大鼠脑缺血再灌注损伤Cathepsin D介导的细胞凋亡与MLK3的关系

发布时间：2018-12-14 22:11

【摘要】：目的本实验通过建立大鼠大脑中动脉脑缺血再灌注模型，并运用Cathepsin D抑制剂pepstatin A对其进行干预，检测Cathepsin D和pMLK3的蛋白表达变化，探讨Cathepsin D与MLK3在大鼠脑缺血再灌注损伤过程中神经细胞凋亡的关系，为研究溶酶体参与脑缺血再灌注损伤神经细胞凋亡提供新的探索，为缺血性脑血管病提供新的治疗靶点。方法 67只清洁级健康雄性Sprague-Dawley大鼠，体重280±20g，随机分为3组：假手术组（sham，n=7），模型组（M，n=30），干预组（I，n=30）：于再灌注前及再灌注后每12h腹腔注射溶酶体Cathepsin D抑制剂pepstatin A2ml/100g（而假手术组及模型组于相应时间腹腔注射生理盐水2ml/100g）。其中模型组和干预组又分为2小时组（M-2h，n=6；I-2h，n=6），6小时组（M-6h，n=6；I-6h，n=6），24小时组（M-24h，n=6；I-24h，n=6），48小时组（M-48h，n=12；I-48h，n=12）。应用改良的Long线栓法制作大鼠大脑中动脉脑缺血再灌注模型，缺血2小时后恢复再灌注，使用Longa’s的5级评分法评价神经功能；使用TTC染色法、TUNEL凋亡检测法及免疫组织化学方法，分别检测脑梗死体积、细胞凋亡数量、CathepsinD与pMLK3的表达变化。结果 1.模型成功率76.14%；模型组48小时相对脑梗死体积为37.36±1.44%，干预组48小时相对脑梗死体积为27.49±2.30%。 2.假手术组脑组织未见梗死灶，，模型组和干预组缺血侧皮质及皮质下可见白色梗死灶，但干预组48小时相对梗死体积较模型组减少（p=0.000），且干预组神经功能缺损评分较模型组也得到明显改善（p0.05）。 3.大鼠脑缺血侧皮质区，假手术组凋亡细胞很少，模型组2小时可以观察到凋亡细胞，6小时、24小时、48小时呈上升趋势（p0.001）；干预组2小时凋亡细胞较模型组2小时无显著改变（p=0.127），干预组6小时、24小时、48小时观察到的凋亡细胞较模型组明显减少（p0.05）。 4.大鼠脑缺血侧皮质区，假手术组可见少量Cathepsin D的表达，模型组Cathepsin D的表达在再灌注2小时、6小时、24小时呈上升趋势，24小时达高峰，48h组较24h组有所下降，但仍居高水平，均比假手术组高（p=0.000）；干预组各时间点Cathepsin D的表达较模型组明显减少（p=0.000）。 5.大鼠脑缺血侧皮质区，假手术组pMLK3少量表达，模型组pMLK3的表达在再灌注2小时即有大量表达，于6小时达高峰，之后逐渐下降，均高于假手术组（p=0.000）；干预组各时间点pMLK3的表达均较模型组明显减少（p0.05）。结论 1.大鼠脑缺血再灌注损伤过程中溶酶体Cathepsin D可能通过上调MLK3的磷酸化而介导神经细胞凋亡。 2.Pepstatin A可能通过抑制Cathepsin D而下调MLK3的磷酸化活性表达，从而减少细胞凋亡，减少脑梗死体积，起到神经保护作用。
[Abstract]:Objective to investigate the changes of Cathepsin D and pMLK3 protein expression in middle cerebral artery (MCAA) ischemia-reperfusion model of rats by using pepstatin A, a Cathepsin D inhibitor, to establish a rat model of cerebral ischemia-reperfusion. To investigate the relationship between Cathepsin D and MLK3 in the process of cerebral ischemia-reperfusion injury in rats, and to explore the role of lysosome in neuronal apoptosis during cerebral ischemia-reperfusion injury, and to provide a new therapeutic target for ischemic cerebrovascular disease. Methods Sixty-seven clean grade healthy male Sprague-Dawley rats, weighing 280 卤20 g, were randomly divided into three groups: sham operation group (sham,n=7), model group (Mannan 30), intervention group (I), and control group (I). Nongma 30: lysosomal Cathepsin D inhibitor pepstatin A2ml/100g was injected intraperitoneally every 12 hours before and after reperfusion (while normal saline 2ml/100g was injected intraperitoneally in sham operation group and model group at the corresponding time). Among them, the model group and the intervention group were divided into two hours group (M-2hnntir 6L), 6-hour group (M-6hnntir 6hnc1hnc6), 24-hour group (M-24hntir 6I-24hncnc6), and 48-hour group (M-48hntrol 12), the model group and the intervention group were divided into three groups: the model group and the intervention group, respectively. I-48h ~ (12). The rat model of middle cerebral artery ischemia reperfusion was established by modified Long thread occlusion method, and the reperfusion was resumed 2 hours after ischemia. The nerve function was evaluated by Longa's 5 grade scoring method. TTC staining, TUNEL apoptosis detection and immunohistochemistry were used to detect the volume of cerebral infarction, the number of apoptosis and the expression of CathepsinD and pMLK3. Result 1. The relative volume of cerebral infarction was 37.36 卤1.44 in the model group and 27.49 卤2.30 in the intervention group at 48 hours. 2. No infarct was found in the cerebral tissue of sham-operation group, and white infarct was found in ischemic cortex and subcortical of model group and intervention group, but the relative infarct volume of 48 hours in intervention group was lower than that in model group (p0. 000). The neurological deficit score in the intervention group was significantly improved than that in the model group (p0.05). 3. There were few apoptotic cells in the cerebral ischemic cortex of rats in the sham-operation group. The apoptotic cells were observed in the model group at 2 hours, and increased at 6 hours, 24 hours and 48 hours (p0.001). There was no significant change in apoptotic cells in the intervention group at 2 hours compared with that in the model group (p0. 127). The apoptotic cells in the intervention group at 6 hours, 24 hours and 48 hours were significantly lower than those in the model group (p0.05). 4. A small amount of Cathepsin D expression was observed in the sham-operated group. The expression of Cathepsin D in the model group increased at 2 hours, 6 hours and 24 hours after reperfusion, and reached its peak at 24 hours. The expression of Cathepsin D in the 48 h group was lower than that in the 24 h group, but it was still at a high level. All of them were higher than those of sham operation group (p0. 000). The expression of Cathepsin D in the intervention group was significantly lower than that in the model group (P < 0. 000). 5. There was a little expression of pMLK3 in the cerebral ischemic cortex and sham-operation group. The expression of pMLK3 in the model group reached its peak at 6 hours after reperfusion and was higher than that in the sham-operation group (p0. 000). The expression of pMLK3 in the model group was higher than that in the sham operation group (P < 0. 000). The expression of pMLK3 in the intervention group was significantly lower than that in the model group at each time point (p0. 05). Conclusion 1. Lysosomal Cathepsin D may induce neuronal apoptosis by up-regulating the phosphorylation of MLK3 during cerebral ischemia-reperfusion injury in rats. 2.Pepstatin A may down-regulate the expression of phosphorylation of MLK3 by inhibiting Cathepsin D, thus reducing apoptosis and cerebral infarct volume, thus playing a neuroprotective role.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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