Nrf2/GPX4信号通路介导的铁死亡在咪达唑仑减轻新生大鼠HIBD中的作用

Role of Nrf2/GPX4 signaling pathway-mediated ferroptosis in midazolam-induced reduction of HIBD in neonatal rats

目的:评价核因子E2相关因子2/谷胱甘肽过氧化物酶4(Nrf2/GPX4)信号通路介导的铁死亡在咪达唑仑减轻新生大鼠缺血缺氧性脑损伤(HIBD)中的作用。方法:健康新生大鼠90只,7日龄,体质量16~20 g,采用随机数字表法分为6组(n=15):假手术组(Sham组)、HIBD组和咪达唑仑低剂量(10 mg/kg)组(L组)、咪达唑仑中剂量(20 mg/kg)组(M组)、咪达唑仑高剂量(40 mg/kg)组(H组)和Nrf2抑制剂ML385组(I组)。采用结扎左颈动脉并在缺氧环境中暴露2 h建立HIBD模型。造模后第2天开始,各咪达唑仑组腹腔注射相应剂量咪达唑仑,Sham组和HIBD组腹腔注射等容量生理盐水,I组腹腔注射咪达唑仑40 mg/kg和Nrf2抑制剂ML38530 mg/kg,均1次/d,连续给药8 d。给药结束后,称重并进行水迷宫实验。水迷宫实验结束后取腹主动脉血样,然后断头取脑。采用ELISA法测定血清铁、IL-6、TNF-α浓度。采用紫外分光光度法和微量法测定海马组织铁和GSH含量。脑组织经HE染色和Nissl染色后观察海马神经元病理学结果;采用免疫荧光染色法测定海马Nrf2/神经元核抗原(NeuN)和GPX4/NeuN的阳性细胞数;采用Western blot法测定海马组织Nrf2、GPX4、4-羟基壬烯酸(4-HNE)表达。结果:与Sham组相比,HIBD组首次到达平台时间延长,穿越原平台位置次数减少,目标象限停留时间缩短,海马组织铁含量升高,GSH含量、Nrf2/NeuN和GPX4/NeuN的阳性细胞数降低,Nrf2和GPX4表达下调,4-HNE表达上调,血清铁、IL-6和TNF-α浓度升高(P<0.05),海马神经元损伤明显;与HIBD组相比,H组、M组和L组首次到达平台时间缩短,穿越原平台位置次数增多,目标象限停留时间延长,海马组织铁含量降低,GSH含量、Nrf2/NeuN和GPX4/NeuN的阳性细胞数升高,Nrf2和GPX4表达上调,4-HNE表达下调,血清铁、IL-6和TNF-α浓度降低(P<0.05),海马神经元损伤减轻;与H组相比,I组首次到达平台时间延长,穿越原平台位置次数减少,目标象限停留时间缩短,海马组织铁含量升高,GSH含量、Nrf2/NeuN和GPX4/NeuN的阳性细胞数降低,Nrf2和GPX4表达下调,4-HNE表达上调,血清铁、IL-6和TNF-α浓度升高(P<0.05),海马神经元损伤加重。结论:咪达唑仑减轻新生大鼠HIBD的机制可能与激活Nrf2/GPX4信号通路,抑制海马神经元铁死亡有关。

Objective To evaluate the role of nuclear factor-erythroid 2-related factor 2(Nrf2)/glutathione peroxidase-4(GPX4)signaling pathway-mediated ferroptosis in midazolam-induced reduction of hypoxic-ischemic brain damage(HIBD)in neonatal rats.MethodsNinety healthy 7-day-old neonatal rats,weighing 16-20 g,were divided into 6 groups(n=15 each)using the random number table method:sham operation group(Sham group),HIBD group,low-dose midazolam(10 mg/kg)group(group L),medium-dose midazolam(20 mg/kg)group(group M),high-dose midazolam(40 mg/kg)group(group H),and Nrf2 inhibitor ML385 group(group I).The HIBD model was developed by ligating the left carotid artery and exposing to a hypoxic condition for 2 h in anesthetized animals.Starting from^(2)nd day after developing the model,the corresponding doses of midazolam were intraperitoneally injected in midazolam groups,the equal volume of normal saline was intraperitoneally injected in Sham and HIBD groups,midazolam 40 mg/kg and Nrf2 inhibitor ML38530 mg/kg were intraperitoneally injected once a day for 8 consecutive days in group I.The rats were weighed and subjected to the Morris water maze test after the end of administration.Blood samples were taken from the abdominal aorta after the end of the Morris water maze test,and then the animals were sacrificed to remove the brain for determination of the concentrations of serum iron,interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)(by enzyme-linked immunosorbent assay),contents of iron and GSH in hippocampal tissues(by ultraviolet spectrophotometry and micro method),the number of Nrf2/neuronal nuclear antigen(NeuN)and GPX4/NeuN positive cells(by immunofluorescent staining),and expression of Nrf2,GPX4,and 4-hydroxynonaenoic acid(4-HNE)in hippocampal tissues and for microscopic examination of the pathological changes of hippocampal neurons in brain tissues(after HE staining and Nissl staining).ResultsCompared with Sham group,the first time to arrival at platform was significantly prolonged,the number of crossing the origional platform was reduced,and the time of staying at the target quadrant was shortened,the iron content in the hippocampal tissues was increased,the content of GSH and the number of Nrf2/NeuN and GPX4/NeuN positive cells were decreased,the expression of Nrf2 and GPX4 was down-regulated,the expression of 4-HNE was up-regulated,the concentrations of serum iron,IL-6 and TNF-αwere increased,and the injury to hippocampal neurons was marked in HIBD group(P<0.05).Compared with HIBD group,the first time to arrival at platform was significantly shortened,the number of crossing the origional platform was increased,and the time of staying at the target quadrant was prolonged,the iron content in the hippocampus tissues was decreased,the content of GSH and the number of Nrf2/NeuN and GPX4/NeuN positive cells were increased,the expression of Nrf2 and GPX4 was up-regulated,the expression of 4-HNE was down-regulated,the concentrations of serum iron,IL-6 and TNF-αwere decreased(P<0.05),and the injury to hippocampal neurons was significantly reduced in H,M and L groups.Compared with group H,the first time to arrival at platform was significantly prolonged,the number of crossing the origional platform was reduced,and the time of staying at the target quadrant was shortened,the iron content in the hippocampus tissue was increased,the content of GSH and the number of Nrf2/NeuN and GPX4/NeuN positive cells were decreased,the expression of Nrf2 and GPX4 was down-regulated,the expression of 4-HNE was up-regulated,the concentrations of serum iron,IL-6 and TNF-αwere increased(P<0.05),and the injury to hippocampal neurons was aggravated in group I.ConclusionsThe mechanism by which midazolam reduces HIBD may be related to activation of the Nrf2/GPX4 signaling pathway and inhibition of hippocampal neuronal ferroptosis in neonatal rats.