常压高浓度氧对新生大鼠脑微血管内皮细胞损伤与Nrf2/HO-1信号通路的影响分析

Effects of normobaric hyperoxia on cerebral microvascular endothelial cells injury and Nrf2/HO-1 signaling pathways in neonatal rats

目的探究常压高浓度氧(NBO)对新生大鼠脑微血管内皮细胞损伤及核因子E2相关因子2/血红素氧合酶-1(Nrf2/HO-1)信号通路的影响。方法取新生SD大鼠45只,采用随机数字表法分为常氧组、NBO组和NBO+Nrf2激活剂组,每组各15只。常氧组大鼠置于普通空气(21%氧气)中饲养,NBO组和NBO+Nrf2激活剂组大鼠置于90%常压氧气饲养,NBO+Nrf2激活剂组每日灌胃5 mg/kg Nrf2激动剂莱菔硫烷。测定脑组织伊文思蓝(EB)含量,采用酶联免疫法检测血管内皮生长因子(VEGF)和基质金属蛋白酶9(MMP-9)含量,干湿重法检测脑组织含水量,HE染色和TUNEL染色观察脑组织病理变化,蛋白质印迹法检测海马组织Nrf2/HO-1信号通路蛋白表达,水迷宫检测大鼠认知功能。结果与常氧组比较,NBO组脑组织EB、VEGF、MMP-9含量及脑组织含水量升高,差异均有统计学意义(P<0.05);与NBO组比较,NBO+Nrf2激活剂组脑组织EB、VEGF、MMP-9含量及脑组织含水量降低,差异均有统计学意义(P<0.05)。病理染色结果显示,常氧组大鼠神经细胞形态及结构完整,未见明显病理变化和细胞凋亡;NBO组神经细胞形态及结构不规则,出现明显的水肿和空泡,并伴有大量的凋亡细胞;NBO+Nrf2激活剂组脑组织病理损伤较NBO组明显减轻。与常氧组比较,NBO组脑组织Nrf2、HO-1蛋白相对表达量降低,差异均有统计学意义(P<0.05);与NBO组比较,NBO+Nrf2激活剂组Nrf2、HO-1蛋白相对表达量升高,差异均有统计学意义(P<0.05)。与常氧组比较,NBO组第2~4天逃避潜伏期延长,穿越平台次数减少,差异均有统计学意义(P<0.05);与NBO组比较,NBO+Nrf2激活剂组第2~4天逃避潜伏期缩短,穿越平台次数增多,差异均有统计学意义(P<0.05)。结论NBO可诱导新生大鼠脑微血管内皮细胞损伤,导致远期认知功能障碍,可能与下调Nrf2/HO-1信号通路表达有关。

Objective To explore the effects of normobaric hyperoxia(NBO) on cerebral microvascular endothelial cells injury and nuclear factor E2-related factor 2/heme oxygenase-1(Nrf2/HO-1) signaling pathways in neonatal rats.Methods A total of 45 neonatal SD rats were enrolled and divided into normoxia group,NBO group and NBO+Nrf2 activator group by random number table method,15 rats in each group.The rats in normoxia group were fed in ordinary air(21% oxygen),while rats in NBO group and NBO+Nrf2 activator group were fed in 90% normobaric oxygen.The rats in NBO+Nrf2 activator group were given intragastric administration of 5 mg/kg Nrf2 agonist sulforaphane daily.The content of Evans blue(EB) in brain tissues was detected,the levels of vascular endothelial growth factor(VEGF) and matrix metalloproteinase-9(MMP-9) were detected by enzyme-linked immunosorbent assay,water content of brain tissues was detected by wet-dry weighting method,the pathological changes of brain tissues were observed by HE staining and TUNEL staining,expressions of Nrf2/HO-1 signaling pathway proteins in hippocampus were detected by Western Blotting,and cognitive function of rats was detected by water maze test.Results Compared with normoxia group,levels of EB,VEGF and MMP-9,and water content of brain tissues were increased in NBO group,the differences were statistically significant(P<0.05).Compared with NBO group,levels of EB,VEGF and MMP-9,and water content of brain tissues were decreased in NBO+Nrf2 activator group,the differences were statistically significant(P<0.05).The results of pathological staining showed that in normoxia group,morphology and structure of nerve cells were intact,and there were no obvious pathological changes or apoptosis.In NBO group,morphology and structure of nerve cells were irregular,and there was obvious edema and vacuoles,with a large number of apoptosis cells.The pathological injury of brain tissues in NBO+Nrf2 activator group was significantly milder than that in NBO group.Compared with normoxia group,relative expression levels of Nrf2 and HO-1 proteins in brain tissue were decreased in NBO group,the differences were statistically significant(P<0.05).Compared with NBO group,relative expression levels of Nrf2 and HO-1 proteins in brain tissue were increased in NBO+Nrf2 activator group,the differences were statistically significant(P<0.05).Compared with normoxia group,escape latency was prolonged,and frequency of crossing the platform was decreased from 2th-4th day in NBO group,the differences were statistically significant(P<0.05).Compared with NBO group,escape latency was shortened,and frequency of crossing the platform was increased from 2th-4th day in NBO+Nrf2 activator group,the differences were statistically significant(P<0.05).Conclusion NBO can induce cerebral microvascular endothelial cells injury in neonatal rats,which leads to long-term cognitive dysfunction.It may be related to the down-regulation of Nrf2/HO-1 signaling pathways.