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目的研究大鼠局部性脑缺血再灌注损伤时,小檗碱(BE)对缺氧诱导因子-1α(HIF-1α)表达及脑神经元凋亡的影响。方法雄性SD大鼠随机分为假手术组、大脑中动脉阻塞再灌注组(MCAO/R组)、假性治疗组(DMSO组)、小檗碱10mg/kg治疗组(BE10组)、小檗碱20mg/kg治疗组(BE20组)、小檗碱40mg/kg治疗组(BE40组)。治疗组在术前48h、24h及术后6h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,制备脑组织切片分别作Nissl染色、免疫组织化学染色、TUNEL标记及免疫荧光双标记。结果BE20、BE40组神经功能较MCAO/R组有明显改善(P<0.05),但BE10组神经学评分与MCAO/R组比较,差异无统计学意义(P>0.05)。不同剂量BE治疗均可以减小梗死灶体积(P<0.05),且呈现剂量依赖性。Nissl染色可见BE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,BE组HIF-1α、Caspase-3、BNIP3、VEGF及TUNEL标记的阳性细胞数减少;免疫荧光双标记显示HIF-1α与BNIP3、Caspase-3及TUNEL阳性颗粒共表达于细胞中。结论BE可能通过降低HIF-1α水平并下调其下游的BNIP3和VEGF的表达,从而减少凋亡因子Caspase-3的作用而发挥神经元保护作用。
Objective To investigate the effect of berberine (BER) on the expression of hypoxia-inducible factor-1α (HIF-1α) and neuronal apoptosis in rats following focal cerebral ischemia reperfusion injury. Methods Male Sprague-Dawley rats were randomly divided into sham-operated group, middle cerebral artery occlusion and reperfusion group (MCAO/R group), sham treatment group (DMSO group), berberine 10mg/kg treatment group (BE10 group), and small sputum. Base 20 mg/kg treatment group (BE20 group), berberine 40 mg/kg treatment group (BE40 group). The treatment group received intraperitoneal injection of drugs at 48h, 24h and 6h before operation to observe the neurobehavioral defects of rats in each group. After 7 days of reperfusion, TTC staining was used to observe the changes of cerebral infarct volume. After 24 hours of reperfusion, brain tissue sections were prepared. Nissl staining, immunohistochemical staining, TUNEL labeling, and immunofluorescence double labeling. Results The neurological function in the BE20 and BE40 groups was significantly improved compared with the MCAO/R group (P<0.05), but the neurological scores in the BE10 group were not significantly different from those in the MCAO/R group (P>0.05). Different doses of BE treatment can reduce infarct volume (P<0.05) and show a dose-dependent manner. Nissl staining showed that the structure of cortical neurons in BE treatment group was clearer, cell body swelling, nuclear pyknosis, nuclear lysis degree were significantly reduced compared with model group and sham treatment group, and lightly-stained area was reduced; immunohistochemical method was observed in BE group HIF. The number of positive cells of -1α, Caspase-3, BNIP3, VEGF, and TUNEL markers was reduced. Immunofluorescence double markers showed that HIF-1α was co-expressed in cells with BNIP3, Caspase-3, and TUNEL positive particles. Conclusion BE may play a protective role in neurons by decreasing the level of HIF-1α and down-regulating the expression of BNIP3 and VEGF downstream, thus reducing the effect of apoptosis factor Caspase-3.