缺血后处理对大鼠脊髓缺血再灌注损伤的保护作用及对钙敏感受体和Caspase-12表达的影响

目的: 探究缺血后处理(ischemic post conditioning,IPC)对大鼠脊髓缺血再灌注损伤(spinal cord ischemia reperfusion injury,SCIRI)的保护作用及其对钙敏感受体(calcium-sensing receptor,CaSR)及内质网应激相关蛋白Caspase-12表达的影响。方法: 将50只SD大鼠随机平均分为5组：假手术组，缺血再灌注模型组（IR组），IR模型+IPC组（IPC组），IR模型+IPC+激动剂组（IPC GdCl3组）和IR模型+抑制剂组（NPS2390组）。假手术组仅显露大鼠腹主动脉而不用动脉夹夹闭，其他各组夹闭腹主动脉25 min后再开放建立SCIRI模型；IPC组再灌注3 min后予3个循环的IPC；IPC-GdCl3组在连续2天每天1次尾静脉注射CaSR激动剂GdCl3后行IPC组操作；NPS2390组提前2天每天1次通过尾静脉注射NPS2390，随后建立IR模型。采用BBB评分法对大鼠后肢运动功能进行评价，采用TUNEL染色检测脊髓组织中神经细胞凋亡数目，采用蛋白质印迹及免疫组织化学定量检测脊髓组织中CaSR及Caspase-12表达。结果： 各模型组大鼠BBB评分均明显低于假手术组（P<0.05）；IPC组、NPS2390组BBB评分均明显高于IR组（P<0.05）；IPC组BBB评分明显高于IPC GdCl3组（P<0.05）。TUNEL染色结果显示，假手术组凋亡细胞极少；IR组较IPC组和NPS2390组凋亡神经细胞数量均明显增多（P<0.05）；IPC组较IPC-GdCl3组凋亡神经细胞数量明显减少（P<0.05）。蛋白质印迹结果表明，建模各组较假手术组CaSR及Caspase-12蛋白表达量明显升高（P<0.05）；IR组CaSR与Caspase-12表达量均明显高于IPC组和NPS2390组（P<0.05）；IPC组较IPC GdCl3组CaSR与Caspase-12表达量明显减少（P<0.05）。免疫组织化学各组比较情况与蛋白质印迹一致。结论： IPC对大鼠SCIRI起到保护作用，且可能与抑制脊髓组织中CaSR及Caspase 12的表达有关。

Protection of ischemic post-conditioning against spinal cord ischemia-reperfusion injury and its effect on the expression of calcium sensing receptor and Caspase-12 in rats

Objective: To investigate the protective effect of ischemic post-conditioning（IPC） on spinal cord ischemia reperfusion injury（SCIRI） in rats and to observe its effect on the expression of calciumsensing receptor（CaSR） and endoplasmic reticulum stress-related proteins Caspase-12. Methods: Fifty SD rats were randomly and averagely divided into 5 groups: sham operation group (Sham group)，ischemia-reperfusion model group（IR group）,IR model+IPC group (IPC group), IR model +IPC+agonist (IPC-GdCl3 group) and IR model+inhibitor group(NPS2390 group). Sham group only exposed abdominal aorta but not clamping, SCIRI models were established after clamping abdominal aortic for 25 min and then openning it in other groups, IPC group was treated with 3 circulatory IPC after 3 min perfusion, IPC-GdCl3 group was treated with IPC group’s treatment after tail vein injection of CaSR agonist GdCl3 for 2 days. CaSR inhibitor NPS2390 was injected intravenously in NPS2390 group once a day for 2 days before establishing IR model. The motor function of hindlimb was evaluated by BBB score, the apoptosis of spinal cord tissue was detected by TUNEL staining, and the expression of CaSR and Caspase-12 was detected by Western blotting and immunohistochemistry. Results: The BBB scores of the model groups were significantly lower than those of the sham group (P<0.05). The scores of the IPC group and the NPS2390 group were higher than those of the IR group(P<0.05). The scores of the IPC group were higher than those of the IPC-GdCl3 group (P<0.05). TUNEL staining showed that almost no apoptotic nerve cells were found in sham group, and the number of apoptosis in IR group were higher than that in IPC group and NPS2390 group (P<0.05). The number of apoptosis in IPC group were lower than that in  IPC-GdCl3 group (P<0.05).Western blotting results showed that the expression of CaSR and Caspase 12 protein in the model group was significantly higher than that in the sham group (P<0.05).The expression of CaSR and Caspase 12 in IR group was higher than that in IPC group and NPS2390 group (P<0.05).The expression of CaSR and Caspase-12 in IPC group was lower than that in IPC GdCl3 group (P<0.05). The results of immunohistochemistry were consistent with Western blotting. Conclusion: IPC exerts a protective effect on SCIRI in rats and this protection may be attributed to inhibition of CaSR and Caspase 12 expression in spinal cord tissues.