地黄苷A对CLP诱导的脓毒症大鼠脑功能障碍和Nrf2/GPX4介导的铁死亡通路的影响
Effects of Rehmannioside A on Brain Dysfunction and Nrf2/GPX4-mediated Ferroptosis Pathway in CLP-induced Sepsis Rats
许静 1霍康 2郭秦乐 3白立曦 1李娜 4杜俊凯1
作者信息
- 1. 西安交通大学第一附属医院急诊中心 陕西西安 710061
- 2. 西安交通大学第一附属医院神经内科 陕西西安 710061
- 3. 西安交通大学第一附属医院重症医学科 陕西西安 710061
- 4. 西安交通大学第一附属医院检验科 陕西西安 710061
- 折叠
摘要
目的:探究地黄苷A(ReA)对盲肠结扎穿孔(CLP)诱导的脓毒症大鼠脑功能障碍的影响及机制.方法:将56只CLP诱导的脓毒症 SD 大鼠随机分为 CLP 组(n=12)、CLP+20ReA 组(n=11)、CLP+40ReA 组(n=11)、CLP+80ReA 组(n=11)和 GPX4-IN-3 组(n=11),将只进行假手术的10只SD大鼠作为Sham组.Sham组和CLP组大鼠灌胃1 mL 1％二甲基亚砜.CLP+20ReA组、CLP+40ReA组、CLP+80ReA组大鼠分别灌胃1mL剂量为20、40和80mg/kg/d的地黄苷A.GPX4-IN-3组大鼠同时灌胃0.5 mL剂量为80 mg/kg/d的地黄苷A和0.5 mL剂量为15 mg/kg/d的铁死亡选择性诱导剂GPX4-IN-3.各组大鼠均给药3 d.通过神经功能评分和Morris水迷宫实验评价大鼠脑功能;采用ELISA法检测大鼠神经元特异性烯醇化酶(NSE)和S100β水平;采用称重法检测大鼠脑组织含水量;采用HE染色和尼氏染色评价大鼠脑损伤情况;采用微量法检测大鼠脑组织还原型谷胱甘肽(GSH)、丙二醛(MDA)水平和Fe2+含量;采用ELISA法检测大鼠脑组织白细胞介素.6(IL-6)和肿瘤坏死因子-α(TNF-α)水平;采用Western blot检测大鼠脑组织核因子E2相关因子2(Nrf2)、Kelch样ECH相关蛋白1(Keap1)和谷胱甘肽过氧化物酶4(GPX4)蛋白水平.结果:与CLP组比较,CLP+20ReA组、CLP+40ReA组和CLP+80ReA组大鼠的神经功能评分和逃避潜伏期降低(P＜0.05),穿越平台次数升高(P＜0.05),神经元损伤减轻,血清NSE和S100β水平降低(P＜0.05),脑组织GSH水平升高(P＜0.05),脑组织含水量、MDA、IL-6和TNF-α水平以及Fe2+含量均降低(P＜0.05),脑组织Nrf2(细胞核)和GPX4的蛋白表达水平升高(P＜0.05),Keap1的蛋白表达水平降低(P＜0.05).与CLP+80ReA组比较,GPX4-IN-3组大鼠的脑功能障碍加重,Nrf2/GPX4通路被抑制(P＜0.05).结论:地黄苷A通过激活Nrf2/GPX4通路抑制铁死亡,从而减轻CLP诱导的脓毒症大鼠脑功能障碍.
Abstract
Objective:To investigate the effect and mechanism of Rehmannioside A(ReA)on brain dysfunction in cecal ligation and perforation(CLP)-induced sepsis rats.Methods:56 CLP-induced sepsis rats were randomly divided into CLP group(n=12),CLP+20ReA group(n=11),CLP+40ReA group(n=11),CLP+80ReA group(n=11),and GPX4-IN-3 group(n=11).Ten rats underwent Sham operation as sham group.Rats in Sham group and CLP group were given 1 mL dimethyl sulfoxide(DMSO).Rats in CLP+20ReA group,CLP+40ReA group and CLP+80ReA group were given 1 mL of 20,40 and 80 mg/kg/d Rehmannioside A,respectively.Rats in GPX4-IN-3 group were simultaneously administrated with 0.5 mL 80 mg/kg/d of Rehmannioside A and 0.5 mL 15 mg/kg/d of the selec-tive inducer of ferroptosis GPX4-IN-3.Each group was given the drug for 3 days.Neural function score and Morris water maze test were used to evaluate brain function.Neuron-specific enolase(NSE)and S100 β levels were detected by ELISA.The water content of brain tis-sue was measured by weighing method.Brain injury was evaluated by HE staining and Nissl staining.The levels of reduced glutathione(GSH)and malondialdehyde(MDA)in brain tissue were detected by micromethods.The levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were measured by ELISA.The content of Fe2+in brain tissue was determined by micromethod.The levels of nuclear factor E2-related factor 2(Nrf2),Kelch-like ECH related protein 1(Keap1)and glutathione peroxidase 4(GPX4)protein in brain tissue were detected by Western blot.Results:Compared with CLP group,the neural function scores and escape latency of CLP+20ReA group,CLP+40ReA group and CLP+80ReA group decreased(P＜0.05),the number of crossing platforms increased(P＜0.05),the neuronal dam-age reduced,the levels of serum NSE and S1 00(3 decreased(P＜0.05),the level of GSH in brain tissue increased(P＜0.05).The water con-tent of brain tissue and levels of MDA,IL-6,TNF-α and the content of Fe2+in brain tissue decreased(P＜0.05),the protein expressions of Nrf2(nucleus)and GPX4 in brain tissue increased(P＜0.05),and the protein expression of Keap1 decreased(P＜0.05).Compared with CLP+80ReA group,the brain dysfunction of GPX4-IN-3 group was aggravated,and the Nrf2/GPX4 pathway was inhibited(P＜0.05).Conclusion:Rehmannioside A inhibits ferroptosis by activating Nrf2/GPX4 pathway,thereby alleviating brain dysfunction in CLP-in-duced sepsis rats.
关键词
脓毒症相关脑病/地黄苷A/铁死亡/核因子E2相关因子2/谷胱甘肽过氧化物酶4/脑功能障碍Key words
Sepsis related encephalopathy/Rehmannioside A/Ferroptosis/Nuclear factor E2 related factor 2/Glutathione peroxi-dase 4/Brain dysfunction引用本文复制引用
基金项目
陕西省自然科学基金-面上项目(2023-JC-YB-736)
陕西省自然科学基础研究计划项目(2018JM7152)
陕西省重点研发计划(2024SF-YBXM-525)
出版年
2024
NETL
NSTL
万方数据