丙磺舒对大鼠急性脊髓损伤后NLRP1炎性小体活化的影响

Effect of probenecid on activation of NLRP1 inflammasome in acute spinal cord injury in rat

[目的]研究丙磺舒(probenecid, PROB)对大鼠急性脊髓损伤后NLRP1炎性小体活化的影响。[方法]成年雄性SD大鼠90只,随机分为Sham组(Sham组)、脊髓SCI组(SCI组)和丙磺舒治疗组(PROB组),参照Rivlin及Tator方法在SCI组和PROB组制作急性脊髓损伤模型。采用BBB评分量表评估大鼠神经功能情况,HE染色观察组织的病理形态变化,Western-blot法和免疫组化法检测Caspase-1、XIAP和IL-1β蛋白表达水平。[结果]Sham组的BBB评分各时间点显著高于SCI组和PROB组;损伤后3、7、14和21 d,PROB组的BBB评分显著高于SCI组,差异均具有统计学意义(P<0.05)。HE染色显示:PROB组与SCI组比较,中性粒细胞浸润、组织水肿减轻,神经元变性坏死减少,脊髓空洞面积减少。Western-blot检测显示:与Sham组比较,SCI组各个时间点的Caspase-1蛋白表达水平明显升高,并且呈现一定时间依赖性,损伤72h表达最高,而PROB组的Caspase-1蛋白表达显著低于SCI组,差异均具有统计学意义(P<0.05);Sham组各个时间点的XIAP蛋白表达水平显著高于SCI组和PROB组,并且SCI组各个时间点的XIAP蛋白表达呈现一定时间依赖性降低,损伤72 h最低,而PROB组的XIAP蛋白表达显著高于SCI组,差异均具有统计学意义(P<0.05)。免疫组化检测显示:与Sham组比较,SCI组的Caspase-1和IL-1β蛋白表达水平明显升高;PROB组显著低于SCI组,差异均具有统计学意义(P<0.05)。[结论]PROB可能通过抑制XIAP的裂解和Caspase-1的活化,进而抑制NLRP1炎性小体的活化,减少脊髓继发性损伤。

[Objective] To explore the effect of probenecid(PROB) on activation of NLRP1 inflammasome in acute spinal cord injury in rat. [Methods] Ninety male adult Sprague-Dawley(SD) rats were randomly divided into sham-operated group(the sham group), spinal cord injury group(the SCI group) and PROB treated group(the PROB group), with the spinal cord injury created by the clip compression method described by Rivlin and Tator in the SCI and PROB group. The locomotor function of hind limbs was evaluated using Basso-Beattle-Bresnahan(BBB) score, and the histopathological changes of spinal cords were observed by HE staining, while the expression levels of Caspase-1、XIAP and IL-1β were detected by western-blot and immunochemistry staining. [Results] The sham group was marked significantly higher BBB scores than the SCI and PROB groups at every time point after operation(P<0.05), whereas the PROB group got significantly higher BBB scores than the SCI group at 3,7, 14 and 21 days postoperatively(P<0.05). In term of histopathological changes, the PROB group revealed significantly alleviated neutrophil infiltration, lightened edema and less neuron necrosis or degeneration, associated with significantly less vacuolar change compared with the SCI group. Regarding to Western-blot assays, the SCI group had significantly elevated Caspase-1 in a time-dependent manner with a peak at 72 hours after injury compared with the sham group(P<0.05), however, the PROB group got significantly less Caspase-1 than the SCI group(P<0.05). The sham group kept significantly higher XIAP level than the other two groups(P<0.05), the SCI group proved a progressive decline of XIAP to the lowest level at 72 hours,whereas the PROB group had significantly higher level of XIAP than the SCI group at each time point(P<0.05). The immunohistochemical assays demonstrated that the SCI group achieved significantly higher protein expression of Caspase-1 and IL-1β than the sham group and PROB group(P<0.05). [Conclusion] The PROB does reduce the secondary spinal cord injury by inhibiting the activation of NLRP1 inflammasome, which is related to the cleavage of XIAP and the activation of Caspase-1.