海马神经细胞铁死亡通过Nrf 2/GPX4信号通路导致脓毒症相关性脑病大鼠认知功能障碍

Hippocampal neuronal ferroptosis involved in cognitive dysfunction in rats with sepsis-related encephalopathy through the Nrf 2/GPX4 signaling pathway

目的评价海马神经细胞铁死亡对脓毒症相关性脑病(SAE)大鼠认知功能障碍的影响及其可能的分子机制。方法①SAE制模条件筛选实验:将42只健康雄性SD大鼠按随机数字表法分为生理盐水(NS)对照组(n=6)及脂多糖(LPS)5、15、30 mg/kg组(均n=12)。通过观察大鼠72 h内存活情况及平均动脉压(MAP)、心率(HR)变化,72 h时僵直时间百分比及血清炎性因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和神经元损伤标志物神经元特异性烯醇化酶(NSE)水平,血清铁和乳酸(Lac)含量,以及海马CA1区细胞形态学改变等指标,明确SAE模型制备条件。②铁离子螯合剂去铁胺(Def)干预实验:根据筛选实验结果,将28只健康雄性SD大鼠按随机数字表法分为NS对照组(n=8)、SAE组(n=10)及Def+SAE组(n=10)。Def+SAE组于制模前12 h开始腹腔注射Def 100 mg/kg,12 h注射1次,共7次;NS对照组和SAE组注射等量NS。采用条件性恐惧实验记录大鼠僵直时间百分比以评价认知功能;采用酶联免疫吸附试验(ELISA)测定血清IL-6、TNF-α及NSE水平;采用化学比色法测定血清Lac和铁含量及海马丙二醛(MDA)和铁含量;采用蛋白质免疫印迹试验(Western Blot)检测海马核因子E2相关因子2(Nrf 2)、谷胱甘肽过氧化酶4(GPX4)、还原型辅酶Ⅱ氧化酶1(NOX1)的蛋白表达;苏木素-伊红(HE)染色后,光镜下观察海马CA1区细胞形态学改变。结果①与NS对照组相比,腹腔注射15 mg/kg LPS可使大鼠MAP、HR明显降低,并呈时间依赖性,72 h时降低最为显著,72 h存活率明显下降,认知功能障碍,血清IL-6、TNF-α、Lac、NSE含量明显升高,血清铁含量显著下降,且光镜下显示海马CA1区锥体细胞形态不规则,部分细胞水肿明显呈空泡状;LPS 5 mg/kg组大鼠生命体征、炎症反应、器官功能及认知功能障碍均不明显;LPS 30 mg/kg组大鼠出现明显的脓毒性休克症状。故选择腹腔注射15 mg/kg LPS 72 h建立SAE模型。②与NS对照组相比,SAE组大鼠僵直时间百分比明显降低,血清IL-6、NSE含量及海马MDA、铁含量明显升高,血清铁含量及海马Nrf 2、GPX4蛋白表达显著降低,海马NOX1蛋白表达明显升高,且光镜下显示海马CA1区锥体细胞形态不规则,胞质深染,表明SAE大鼠海马区氧化应激水平升高,神经元退行性改变明显,认知功能受损。与SAE组相比,Def+SAE组大鼠僵直时间百分比明显增加〔(63.4±6.4)%比(47.6±6.0)%,P<0.05〕,血清IL-6、NSE、铁含量及海马MDA和铁含量显著降低〔血清IL-6(ng/L):73.14±8.31比99.86±12.37,血清NSE(μg/L):3.67±0.51比5.92±0.79,血清铁(mg/L):68.43±8.12比134.60±15.63,海马MDA(mol/g):4.62±0.90比6.62±0.84,海马铁(μg/g):155.32±17.86比221.54±27.54,均P<0.05〕,海马Nrf 2、GPX4蛋白表达均明显升高〔Nrf 2/β-actin:0.41±0.07比0.18±0.03,GPX4/β-actin:0.74±0.09比0.40±0.06,均P<0.05〕,海马NOX1蛋白表达明显降低(NOX1/β-actin:0.62±0.08比1.11±0.16,P<0.05),且光镜下显示锥体细胞形态较SAE组明显改善,说明Def+SAE组大鼠海马区氧化应激水平降低,神经元退行性变显著减缓,认知功能明显改善。结论SAE大鼠认知功能受损,海马神经细胞损伤明显、铁死亡增多;Def预处理可显著减少SAE大鼠海马区铁沉积,减轻神经细胞铁死亡,改善认知功能障碍,该作用可能与激活Nrf 2/GPX4信号通路有关。

Objective To evaluate the effects of ferroptosis in hippocampal neurons on cognitive dysfunction in rats with sepsis-associated encephalopathy(SAE)and its potential molecular mechanisms.Methods①Screening experiment of SAE modeling conditions:42 healthy male Sprague-Dawley(SD)rats were divided into normal saline(NS)control group(n=6)and lipopolysaccharide(LPS)5,15,30 mg/kg groups(each n=12)according to the random number table method.The SAE modeling conditions were determined by survival and the changes in mean arterial pressure(MAP)and heart rate(HR)within 72 hours,the percentage of stiffness status,the levels of serum inflammatory factors including interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α),neuron specific enolase(NSE,a marker of neuronal injury),serum iron and lactic acid(Lac)contents,and the morphological changes in CA1 of hippocampus after 72 hours.②Deferoxamine(Def)intervention experiment:according to the results of screening experiments,28 healthy male SD rats were divided into NS control group(n=8),SAE group(n=10)and Def+SAE group(n=10)according to the random number table method.In the Def+SAE group,100 mg/kg Def was injected intraperitoneally 12 hours before the modeling,once every 12 hours,with a total of 7 times;the rats in the NS control group and SAE group were injected with the same amount of NS.Then the cognitive function of rats was evaluated by fear conditioning test for the percentage of stiffness status;serum IL-6,TNF-αand NSE levels were determined by enzyme-linked immunosorbent assay(ELISA);the levels of serum Lac,serum iron and hippocampal malondialdehyde(MDA)and iron contents were determined by chemical colorimetric;the protein expressions of nuclear factor E2 related factor 2(Nrf 2),glutathione peroxidase 4(GPX4)and NAPDH oxidase 1(NOX1)in hippocampus were determined by Western Blot;morphological changes in hippocampal CA1 were observed after hematoxylin and eosin(HE)staining.Results①Compared with the NS control group,intraperitoneal injection of 15 mg/kg LPS could significantly reduce the MAP and HR as time prolonged,and the reduction was most significant at 72 hours.The 72-hour survival rate was significantly reduced and cognitive function was impaired.The levels of serum IL-6,TNF-α,Lac and NSE were increased while the serum iron content was decreased significantly.The morphology of vertebral cells in hippocampal CA1 was irregular and some of the cells were obviously vacuolated.In the LPS 5 mg/kg group,there were no significant changes in vital signs,inflammation,organ function or cognitive dysfunction,while the symptoms of septic shock were apparent in the LPS 30 mg/kg group.Therefore,SAE model was reproduced by intraperitoneal injection of 15 mg/kg LPS for 72 hours.②Compared with the NS control group,the percentage of stiffness in the SAE group was significantly reduced.The levels of serum IL-6,NSE and hippocampal MDA,iron were significantly increased.The serum iron contents and hippocampal Nrf 2 and GPX4 protein expressions were significantly reduced,while the hippocampal NOX1 protein expression was significantly increased.The morphology of vertebral cells in hippocampal CA1 was irregular and the cytoplasm was deeply stained.The results indicated that the level of oxidative stress in the hippocampus of SAE rats was increased,the neuron degenerations were obvious,and the cognitive function of rats were impaired.Compared with the SAE group,the percentage of stiffness in the Def+SAE group was significantly increased[(63.4±6.4)%vs.(47.6±6.0)%,P<0.05].The levels of serum IL-6,NSE,iron and hippocampal MDA,iron were significantly reduced[serum IL-6(ng/L):73.14±8.31 vs.99.86±12.37,serum NSE(μg/L):3.67±0.51 vs.5.92±0.79,serum iron(mg/L):68.43±8.12 vs.134.60±15.63,hippocampal MDA(mol/g):4.62±0.90 vs.6.62±0.84,hippocampal iron(μg/g):155.32±17.86 vs.221.54±27.54,all P<0.05].The hippocampal protein expressions of Nrf 2 and GPX4 were significantly increased[Nrf 2/β-actin:0.41±0.07 vs.0.18±0.03,GPX4/β-actin:0.74±0.09 vs.0.40±0.06,all P<0.05]while the hippocampal NOX1 protein expression was significantly reduced(NOX1/β-actin:0.62±0.08 vs.1.11±0.16,P<0.05).The vertebral cells was significantly improved as compared with the SAE group.These findings showed that the oxidative stress level in hippocampus of the Def+SAE group was reduced,neuron degeneration was significantly alleviated,and the cognitive function of the rats was significantly improved.Conclusions The cognitive function of rats with SAE was significantly impaired,the hippocampal neurons were obviously damaged and ferroptosis was increased.Def pretreatment could significantly reduce iron deposition and ferroptosis in hippocampal neurons of SAE rats and improve cognitive dysfunction,which may be related to activation of Nrf 2/GPX4 signaling pathway.