重组人源Kunitz型蛋白酶抑制剂治疗大鼠脑皮质缺血/再灌注损伤

An in vivo study of neural protective effect of recombinant human KD/APP on rats with cerebral ischemia/reperfusion injury

目的旨在研究人源重组丝氨酸酶抑制剂rh KD/APP治疗大鼠脑皮质缺血/再灌注损伤的临床前景。方法构建大鼠大脑中动脉(MCAO)局灶性脑缺血/再灌注(I/R)的动物模型,分别用三个剂量rh KD/APP(4、8、16 mg/kg)的干预进行临床治疗,设神经保护药物尼莫地平治疗作为阳性对照,各干预治疗组分别进行临床评分、神经病理损伤、脑水肿程度的评估和比较,评价rh KD/APP在大鼠脑缺血/再灌注神经保护的临床疗效。同时,分组检测缺血/再灌注脑皮质的自由基氧化程度的三个指标(抗氧化酶SOD活性、Na+-K+-ATP酶活性、膜脂过氧化产物丙二醇MDA含量)、反映中性粒细胞呼吸爆发的髓过氧化物酶MPO活性、血管内皮表达粘附因子ICAM-1和E-selectin的表达水平以及损伤区神经元凋亡的状况。结果低、中、高剂量组rh KD/APP干预均能改善脑缺血/再灌注损伤大鼠的临床症状、脑水肿和神经病理损伤,且呈一定量效关系。与缺血/再灌注模型组大鼠比较,各个剂量的rh KD/APP干预均能提高损伤区脑组织抗氧化酶SOD和Na+-K+-ATP酶活性,降低丙二醇MDA含量,降低髓过氧化酶MPO活性,下调粘附因子ICAM-1和E-selectin的表达,拮抗受损神经元的凋亡。与尼莫地平治疗组比较,在降低MPO活性上,三个剂量rh KD/APP均显示出显著优势(P<0.05)。其余各个检测指标,rh KD/AP干预组与尼莫地平治疗组之间比较,差异无显著性。结论重组人源rh KD/APP丝氨酸酶抑制剂能够对大鼠脑缺血/再灌注神经损伤发挥保护作用,这些保护作用可能通过清除自由基、抑制中性粒细胞激活和炎性损伤,拮抗神经元凋亡等多层面综合完成。因此,人源Kunitz型丝氨酸蛋白酶抑制剂可成为一个潜在的神经保护药物。

Objective The amyloid β-protein precursor contains a domain highly homologous to Kunitz-type serine protease inhibitors. We have successfully established and characterized the recombinant human rh KD/APP in vitro.The aim of this study is to investigate the potential neuroprotective role of rh KD/APP on cerebral ischemia/reperfusion injury in rats. Methods Rats pretreated with rh KD/APP（ 4,8,16 mg/kg） were subjected to prepare models of cerebral ischemia/reperfusion（ I/R） injury and those rats treated with Nimodipine were used as positive control. Comparison of the scores of neurological deficits,TTC-stained infarct volume and cerebral water content between the groups was performed.The activities of SOD,Na^＋-K^＋-ATPase and the content of MDA in the cortex tissues were measured and the activities of serum myeloperoxidase（ MPO） enzyme were also compared. The expressions of adhesion molecules（ ICAM-1 and Eselectin） were compared by immunohistochemistry. End-labeling of nuclear DNA fragmentation（ TUNEL） and qualification of caspase-3,Bcl-2 and Bax were also employed to evaluate the local apoptosis in cortex tissues. Results By pretreatment with the rh KD/APP at three doses,cerebral infarct volume,water content and neurological deficits were all reduced. The activities of SOD,and Na^＋-K^＋-ATPase were increased,the contents of MDA were decreased in the cortex tissues,and the serum MPO activity was reduced. The expressions of adhesion molecules were downregulated and the apoptotic signaling of neurons were inhibited. All the changes induced by rh KD/APP treatment in the ischemia/reperfusion injury models showed statistical significance compared with the control rats. However,no significant difference was shown between the rh KD/APP group and Nimodipine group excepted for the reduced MPO in sera. Conclusions The result of this study suggest that rh KD/APP has neuroprotective effect on the cerebral ischemia/reperfusion injury through inhibiting multiple signaling pathways and is promising to be a potential neuroprotective drug.