| Objective：Traumatic spinal cord injury (SCI) is a devastating medical problem for humanhealth. It is difficult for finding an effective therapy because of its complexpathophysiology. Inflammatory responses and are a major component of secondaryinjury and play a central role in mediating the pathogenesis of acute spinal cord injury.Lots of efforts have been focused on the secondary phase of SCI, which aim toameliorate the secondary injury and to preserve the necessary anatomic substrates forfunctional recovery to take place. Butein, a plant polyphenol, is one of the major activecomponent of the Caragana have been traditionally used for treatment of pain, parasite,and thrombotic disease in China. These beneficial pharmacological effects includeantioxi-dant and anti-inflammatory activities. Recently, butein has been shown to beneuro-protective by the direct inhibition of IKK/NF-κB pathway in vitro. However it isstill no paper about treatment with butein in SCI. In this study, Rats received a subduralinjection with butein post SCI at T12segment. we investigated the effects of butein onIKK/NF-κB pathway in rats after SCI, which incuded: the locomotor function of hindlimb, the degree of tissue edema, the activation of MPO, iNOS and MCP-1, theexpression of caspase-3, and Bax, the amount of apoptotic cells, the expression ofNF-κB and the activation of its inhibitor I-κBα at24hours after injury. Overall, thefindings demonstrated the neuro-protective effects of butein in SCI in vivo and itspossible mechanism. Our results further imply that butein targeting the IKK/NF-κBpathway may be useful in the therapy of SCI. Method1. Moderately severe injury (25g×cm force) using the modified Allen weight-dropmethod was induced in female adult Sprague-Dawley rats following laminectomy atT12.2. 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,50μl/kg), the Butein-treated group (SCI followed by asubdural injection of Butein,0.5mg/kg).3. Rats received a subdural injection of DMSO or Butein at0.5cm below theinjury site at15min after SCI.4.The changes of locomotor, sensory and reflex function of hind limbs after acutespinal cord injury were investigated. A subdural injection of butein was applied in ratsat15min after SCI. Locomotor function of hind limbs of rats was evaluated using theBBB open-field locomotor test on the first, seventh, forteenth, twenty-first dayspost-injury. The sensory and reflex function of hind limbs of rats was evaluated usingthe Combine Behavioral Score on the first, seventh, forteenth, twenty-first dayspost-injury.5. The infiltration of inflammatory cells and the apoptosis in spinal cord at24hrafter SCI were investigated. A subdural injection of Butein was applied in rats at15minafter SCI. The animals were euthanized at24hr after SCI, and10-mm injured spinalcord segments were dissected. The degree of tissue edema was tested by dry/wetweight ratio. The expression of iNOS and Bax in spinal cords was tested by Westernblotting and Immunohistochemistry. MCP-1、Caspase-3was tested by Western blottingin injured spinal cords. MPO activity in injured spinal cords was tested by MPO activitykit. For detection of apoptotic cells, a terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) stain was performed using the OneStep Cell Death Detection Kit-Fluorescein.6. The effect of butein targeting the IKK/NF-κB signal pathway on spinal cordafter SCI was investigated.. A subdural injection of Butein was applied in rats at15minafter SCI. The animals were euthanized at24hr after SCI, and10-mm injured spinalcord segments were dissected. IKKβkinase activity kit was used to test the kinaseactivity in spinal cords. The expression of NF-Κb and phosphorylating I-κBαin spinalcords removing from rats at24hr after SCI was tested by Western blotting.Result:1. Serious hind legs dysfunction was seen both in SCI group and butein group on the first day after SCI with BBB score1.2±0.25and1.1±0.14respectively, significantlydifferent compared with rats in sham group（p<0.01）. The treatment by butein also ledto a significant improvement in locomotor functional recovery at14days after injurycompared with that in SCI group (P <0.05). At14days post-injury, rats in the SCIgroup only showed the return of isolated movements in three joints (hip, knee, andankle) with a mean score of6.6±0.42. In contrast, rats in the butein treatment groupexhibited a return of paw placement and weight-bearing walking with a mean score of11.2±0.54. The sensory and reflex function of hind limbs after spinal cord injury wasevaluated by the combine behavioral score. Serious hind legs dysfunction was seen bothin SCI group and butein group on the first day after SCI with the combine behavioralscore, significantly different compared with rats in sham group（p<0.01）. The combinebehavioral score in butein treatment group equivalent52.7%of the sham group showeda significant improvement in functional recovery at21days after injury compared withthat in SCI group (P <0.05), the combine behavioral score of which was equivalent to23.4%of the sham group.2. The degree of tissue edema, MCP-1, iNOS and MPO activity in SCI group wassignificantly increased at24hr after injury compared with rats in sham group.Treatment with butein significantly reduced the increase. TUNEL-positive cells in theSCI group were significantly increased, and butein treatment greatly reduces thequantity of TUNEL-positive cells in spinal cord tissues. The expression of activatedCaspase-3and Bax in spinal cord tissues obtained from SCI group was significantlyincreased compared with that in sham group, and butein treatment obviously reducedthe expression of activated Caspase-3and Bax in spinal cord tissues tested byImmunohistochemistry and Western blot analysis.3. The IKKβkinase activity in SCI group significantly increased at24hr after SCIwith0.84±0.027U/mg compared with rats in sham group.Treatment with Butein maysignificantly reduce the increase in IKKβkinase activity(P<0.05).The expression ofphosphorylating I-κBαsignificantly increased at24hr after injury compared with thosein sham group(P<0.01), and the increase was significantly reduced by treatment withbutein(P<0.05). At24hours after SCI, little NF-κB immunoreactivity was detected inthe sham surgery group. Rats in SCI group exhibited a significantly increased ratio ofNF-κB P65positive cells compared with that in the sham surgery animals (P <0.05).Butein treatment significantly reduced the expression of NF-κB P65(P <0.05).Conclusion: 1. The findings that Butein significantly ameliorated spinal cord injury-inducedhind dysfunction after acute spinal cord injury demonstrated Butein has therapeuticimplications on spinal cord injury.2.Treatment with butein can attenuate the degree of tissue edema and theinfiltration of inflammatory cells and decrease the amount of apoptotic cells in SCI.Butein maybe disturb the infiltration of neutrophils mediated by MCP-1. Buteinsignificantly suppressed the expression of iNOS, activated caspase-3and Bax andshowed the neuroprotective effect. Butein may play an important role on theanti-inflammatory and anti-apoptotic effects with experimental SCI.3. Treatment with butein may reduce the increase in IKKβkinase activity andinhibit IKK/NF-κB pathway after SCI and imply the molecular basis of its applicationin the treatment of spinal cord injury. |
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