Nrf2-ARE信号通路对脑出血灶周神经保护作用的机制研究

Mechanisms underlying the perifocal neuroprotective effect of the Nrf2-ARE signaling pathway after intracranial hemorrhage

目的研究大鼠脑出血后Nrf2-ARE通路HO-1对出血灶周神经保护作用及其相关机制探讨。方法采用SD大鼠基底节自体股动脉血注射法建立脑出血模型,分4组:单纯脑出血(ICH)组、莱菔硫烷(SFN)组、维甲酸(RA)组、对照组,观察不同时间点大鼠神经功能评分,取脑组织行免疫荧光检查、Western blot、RT-PCR检测Nrf2、HO-1、NF-κB、TNF-α表达。对所得数据进行统计分析,观察这些指标的变化。结果 SFN组与ICH组比较,神经功能障碍明显减轻,出血灶周Nrf2因子、HO-1抗氧化蛋白表达均升高,于3 d达高峰值,持续表达至7 d;SFN组NF-κB、TNF-α表达下降;RA组大鼠死亡率最高,神经功能障碍较ICH组及SFN组均严重,Nrf2因子、HO-1抗氧化蛋白表达均较SFN组和ICH组低,持续性抑制作用可能持续7 d左右,RA处理组NF-κB、TNF-α表达升高,炎性反应持续时间可能超过7 d。结论 RA抑制Nrf2解离及转入核,抑制Nrf2-ARE信号通路的抗炎性作用,SFN激活Nrf2-ARE信号通路可提高HO-1抗氧化酶表达,减轻脑出血后灶周炎性反应,具有神经保护作用,提示Nrf2-ARE信号通路将是治疗脑出血灶周炎性损伤的新方向。

Objective The aim of this study is to perform sulforaphane （SFN） activating the Nrf2-ARE signaling pathway and retinoic acid（RA） inhibiting the Nrf2-ARE signaling pathway to examine the neuroprotective effect of heme ox- ygenase（HO-1 ）and to explore the underlying mechanisms on perifocal tissues after ICH. Methods SD rats with autolo- gous femoral arterial blood injection into the basal ganglia were applied to establish ICH models and were divided into four groups：ICH,SFN, RA and DMSO group. We observe neurological score of the rats in different groups at different time points and collect brain tissues for immunofluorescence,Western blot, and RT-PCR to detect the expression of Nrf2, HO-1, nuclear factor-KB（NF-KB） and tumor necrosis factor-a（ TNF-α）. And then,we observe the changes of the metrics by statis- tical analysis. Results In the SFN group, rats showed that neurological dysfunction was significantly reduced, and the ex- pressions of Nrf2 and HO-1 in tissues surrounding the hemorrhage were increased ,reached a peak value on day 3 ,followed by sustained expression until day 7. And the level of NF-KB and TNF-α were reduced compared to the ICH group. The RA group exhibited the highest mortality, more severe neurological dysfunction and lower levels of Nrf2 and HO-1 than the SFN and ICH groups. The inhibitory effect persisted for approximately seven days. Compared to the ICH group ,the NF-KB and TNF-~t expression in the RA groups was increased, and the inflammatory response persisted for more than seven days. Con- clusion RA inhibits Nrf2 dissociation and translocation into the nucleus, thereby suppressing the anti-inflammatory effect of the Nrf2-ARE signaling pathway. The activation of the Nrf2-ARE signaling pathway by SFN can elevate the expression of the antioxidant enzyme HO-1 ,reduce the perifocal inflammatory response after ICH,and thus play a neuroprotective role. The results suggest that the Nrf2-ARE signaling pathway may serve as a new target for treatment of perifocal inflammatory injury caused by ICH.