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

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1 mL剂量为20、40和80 mg/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Fe^(2+)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Fe^(2+)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。

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 selective 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 S100β levels were detected by ELISA.The water content of brain tissue 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 Fe^(2+)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 damage reduced,the levels of serum NSE and S100βdecreased(P<0.05),the level of GSH in brain tissue increased(P<0.05).The water content of brain tissue and levels of MDA,IL-6,TNF-α and the content of Fe^(2+)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-induced sepsis rats.