腺苷酸活化蛋白激酶对大鼠脊髓缺血再灌注损伤的影响及可能机制

Effect of adenylate-activated protein kinase on spinal cord ischemia-reperfusion injury in rats and its possible mechanism

目的探讨腺苷酸活化蛋白激酶(AMPK)对大鼠脊髓缺血再灌注损伤的影响及可能机制。方法将48只健康雄性SD大鼠随机分为假手术组、模型组、生理盐水组和AMPK抑制剂组,每组12只。模型组、生理盐水组和AMPK抑制剂组制备大鼠脊髓缺血再灌注损伤模型,AMPK抑制剂组大鼠建模前腹腔注射Compound C 20 mg/kg,生理盐水组建模前给予等量生理盐水。于建模后6、12、24和48 h用Basso、Beattie、Bresnahan(BBB)评分法评估大鼠神经运动功能,末次评估完成后处死大鼠并取脊髓组织标本,用HE染色法检测大鼠脊髓组织病理变化,TUNEL法检测大鼠脊髓组织细胞凋亡情况,蛋白质印迹法检测大鼠脊髓组织中AMPK、p-AMPK、NF-κB、IκBα和p-IκBα蛋白表达,ELISA法检测大鼠脊髓组织中白细胞介素-1β(IL-1β)、IL-6和肿瘤坏死因子-α(TNF-α)浓度。结果建模后6、12、24和48 h,模型组、生理盐水组和AMPK抑制剂组BBB评分均低于假手术组,而AMPK抑制剂组BBB评分高于模型组和生理盐水组,差异均有统计学意义(P <0.05)。假手术组脊髓组织结构清晰完整,未见神经元凋亡;模型组和生理盐水组大鼠脊髓组织出现神经元变性和坏死、炎性细胞浸润,可见大量神经元凋亡;AMPK抑制剂组大鼠脊髓组织病理改变减轻,神经元凋亡减少。模型组、生理盐水组和AMPK抑制剂组大鼠脊髓组织中p-AMPK、NF-κB和p-IκBα蛋白相对表达量均高于假手术组,AMPK和IκBα蛋白相对表达量均低于假手术组,差异均有统计学意义(P <0.05);AMPK抑制剂组p-AMPK、NF-κB和p-IκBα蛋白相对表达量与模型组和生理盐水组相比均降低,差异均有统计学意义(P <0.05),AMPK和IκBα蛋白相对表达量均升高,差异均有统计学意义(P <0.05)。模型组、生理盐水组和AMPK抑制剂组大鼠脊髓组织中IL-1β、IL-6和TNF-α浓度均高于假手术组,AMPK抑制剂组大鼠脊髓组织中IL-1β、IL-6和TNF-α浓度与模型组和生理盐水组相比均降低,差异均有统计学意义(P <0.05)。结论 AMPK可能参与大鼠脊髓缺血再灌注损伤,其机制可能与NF-κB通路介导的炎性反应有关;抑制AMPK活性可减轻大鼠脊髓缺血再灌注损伤、保护神经元。

Objective To investigate the effect of adenylate-activated protein kinase(AMPK) on spinal cord ischemiareperfusion injury in rats,and explore the possible mechanism. Methods Forty-eight healthy male SD rats were randomly divided into sham group,model group,saline group and AMPK inhibitor group(n=12). The rat spinal cord ischemiareperfusion injury model was constructed in model group,saline group and AMPK inhibitor group. Before modeling,the rats in AMPK inhibitor group were injected intraperitoneally with Compound C 20 mg/kg,while ones in the saline group were given the same amount of saline. After modeling 6,12,24 and 48 h,the neurological function was evaluated by Basso,Beattie and Bresnahan(BBB) scale. The spinal cord tissues in the rats were collected after the last BBB evaluating. Histopathological changes of rat spinal cord tissue were detected by HE staining. The cell apoptosis in the spinal cord tissue in the rats was detected by TUNEL assay. The expressions of AMPK,p-AMPK,NF-κB,IκBα and p-IκBα proteins in the spinal cord tissues in the rats were detected by Western blotting. The concentrations of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α) in the spinal cord of the rats were measured by ELISA. Results At 6,12,24 and 48 h after modeling,the BBB scores in model,saline and AMPK inhibitor groups were higher than those in the sham group,and the BBB scores in the AMPK inhibitor group were lower than those in model and saline groups,all with significantly differences(P < 0.05). In the sham group,the structure of spinal cord was clear and intact,and no neuron apoptosis was observed. The neurons degeneration and necrosis and inflammatory cells infiltration and a large number of neurons apoptosis were observed in the spinal cord tissues of rats in model and saline groups,while the pathological changes of spinal cord tissues in the AMPK inhibitor group were alleviated and the apoptosis of neurons was reduced. The relative expression levels of p-AMPK,NF-κB and p-IκBα proteins in the spinal cord tissue in model,saline and AMPK inhibitor groups were higher than those in the sham group,while the relative expression levels of AMPK and IκBα proteins were lower than those in the sham group,all with significantly differences(P < 0.05). The relative expression levels of p-AMPK,NF-κB and p-IκBα proteins in the spinal cord tissue in the AMPK inhibitor group were lower than those in model and saline groups,while the relative expression levels of AMPK and IκBα proteins were higher than those in model and saline groups,all with significantly differences(P < 0.05). The levels of IL-1β,IL-6 and TNF-α in the spinal cord tissue in model,saline and AMPK inhibitor groups were higher than those in the sham group,and the levels of IL-1β,IL-6 and TNF-α in the spinal cord tissue in the AMPK inhibitor group were lower than those in model and saline groups,all with significantly differences(P < 0.05). Conclusion AMPK may be involved in spinal cord ischemia-reperfusion injury in rats,and its mechanism may be related to inflammatory response mediated by NF-κB pathway. Inhibiting AMPK activity can alleviate spinal cord ischemia-reperfusion injury and protect the neurons.