Hippocampal neuronal ferroptosis involved in cognitive dysfunction in rats with sepsis-related encephalopathy through the Nrf2/GPX4 signaling pathway

Abstract

To evaluate the effects of ferroptosis in hippocampal neurons on cognitive dysfunction in rats with sepsis-associated encephalopathy (SAE) and its potential molecular mechanisms. (1) Screening 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. (2) Deferoxamine (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-α and 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 (Nrf2), 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. (1) Compared 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. (2) Compared 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 Nrf2 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 Nrf2 and GPX4 were significantly increased [Nrf2/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. 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 Nrf2/GPX4 signaling pathway.