation of inflammatory gene expression and it regulates many cytokines mediatingthe inflammatory responses in central nervous system (CNS) injury. Aberrant activationof NF-κB promotes neuronal death in SCI. Inhibition of NF-κB may reduce these genesexpression, attenuate inflammation and improve functional recovery after spinal cordinjury. Inhibitor of NF-κB (I-κB) kinases complex (IKKs) is a central component of thesignaling cascade that controls NF-κB-dependent inflammatory gene transcription.IKKβ, the main catalytic subunit of IKKs, can activate the NF-κB pathway byphosphorylating I-κB and the NF-κB subunit P65/RelA at Ser536, which made thisenzyme an attractive target for therapeutic intervention. BMS-345541is a selectiveinhibitor of IKKβ. Previous study has been found that BMS-345541may ameliorateischemic brain damage by inhibiting IKKβactivity and NF-κB activation, reducingexpression of inflammatory gene and apoptosis of neuron. However it is still poorlyunderstood about treatment with BMS-345541in SCI. The aim of this study was toexamine whether BMS-345541modulate the IKK/NF-κB signal pathway which wasactivated in SCI. We also sought to elucidate the changes of NF-κB expression andIKKβkinase activity as well as locomotor function of hind limb in rats with acute spinalcord injury. We also want to know the neuroprotective effects of BMS-345541and possible mechanisms by which BMS-345541acts against inflammatory cells infiltrationand apoptosis in rat SCI models.Method:1. Moderately severe injury (25g×cm force) using the modified weight-dropmethod was induced in female Sprague-Dawley rats following laminectomy at T12.2. Rats were randomly assigned to one of the following three groups beforeoperation: the sham group (laminectomy only without SCI), the SCI group (SCI with asubdural injection of DMSO), the BMS-treated group (SCI followed by a subduralinjection of BMS-345541,0.5mg/kg).3. Rats received a subdural injection at0.5cm below the injury site before or afterSCI.4. IKKβkinase activity and the expression of phosphorylating I-κBαafter SCI inrats were investigated. A subdural injection of BMS-345541was used at10minutesbefore injury. The animals were euthanized at0.5hr、4hr、12hr、24hr and72hr after SCI,and10-mm injured spinal cord segments were dissected. IKKβkinase activity kit wasused to test the kinase activity in spinal cords. The expression of phosphorylating I-κBαin spinal cords removing from rats at12hr after SCI was tested by Western blotting.5. The changes of NF-κB expression in spinal cord at24hr after SCI and locomotorfunction of hind limbs in two weeks after acute spinal cord injury were investigated. Asubdural injection of BMS-345541was applied in rats at15min after SCI. The animalswere euthanized at24hr after SCI, and10-mm injured spinal cord segments weredissected. The expression of NF-κB in spinal cords was tested by Western blotting andImmunohistochemistry. Locomotor function of hind limbs was evaluated using the BBBopen-field locomotor test in seven rats of each group at1-14days post-injury.6. The infiltration of inflammatory cells and the apoptosis in spinal cord at24hrafter SCI were investigated. A subdural injection of BMS-345541was applied in rats at15min after SCI. The animals were euthanized at24hr after SCI, and10-mm injuredspinal cord segments were dissected. The expression of caspase-3, Bcl-2and Bax inspinal cords was tested by Western blotting and Immunohistochemistry. ICAM-1wastested by Western blotting in injured spinal cords. MPO activity in injured spinal cordswas tested by MPO activity kit. For detection of apoptotic cells, a terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) stainwas performed using the One Step Cell Death Detection Kit-Fluorescein. Result:1. The IKKβkinase activity increased at0.5hr after SCI, and the increase becamesignificantly at4hr with1.05±0.043U/mg, and peaked at12hr after SCI with1.47±0.037U/mg. It is0.71±0.027U/mg at24hr after SCI. And the increase wassignificantly reduced by treatment with BMS-345541(P<0.05) at4hr and12hr after SCI.The expression of phosphorylating I-κBαsignificantly increased at12hr. after injurycompared with those in sham group(P<0.01), and the increase was significantly reducedby treatment with BMS-345541(P<0.05).2. At24hours after SCI, little NF-κB immunoreactivity was detected in the shamsurgery group, and the ratio of NF-κB P65positive cells was1.02±0.32%. Rats in SCIgroup exhibited a significantly increased ratio of NF-κB P65positive cells comparedwith that in the sham surgery animals (P <0.05), accounting for34.65±0.23%.BMS-345541treatment significantly reduced the expression of NF-κB P65(P <0.05),and the ratio of positive cells was10.87±0.56%. This treatment also led to a significantimprovement in locomotor functional recovery at14days after injury compared withthat in SCI group (P <0.05). At14days post-injury, rats in the SCI group only showedthe return of isolated movements in three joints (hip, knee, and ankle) with a mean scoreof6.8±0.4. In contrast, rats in the BMS-345541treatment group exhibited a return ofpaw placement, with a mean score of10.4±0.5.3. ICAM-1and MPO activity in SCI group was significantly increased at24hr.after injury compared with rats in sham group. Treatment with BMS-345541significantly reduced the increase. TUNEL-positive cells in the SCI group weresignificantly increased, and BMS-345541treatment greatly reduces the quantity ofTUNEL-positive cells in spinal cord tissues. The expression of activated Caspase-3inspinal cord tissues obtained from SCI group was significantly increased compared withthat in sham group, and BMS-345541treatment obviously reduced the expression ofactivated Caspase-3in spinal cord tissues tested by Immunohistochemistry and Westernblot analysis. BMS-345541inhibited the expression of Bax but not Bcl-2in spinal cordtissues after SCI.Conclusion:1. The IKKβkinase activity after SCI significantly increased at4hr and peaked at12hr. It means that the therapeutic window of inhibition of IKKβkinase activity is up to4hr after the onset of SCI. Treatment with BMS-345541may reduce the increase inIKKβkinase activity after SCI and imply its potential effect on the therapy of SCI. 2. The findings demonstrated that BMS-345541significantly ameliorated spinalcord injury-induced hind dysfunction by inhibiting the expression of NF-κB after acutespinal cord injury3. Treatment with BMS-345541can attenuate the infiltration of inflammatory cellsand inhibit the apoptosis in SCI. BMS-345541maybe disturb the infiltration ofneutrophils mediated by ICAM-1through the IKK/NF-κB pathway. BMS-345541significantly suppressed the expression of activated caspase-3and Bax and showed theneuroprotective effect. BMS-345541may play an important role on theanti-inflammatory and anti-apoptotic effects with experimental SCI. Our results implythe IKK inhibitors may be useful in the therapy of spinal cord injury.