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目的研究大鼠局部脑缺血再灌注损伤时,缺氧诱导因子-1α(HIF-1α)抑制剂2-甲氧雌二醇(2ME2)对损伤脑组织的影响。方法雄性SD大鼠60只随机分为假手术组、大脑中动脉阻塞再灌注组(MCAO组)、假性治疗组(DMSO组)、2ME2治疗组(2ME2组)。治疗组在术后1h腹腔注射相应剂量药物。观察各组大鼠神经行为学缺陷;再灌注7d,TTC染色观察脑梗死体积变化;再灌注24h,Western blotting检测HIF-1α及其下游基因的表达变化;制备脑组织切片分别作Nissl染色、免疫组织化学染色及原位缺口末端标记(TUNEL)。结果2ME2组神经功能较MCAO及DMSO组有明显改善(P<0.05),同时,2ME2组梗死面积明显减小(P<0.05)。Western blotting结果显示,HIF-1α的表达经2ME2治疗后降低,其下游基因VEGF、BNIP3及Caspase-3的表达有同样的变化。Nissl染色可见2ME2治疗组皮质神经元结构较清晰,胞体肿胀、核固缩、核溶解程度较模型组及假性治疗组明显减轻,淡染区域减小;免疫组织化学法观察到2ME2组HIF-1α、Caspase-3、BNIP3、VEGF及TUNEL标记的阳性细胞数明显减少(P<0.05)。结论2ME2可能通过降低HIF-1α水平并下调其下游的BNIP3和VEGF的表达,降低血脑屏障的通透性并减少凋亡因子Caspase-3的作用,发挥神经元的保护作用。
Objective To investigate the effect of hypoxia inducible factor-1α (HIF-1α) 2-methoxyestradiol (2ME2) on brain injury following focal cerebral ischemia-reperfusion injury in rats. Methods Sixty male Sprague Dawley rats were randomly divided into sham operation group, middle cerebral artery occlusion reperfusion group (MCAO group), sham operation group (DMSO group) and 2ME2 treatment group (2ME2 group). The treatment group was intraperitoneal injection of the corresponding dose of medicine 1h after operation. The neurobehavioral defects of the rats in each group were observed. At 7 days after reperfusion, the changes of infarct volume were observed by TTC staining. The expressions of HIF-1α and its downstream genes were detected by Western blotting after reperfusion for 24 hours. Nissl staining and immunohistochemistry Histochemical staining and in situ nick end labeling (TUNEL). Results The neurological function of 2ME2 group was significantly improved compared with that of MCAO and DMSO groups (P <0.05). Meanwhile, the infarct size of 2ME2 group was significantly decreased (P <0.05). The results of Western blotting showed that the expression of HIF-1α was decreased after treatment with 2ME2, and the expression of VEGF, BNIP3 and Caspase-3 in the downstream cells changed in the same way. Nissl staining showed that the structure of cortical neurons in 2ME2 group was clearer, the cell body swelling, nuclear pyknosis and nuclear lysis were significantly reduced compared with the model group and the sham-operation group, and the light-stained area was reduced. Immunohistochemistry showed that HME- The number of positive cells of 1α, Caspase-3, BNIP3, VEGF and TUNEL markedly decreased (P <0.05). Conclusion 2ME2 may exert the neuronal protective effect by decreasing the level of HIF-1α, down-regulating the expression of BNIP3 and VEGF downstream of it, decreasing the permeability of blood-brain barrier and decreasing the effect of apoptosis factor Caspase-3.