Calpain抑制剂-3对新生大鼠脑缺氧缺血后海马CA1区Caspase-3表达和神经元凋亡的影响

目的：钙依赖中性蛋白酶Calpain的活化可引起神经元凋亡,其抑制剂3(MDL28170)对成年动物脑缺血有治疗作用,但尚不清楚MDL28170对新生动物缺氧缺血性脑损伤(HIBD)是否也有治疗作用。本研究观察了MDL28170对新生大鼠HIBD后海马CA1区Caspase3表达及细胞凋亡的影响,探讨其神经保护作用机制。方法：将7日龄新生SD大鼠随机分为HIBD模型组(n=40)、干预组(n=40)及对照组(n=8),干预组在HI后0h、2h、4h予腹腔注射MDL2817050mg/kg,模型组和对照组同时予腹腔注射生理盐水。干预组和模型组大鼠在HI后6h、12h、24h、48h、72h各处死8只,用免疫组化染色和脱氧核糖核酸末端转移酶介导的原位缺口末端标记法(TUNEL)分别检测海马CA1区Caspase3的表达和神经凋亡。结果：HIBD组大鼠损伤侧海马CA1区Caspase3阳性细胞在HI后6h时较对照组增多(13.4±3.5/HPvs2.6±0.6/HP,P=0.028),于48h(27.1±4.1/HP)达到高峰,72h仍高于对照组(22.6±4.8/HP,P<0.001);TUNEL阳性细胞于HI后6h开始增多,12h时与对照组比较差别有显著性(25.0±1.7/HPvs2.3±1.5/HP,P<0.001),48h(67.8±2.6/HP)到达高峰,72h仍处于较高水平(44.3±6.8/HP)。MDL28170干预可明显减少Caspase3及TUNEL阳性细胞数量,与模型组比较48h内差异有显著性,72h以后作用不明显。结论：MDL28170可通过抑制海马CA1区Caspase3的表达,减少脑细胞凋亡,起到脑保护作用。

Effect of Calpain inhibitor-3 on Caspase-3 expression and neuronal apoptosis in-hippocampal CA1 region of neonatal rats after hypoxic-ischemic brain damage

OBJECTIVE: Activation of calcium-dependent neutral proteinase, Caplain, can induce apoptosis of neuron and Caplain inhibitor-3 (MDL28170) has protective effects against brain ischemia in adult animals. Whether it also has protective effects on neonatal animals with hypoxic-ischemic brain damage (HIBD) is has not been determined. In this study, the effect of MDL28170 on Caspase-3 expression and apoptosis in hippocampal CA1 region of neonatal rats after HIBD was examined to explore its neural protective effect on neonatal animals and the possible underlying mechanism. METHODS: Seven-day-old Sprague-Dawley rat pups were randomly assigned into three groups: HIBD (n=40), MDL (n=40) and Control (n=8). The pups in the first two groups were subjected to unilateral ligation of the right carotid artery followed by 2 hrs of hypoxia (8% O_2). The pups in the MDL group were intraperitoneally injected with MDL28170 (50-mg/kg) at 0, 2 and 4 hrs after hypoxia-ischemia(HI), while those in the Control and HIBD groups were intraperitoneally injected with normal saline instead. The rats in the HIBD and MDL groups were sacrificed at 6, 12, 24, 48 and 72 hrs after HI (8 rats in each group at each time point). Immunohistochemistry and terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling staining (TUNEL) were used to detect the Caspase-3 expression and neuronal apoptosis in hippocampal CA1 region. RESULTS: In the HIBD group, Caspase-3 expression in hippocampal CA1 region increased at 6 hrs after HI compared with the Control group (13.4±3.5/HP vs 2.6±0.6/HP,P=0.028), peaking at 48 hrs (27.1±4.1/HP) and remaining higher at 72 hrs (22.6±4.8/HP,P<0.001). The number of TUNEL-positive neurons in the HIBD group increased at 6 hrs after HI, and were significantly greater than those of the Control group at 12 hrs (25.0± 1.7/HP vs 2.3±1.5, P<0.001). At 48 hrs after HI, the number of TUNEL-positive neurons peaked (67.8±2.6/HP) and remained greater at 72 hrs (44.3±6.8/HP). The treatment with MDL28170 attenuated the Caspase-3 expression and reduced the number of TUNEL-positive neurons within 48 hrs after HI when compared with the HIBD group (P<0.05), but the effect significantly decreased at 72 hrs. CONCLUSIONS: MDL28170 can inhibit the Caspase-3 expression and reduce the apoptotic cells in hippocampal CA1 region, thereby yielding protective effects against HIBD in neonatal rats.