乌司他丁对大鼠脑缺血再灌注损伤后的保护作用及其机制研究

A Study on the Protective Effect and Its Mechanism of Ulinastatin in Cerebral Ischemia Reperfusion Injury

目的:探究乌司他丁在脑缺血再灌注损伤中的脑保护作用机制。方法:原代分离培养雄性SD大鼠脑皮质细胞,部分细胞经siRNA沉默HSP70基因。细胞先以无糖培养基在低氧条件下培养,12 h后复糖复氧模拟体外缺血再灌注损伤,并实施乌司他丁预处理干预,流式细胞术检测各组细胞的凋亡率,western-blotting检测Bcl-2,Bax,HSP70,JNK和p-JNK蛋白的表达。结果:与对照组比较,模型组脑组织细胞凋亡率明显增多(P<0.05)、Bcl-2和Bax的表达量均有上调,Bcl-2/Bax的比值显著降低(P<0.01)、HSP70的表达无显著变化;与模型组比较,乌司他丁处理组脑组织细胞凋亡率明显降低(P<0.05)、Bax的表达量显著下调(P<0.05),Bcl-2/Bax的比值显著上调(P<0.05),HSP70的表达显著上调(P<0.05),JNK的表达无显著变化、p-JNK则显著下调(P<0.05)。HSP70沉默后乌司他丁的脑保护作用消失,对以上蛋白的表达无显著影响。结论:乌司他丁可能是通过上调HSP70表达进而抑制JNK信号转导通路对缺血再灌注引起的脑损伤起保护作用。

Objective： To explore the brain protection mechanism of ulinastatin in cerebral ischemia reperfusion injury. Methods：cortical cells of SD rat were isolated and cultured. HSP70 gene of some nerve cells was silenced by siRNA. Cortical cells were subjected to the oxygen-glucose deprivation and recovery of oxygen-glucose, which simulated in vitro ischemia reperfusion injury, and pretreated with ulinastatin. Flow cytometry was used to detect the apoptosis rate of cells, and western-blotting was employed to measure the protein expression of Bcl-2, Bax, HSP70, JNK, and p-JNK. Results： Compared with that of the control group, the apoptosis rate of cortical cells of the model group was significantly increased（P〈0.05）, and the protein expression of Bcl-2 and Bax were upregulated obviously（P〈0.01）, but the ratio of Bcl-2/Bax was significantly decreased（P〈0.05）, the protein expression of HSP70 presented no significant changes.The apoptosis rate of cortical cells of ulinastatin pretreated group was significantly decreased（P〈0.05）, and the protein expression of Bax was downregulated remarkablely compared with the model group（P〈0.05）, and the ratio of them become upregulated gradually（P〈0.05）. Compared with the model group, the protein expression of HSP70 was significantly upregulated（P〈0.05）, and the protein expression of JNK presented no significant changes, while the protein expression of p-JNK was remarkablely downregulated（P〈0.05）. The protective effect of ulinastatin on cortical cells of cerebral ischemia reperfusion rats disappeared after HSP70 knockout, and the effect of ulinastatin on the protein expression of above genes present no significant changes. Conclusion： Protective effect of ulinastatin on cortical cells of cerebral ischemia reperfusion rats may be associated with the upregulation of HSP70 and the downregulation of p-JNK.