大黄酚对局灶性脑缺血再灌注小鼠缺血半暗带区HIF-1α与VEGF表达的影响

目的 研究大黄酚(chrysophanol, CHR)对局灶性脑缺血再灌注小鼠缺血半暗带区缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)及血管内皮生长因子(vascular endothelial growth factor, VEGF)表达的影响,探究CHR对脑缺血再灌注损伤的长期脑保护机制。方法 采用数字表法随机将18只健康雄性2月龄C57BL小鼠分为3组:假手术(Sham)组、大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)组、CHR组(自造模当日至脑缺血再灌注后14 d按0.1 mg·kg^-1·d^-1腹腔注射CHR),每组6只。线栓法制作小鼠MCAO模型,缺血45 min后拔出线栓进行再灌注。于再灌注14 d将小鼠处死、取脑,通过免疫荧光染色方法检测脑组织冰冻切片缺血半暗带区内HIF-1α及VEGF水平,并通过免疫荧光双标染色方法确定HIF-1α、VEGF是否在神经元中表达。结果 1)Sham组小鼠脑内偶见HIF-1α阳性细胞。MCAO组小鼠脑缺血半暗带区HIF-1α的表达水平比Sham组显著升高(P<0.05)。经CHR治疗14 d的脑缺血再灌注小鼠半暗带区内HIF-1α的表达水平比MCAO组显著减少(P<0.05)。2)Sham组小鼠脑内可见大量的VEGF阳性细胞。MCAO组小鼠脑缺血半暗带区VEGF的表达水平比Sham组显著降低(P<0.05)。经CHR治疗14 d的脑缺血再灌注小鼠半暗带区内VEGF的表达水平比MCAO组显著增加(P<0.05)。3)在小鼠脑缺血半暗带区,HIF-1α或VEGF分别与神经元标志物NeuN共定位。结论 CHR可能通过下调HIF-1α的表达水平、上调VEGF的表达水平,减少神经元损伤,从而对脑缺血再灌注损伤发挥长期神经保护作用。

Effects of chrysophanol on HIF-1α and VEGF expressions in mice with focal cerebral ischemia/reperfusion injury

Objective To investigate the effects of chrysophanol (CHR) on the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the penumbra of focal cerebral ischemia/reperfusion mice and further, to explore the long-term protective effect mechanism of CHR against cerebral ischemia/reperfusion injury.Methods According to the random number table, 18 healthy male 2-month-old C57BL mice were divided into 3 groups: sham operation (Sham) group (n=6), middle cerebral artery occlusion (MCAO) group (n=6), and CHR group (CHR in dose of 0.1 mg/kg was intraperitoneally injected in CHR group mice once a day for 14 days after reperfusion, n=6). The MCAO for 45 min was induced by thread embolism and the reperfusion lasted for 14 days. On the 14th day after reperfusion, the brain tissue was obtained. The expressions of HIF-1αand VEGF in the brain ischemic penumbra were observed with immunofluorescence labeling. The colocalization of HIF-1α or VEGF with neuron marker NeuN was determined with double labeling immunofluorescence. Results 1) There was little HIF-1α positive cell in Sham group. Compared with Sham group, the HIF-1α positive cells in the penumbra of MCAO group obviously increased on the 14th day after reperfusion (P<0.05). Compared with MCAO group, the HIF-1α positive cells decreased significantly in the penumbra of CHR group on the 14th day after reperfusion (P<0.05). 2) There were many VEGF positive cells in Sham group. Compared with Sham group, the VEGF positive cells in the penumbra of MCAO group obviously decreased on the 14th day after reperfusion (P<0.05). Compared with MCAO group, the VEGF positive cells increased significantly in the penumbra of CHR group on the 14th day after reperfusion (P<0.05). 3) In the penumbra of ischemic brain of mice, HIF-1α or VEGF was colocalized with neuron marker NeuN, respectively. Conclusion CHR may inhibit the expression of HIF-1α protein, upregulate the expression of VEGF protein, and reduce neuronal damage, thereby exerting long-term neuroprotection against cerebral ischemia-reperfusion injury.