SPP1在脊髓损伤小鼠神经病理性痛内源性保护机制中的作用:与VEGF/Akt信号通路的关系

Role of SPP1 in endogenous protective mechanism underlying neuropathic pain in mice with spinal cord injury:relationship with VEGF-AKT signaling pathway

目的评价分泌磷蛋白1(SPP1)在脊髓损伤小鼠神经病理性痛内源性保护机制中的作用及其与血管内皮生长因子(VEGF)/蛋白激酶B(Akt)信号通路的关系。方法清洁级健康雌性昆明小鼠72只,7~8周龄,体重30~35 g,采用随机数字表法分为4组(n=18):假手术组(Sham组)、脊髓损伤致神经病理性痛组(NP组)、脊髓损伤致神经病理性痛+SPP1-siRNA组(NS-siRNA组)和脊髓损伤致神经病理性痛+空载病毒组(NP-EV组)。采用半横断脊髓的方法制备小鼠神经病理性痛模型。NS-siRNA组和NP-EV组鞘内注射7μl AAV-SPP1-siRNA-GFP腺病毒或空载腺病毒AAV-vector-GFP后5 d制备模型。于术后1、2和3周时每组随机取6只小鼠测定机械缩足反应阈(PWMT)和热刺激甩尾潜伏期(TFL),随后处死取同侧损伤部位脊髓组织,采用RT-PCR法检测SPP1 mRNA表达,Western blot法检测SPP1、VEGF、Akt和p-Akt的表达。结果与Sham组比较,NP组、NS-siRNA组和NP-EV组术后1、2和3周时PWMT降低,TFL缩短,脊髓SPP1 mRNA、SPP1、VEGF和p-Akt表达上调(P<0.05);与NP组比较,NS-siRNA组术后1、2和3周时PWMT降低,TFL缩短,脊髓SPP1 mRNA、SPP1、VEGF和p-Akt表达下调(P<0.05);与NS-siRNA组比较,NP-EV组术后1、2和3周时PWMT升高,TFL延长,脊髓SPP1 mRNA、SPP1、VEGF和p-Akt表达上调(P<0.05)。结论SPP1参与了脊髓损伤小鼠神经病理性痛的内源性保护机制,可能与激活脊髓VEGF/Akt信号通路有关。

Objective To evaluate the role of secreted phosphoprotein 1(SPP1)in endogenous protective mechanism underlying neuropathic pain(NP)in mice with spinal cord injury and the relationship with the vascular endothelial growth factor(VEGF)/kinase B(Akt)signaling pathway.Methods Seventy-two clean-grade healthy female Kunming mice,aged 7-8 weeks,weighing 30-35 g,were divided into 4 groups(n=18 each)using a random number table method:sham group(Sham group),NP caused by spinal cord injury group(NP group),NP caused by spinal cord injury+SPP1-siRNA group(NS-siRNA group),and NP caused by spinal cord injury+adeno-associated virus vector group(NP-EV group).A model of NP was established by a semi-transecting of the spinal cord.AAV-SPP1-siRNA-GFP adenovirus and AAV-vector-GFP adenovirus 7μl were intrathecally injected in NS-siRNA group and NP-EV group,respectively,and 5 days later the model was established.At 1,2 and 3 weeks after operation,6 mice in each group were randomly selected to measure paw withdrawal threshold to mechanical stimulation(PWMT)and tail flick latency(TFL)to thermal stimuli.And then the mice were sacrificed and the ipsilateral injured spinal cord tissues were taken for determination of the expression of SPP1 mRNA(by real-time polymerase chain reaction)and expression of SPP1,VEGF,Akt and phosphorylated Akt(p-Akt)(by Western blot).Results Compared with group Sham,PWMT was significantly decreased,TFL was shortened,and the expression of SPP1 mRNA,SPP1,VEGF and p-Akt protein was up-regulated at 1,2 and 3 weeks after operation in NP,NS-siRNA and NP-EV groups(P<0.05).Compared with group NP,PWMT was significantly decreased,TFL was shortened,and the expression of SPP1 mRNA,SPP1,VEGF and p-Akt protein was down-regulated at 1,2 and 3 weeks after operation in group NS-siRNA(P<0.05).Compared with group NS-siRNA,PWMT was significantly increased,TFL was prolonged,and the expression of SPP1 mRNA,SPP1,VEGF and p-Akt protein was up-regulated at 1,2 and 3 weeks after operation in group NS-siRNA(P<0.05).Conclusion SPP1 is involved in the endogenous protective mechanism underlying NP in mice with spinal cord injury,which may be related to the activation of the VEGF/AKT signaling pathway.