Baicalein Alleviates Lithium-Pilocarpine-Induced Status Epilepticus by Regulating DNMT1/GABRD Pathway in Rats

Abstract

Background: Epilepsy is a common disease of the nervous system. Recent advances in epigenetics have revealed DNA methylation as a key mechanism in epilepsy pathogenesis, particularly through dysregulation of GABAergic signaling. Baicalein has been shown to have anticonvulsant and neuroprotective effects. However, its epigenetic regulatory effects on GABA receptor function remain unexplored.

Methods: The status epilepticus (SE) model was induced by lithium chloride-pilocarpine (LiCl-PILO) in Sprague-Dawley (SD) rats. The rats were divided into control group, epileptic SE group and baicalein intervention group. Morris water maze (MWM) test, Nissl staining, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) were used to detect cognitive functions and neuronal damage. Online sites, chromatin immunoprecipitation (ChIP) and western blotting were used to identify DNA methyltransferase 1 (DNMT1)-mediated methylation of gamma-aminobutyric acid type A receptor subunit delta (GABRD) promoter region.

Results: Baicalein treatment significantly prolonged the latency of SE onset and seizure onset, and improved the development of epilepsy. Meanwhile, baicalein improved the cognitive impairment in rats induced by LiCl-PILO. After treatment with baicalein, a sustained elevation in the number of neurons and NeuN levels was observed, along with a decrease in the contents of tumor necrosis factor -alpha (TNF-α), interleukin-1β (IL-1β), and ionized calcium-binding adapter molecule 1 (Iba-1) in the hippocampus. Mechanistically, baicalein interacted with DNMT1 to suppress GABRD promoter region methylation, thus increasing GABRD protein level in the hippocampus of rats induced by LiCl-PILO.

Conclusion: This study identifies DNMT1/GABRD axis as a novel epigenetic target for epilepsy intervention. Baicalein's ability to enhance tonic inhibition through demethylation of GABRD provides a groundbreaking strategy for drug-resistant epilepsy.

Keywords: Baicalein; DNMT1; GABRD; epilepsy; neuro.

Graphical abstract

Figure 1.

Effect of Baicalein on behavioral seizures induced by SE. A: chemical structure of baicalein. B: seizure scores after were decreased baicalein intervention (n = 10). C: seizure latency was increased after baicalein intervention (n = 10). D and E: seizure rate and seizure duration were improved after baicalein intervention (n = 10). *** p < 0.001，** p < 0.01, * p < 0.05.

Figure 2.

Effects of Baicalein on SE induced cognitive deficits. A: swimming velocity during the spatial probe test after baicalein intervention (n = 10). B: the escape latency was reduced after baicalein intervention in comparison with SE group (n = 10). C-E: number of platform crossings, the time spent and distance spent in the target quadrant were increased after baicalein intervention (n = 10). *** p < 0.001，** p < 0.01, * p < 0.05.

Figure 3.

Effects of Baicalein on SE induced hippocampal neuron injury. A: Nissl staining of neuron in hippocampal tissues of each group rats (n = 5). Scale bar = 50 μm. B: quantitative analysis of Nissl-positive neurons in the CA1 region in each group. Compared with the SE group, baicalein increased the number of Nissl-positive hippocampal neurons in CA1 region. C: immunofluorescence staining of NeuN (green) in hippocampal CA1 region of rats in each group (n = 5). The nuclei were labeled with DAPI (blue). Scale bar = 50 μm. D: Western blotting showed that the content of caspase-3 in hippocampal tissues was reduced after baicalein treatment (n = 5). Quantitative analysis of caspase-3 protein in the CA1 region in each group. *** p < 0.001，** p < 0.01, * p < 0.05.

Figure 4.

Effect of Baicalein on hippocampal neuroinflammation induced by SE. A and B: the contents of TNF-α and IL-1β in hippocampal tissues of each group were analyzed by ELISA (n = 5). C: quantitative analysis of the number of microglia in the CA1 region in each group. Compared with the SE group, the intervention group with baicalein showed a significant decrease in the number of microglia (iba-1 positive cells). D: immunofluorescence staining of iba-1 (red) in hippocampal tissue CA1 region of rats in each group (n = 5). The nuclei were labeled with DAPI (blue). Scale bar = 50 μm. *** p < 0.001，** p < 0.01, * p < 0.05.

Figure 5.

Effect of Baicalein on DNMT1/GABRD axis in SE hippocampus. A: Western blotting showed that baicalein treatment resulted in the upregulation of GABRD protein level and the inhibition of DNMT1 protein level in HT22 cells. B: quantitative analysis of GABRD protein in HT22 cells after baicalein treatment. C: MethPrimer predicted potentially hypermethylated CpG islands (blue) within GABRD promoter region. D: ChIP evaluating the recruitment of DNMT1, DNTM3A, DNMT3B at GABRD promoter region. DNMT1 was the main regulator of methylation in the GABRD promoter region. E: Western blotting showed that DNMT1 overexpression resulted in the inhibition of GABRD protein level, while DNMT1 silencing resulted in the upregulation of GABRD protein level in HT22 cells. Quantitative analysis of GABRD level in HT22 cells after DNMT1 upregulation or inhibition. F: quantitative analysis of DNMT1 protein in HT22 cells after baicalein treatment. G: IHC analysis GABRD and DNMT1 level in hippocampal tissue CA1 region of each group rats (n = 5). Compared with the SE group, baicalein decreased DNMT1expession and increased GABRD expression in CA1 region. Scale bar = 50 μm. *** p < 0.001，** p < 0.01, * p < 0.05.