Quercetin Administration Following Hypoxia-Induced Neonatal Brain Damage Attenuates Later-Life Seizure Susceptibility and Anxiety-Related Behavior: Modulating Inflammatory Response

Abstract

Background: Neonatal seizures commonly caused by hypoxia could lead to brain injury and cognitive deficits. Quercetin could cross the blood brain barrier and exerts neuroprotective effects in many neurological disease settings. In this study, we aim to investigate the role of quercetin in attenuating cognitive impairment following hypoxia-induced neonatal seizure (HINS).

Method: Sprague-Dawley rats at P7 were exposed to a premixed gas in a hypoxic chamber to induce brain injury, and then continuously administered with quercetin for 21 days. Pentylenetetrazol kindling was used to induce seizures in the evolution. After the hypoxic lesion was stablished, anxiety-related behavior of rats after HINS was assessed using open field test. Memory impairment of rats after HINS was evaluated using novel object-recognition test and elevated plus maze test. The serum and hippocampal concentrations of TNF-a, iNOS, IL-6 MCP-1, and IL-1β were measured using ELISA. The mRNA expression levels of TNF-a, iNOS, IL-6 in the hippocampus were determined using qRT-PCR. The protein levels of TLR4, NF-κB p65, and p-NF-κB p65 in the hippocampus were determined using Western blot.

Results: Quercetin administration significantly reduced later-life seizure susceptibility, anxiety-related behavior, and memory impairments in the rats following the HINS when compared to the HINS group without treatment. Both serum and hippocampal proinflammatory cytokines levels were significantly elevated in the rat after HINS. TLR4 protein expressions were increased in the HINS group when compared to control group, and decreased in the group of quercetin. The protein level of p-NF-κB p65 was significantly lower in the quercetin group compared to the HINS group.

Conclusion: We demonstrated that Quercetin significantly reduced susceptibility to later-life seizures. Quercetin could downregulate inflammatory response through TLR4/ NF-κB pathway, thereby attenuating HINS-induced anxiety, hippocampal memory impairment, and cognitive impairment in later life following HINS.

Keywords: NF-κB; TLR4; hypoxia-induced neonatal seizure; inflammation; quercetin.

Figure 1

Quercetin administration following HINS attenuated later-life seizure susceptibility in rats. (A) Effect of quercetin on body weight following HINS. (B) Maximum seizure stages during 20 min after PTZ injections in different experimental groups. (C) The averaged days to reach fully kindled situation in different experimental groups. (D) Total MS durations were recorded in different experimental groups. (E) Total GS durations were recorded in different experimental groups. (F) Stage 5 latency were recorded in different experimental groups. Eight rats were used to recorded in each group. Mean ± SD. **p < 0.01, ***p < 0.001 compared to control. #p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to HINS.

Figure 2

Quercetin administration following HINS attenuated anxiety-related behavior of rats in Open-Field test. Total traveled distance (A) and number of central crossings (B) over 10 min exploring the arena were recorded. Eight rats were used to recorded in each group. Mean ± SD. ***p < 0.001 compared to control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to HINS.

Figure 3

Quercetin administration following HINS attenuated memory impairments of rats in novel object-recognition test (NOR) and elevated plus maze test (EPM). The percentage of OAE (A) and OAT (B) in EPM were recorded. (C) NOR was evaluated by calculating the novel object index. Eight rats were used to record in each group. Mean ± SD. ***p < 0.001 compared to control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to HINS.

Figure 4

Quercetin administration following HINS attenuated serum inflammatory cytokines. ELISA was used to measure the concentrations of IL-6 (A), TNF-α (B), MCP-1 (C), and IL-1β (D) in serum of rats from different groups. Eight rats were used in each group. Mean ± SD. ***p < 0.001 compared to control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to HINS.

Figure 5

Quercetin administration following HINS attenuated hippocampal inflammatory responses of rats. ELISA was used to measure the concentrations of iNOS (A), TNF-α (B), and IL-6 (C) in the hippocampus of rats from different groups. RT-qPCR was used to measure the mRNA expressions of iNOS (D), TNF-α (E), and IL-6 (F) in the hippocampus of rats from different groups. Eight or 3 rats were used in each group. Mean ± SD. **p < 0.01, ***p < 0.001 compared to control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to HINS.

Figure 6

Quercetin administration following HINS down-regulated hippocampal TLR4/NF-κB signaling. The hippocampal protein expressions of TLR4, p-p65, and p65 (A) were measured by western blotting. The expressions were normalized to control (B,C). Three rats were used in each group. Mean ± SD. **p < 0.01, ***p < 0.001 compared to control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to HINS.