[Role of carbohydrate response element-binding protein/hypoxia-inducible factor-1α signaling pathway in...

To investigate the role of the carbohydrate response element-binding protein/hypoxia-inducible factor-1α (ChREBP/HIF-1α) signaling pathway in sepsis-associated encephalopathy (SAE) in rats.

1) Experiment 1: Forty-two healthy male Sprague-Dawley rats were randomly assigned to a control group (n=6), a 24-hour LPS group (n=12), a 48-hour LPS group (n=12), and a 72-hour LPS group (n=12). The control group was injected with an equal volume of normal saline. Rats in the LPS groups were treated with intraperitoneal lipopolysaccharide (LPS, 10 mg/kg) for 24, 48, or 72 hours to induce SAE. Cognitive function was evaluated by behavioral tests. Histopathological changes in the hippocampus were observed under light microscopy. Levels of pro-inflammatory mediators and glycolytic metabolites in the hippocampus were determined by enzyme-linked immunosorbent assay (ELISA). Hippocampal protein expressions of ChREBP and HIF-1α were measured by Western blotting to assess their association with SAE progression. 2) Experiment 2: Forty-eight healthy male Sprague-Dawley rats were randomly assigned to a control group (n=12), an LPS-induced SAE group (SAE group, n=12), an SAE+ChREBP-specific antagonist ChREBPα/14-3-3 regulator-1 (CR1) group (n=12), and an SAE+CR1+high-glucose (Glu) group (n=12). The control group was injected with an equal dose of vehicle. SAE was induced in the SAE group by intraperitoneal injection of LPS (10 mg/kg). In the SAE+CR1 group, CR1 (15 mg/kg) was intraperitoneally administered once every 24 hours after SAE induction. In the SAE+CR1+Glu group, CR1 (15 mg/kg) was administered once every 24 hours after SAE induction in combination with high-concentration Glu (1 g/kg, once every 6 hours). Cognitive function was assessed by behavioral tests. Histopathological changes in the hippocampus were observed under light microscopy. The changes of microglia in the hippocampus were observed under a fluorescence microscope. Blood-brain barrier permeability, inflammatory mediator, glycolytic metabolism, and neuronal injury markers levels were measured by ELISA. Expressions of glycolytic enzymes and apoptosis-related proteins in the hippocampus were determined by Western blotting to evaluate the relationship between SAE and the ChREBP/HIF-1α signaling pathway.

1) Results of experiment 1: Compared with the control group, from LPS exposure for 48 hours on, the freezing time ratio and discrimination index reduced, the expressions of ChREBP and HIF-1α increased, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10 levels and lactic acid/pyruvate ratio (LPR) elevated, and the number of degenerative neurons increased and neuronal number decreased (all P<0.05). These changes were accompanied by disordered neuronal arrangement, reduced Nissl bodies, and increased nuclear pyknosis and hyperchromasia. Therefore, intraperitoneal injection of 10 mg/kg LPS for 48 hours was selected as the condition for SAE model establishment in subsequent experiments. 2) Results of experiment 2: Compared with the control group, rats in the SAE group exhibited significant cognitive impairment and neuroinflammation, as evidenced by decreased freezing time ratio and discrimination index, increased hippocampal Evans blue, IL-6, TNF-α, IL-10 levels and LPR, elevated peripheral blood levels of neuron specific enolase (NSE) and S100β, increased neuronal degeneration and reduced neuronal density, and the obvious microglial activation and M1 polarization in the hippocampus, as well as the upregulated protein expressions of ChREBP, HIF-1α, and cleaved caspase-3 (all P<0.05). Compared with the SAE group, CR1 treatment significantly improved the cognitive function, metabolism, inflammation, cell polarization, and apoptosis in rats. The freezing time ratio and the discrimination index were increased [freezing time ratio: (15.1±2.2)% vs. (6.9±1.6)%, discrimination index: (53.0±5.6)% vs. (41.0±6.0)%, both P<0.05]. The hippocampal Evans blue, IL-6, TNF-α levels and LPR were reduced [Evans blue (mg/g): 2.09±0.26 vs. 2.94±0.42, IL-6 (ng/g): 96.56±11.50 vs. 176.50±21.20, TNF-α (ng/g): 176.50±21.20 vs. 298.66±34.60, LPR: 14.76±3.65 vs. 25.62±3.44, all P<0.05]. The activation of microglia and the degree of M1 polarization were reduced. Serum NSE and S100β levels were also reduced [NSE (ng/L): 20.37±2.65 vs. 31.22±4.13, S100β (ng/L): 100.83±11.25 vs. 165.55±21.26, both P<0.05]. Neuronal degeneration was alleviated and neuronal density increased. The protein expressions of ChREBP, HIF-1α, and cleaved caspase-3 were all downregulated (all P<0.05). However, these effects of CR1 were reversed by high-Glu intervention.

LPS exposure upregulates the expressions of ChREBP and HIF-1α and promotes neuroinflammation and cognitive dysfunction. A ChREBP-specific antagonist attenuates pro-inflammatory microglial polarization and neuroinflammation by inhibiting the ChREBP/HIF-1α pathway, thereby alleviating hippocampal histopathological injury and cognitive dysfunction in SAE.