西洛他唑对大鼠脑缺血再灌注损伤后PARP／AIF介导的细胞凋亡途径的影响

Effect of cilostazol pretreatment on poly （ADP-ribose） polymerase/apoptosis-inducing factor pathway induced nerve cell apoptosis in rats after focal cerebral ischemia/reperfusion

目的研究大鼠局灶性脑缺血再灌注损伤后多聚腺苷二磷酸核糖聚合酶（PARP）和凋亡诱导因子（AIF）在海马CAl区的表达，探讨西洛他唑预处理能否通过PARP／AIF途径发挥脑保护作用。方法将135只雄性SD大鼠按随机数字表法分为3组：假手术组、模型组、西洛他唑组，每组45只。采用线栓法阻塞大鼠大脑中动脉制作局灶性脑缺血再灌注损伤模型。西洛他唑组造模前灌胃给予30mg／kg剂量西洛他唑（2次）。每组根据再灌注时间点不同（6h、24h、72h）分为3个亚组，每亚组15只。应用TUNEL法检测神经细胞凋亡变化，Western blotting法检测AIF、腺苷二磷酸核糖（PAR）在不同时间点的变化．RT-PCR法检测AJFmRNA的表达变化。结果大鼠局灶性脑缺血再灌注损伤后出现AIF核移位。与假手术组比较，模型组凋亡细胞数明显增加，AIF、PAR含量及AIFmRNA表达明显增加，24h时最显著，差异均有统计学意义（P〈0．05）。西洛他唑组再灌注6h、24h、72h各亚组凋亡细胞数较模型组中各亚组明显减少，AIF、PAR含量较模型组各亚组明显降低，AIFmRNA表达亦明显减少，24h时最显著，差异有统计学意义（P〈0．05）。结论西洛他唑对大鼠脑缺血再灌注损伤有一定保护作用，其抗神经细胞凋亡的机制之一可能是通过抑制脑缺血损伤引起的PARP的过度活化及AIF的易位而实现的。

Objective To study the expressions of poly（ADP-ribose） polymerase （PARP） and apoptosis-inducing factor （AIF） in the hippocampal CA1 region of rats after focal cerebral ischemia reperfusion injury, and elucidate the neuroprotective effect of cilostazol pretreatment through PARP/AIF pathway. Methods One hundred and thirty-five male SD rats were randomly divided into three groups （n=45） for experiments： sham-operated group, ischemia/reperfusion （I/R） vehicle group and cilostazol pretreatment group. Embolization thread was inserted into all rats except those in the sham-operated group for 2 hours to establish middle cerebral artery occlusion models; and then, reperfusion for 6, 24, 72 hours was performed, and rats of each group were sub-divided into three groups according to reperfusion times （n=15）. Cilostazol pretreatment group was given cilostazol （30 mg/kg, twice orally） before surgery. The apoptosis cells of the hippocampus were detected by Terminal deoxynucleotidyl Transferase- mediated dUTP nick end-labeling （TUNEL）. Western blotting was used to test the dynamic changes of AIF and polyadenosine diphosphate ribose （PAR） levels at different times. The mRNA expression of AIF was assessed by real time-PCR. Results There appeared subsequent translocation of AIF after focal cerebral ischemia reperfusion injury in rats. As compared with those in the sham-operated group, theapoptotic cells statistically increased; AIF and PAR contents and AIF mRNA expression significantly increased in the FR vehicle group reaching its peak level at 24 h ofreperfusion （P〈0.05）. As compared with of the I/R model subgroups, the cilostazol pretreatment subgroups had significantly decreased AIF and PAR contents, AIF mRNA expression and number of apoptosis cells （P〈0.05）; 24 h of reperfusion subgroup had the most obvious effect （P〈0.05）. Conclusion Cilostazol pretreatment in rats after ischemia-reperfusion injury has certain neroprotective effect, whose resistance roles in nerve cell apoptosis may be implemented by inhibiting excessive PARP activation and AIF translocation caused by cerebral ischemia injury.