Creb1 aggravates glutamate-induced excitotoxicity and KA-induced seizure activity by transcriptionally activating...

Epilepsy is a chronic neurological disorder, more common in children and the elderly. Although treatments have advanced, significant challenges remain. This study focuses on its molecular mechanisms to provide new insights and strategies for its treatment. Gene expression omnibus (GEO) database analysis, enrichment analysis, and the establishment of protein-protein interaction (PPI) networks were conducted for gene screening. HT-22 cells were treated with glutamate (Glu) to establish an in vitro epilepsy model. Gene knockdown and overexpression were achieved through cell transfection. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while gene expression at the protein and messenger RNA (mRNA) levels was detected by western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to analyze apoptosis and mitochondrial membrane potential. Lactate dehydrogenase (LDH), reactive oxygen species (ROS), and Fe²⁺ levels were measured using commercial kits. Kainic acid (KA) was administered intraperitoneally to mice to establish an epilepsy animal model, which was used to verify the role of the gene in epilepsy development. Transcription factor binding sites in target gene promoters were predicted using the JASPAR database and validated by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. Tissue inhibitor of metalloproteinases 1 (Timp1) was highly expressed in tissue samples from the epilepsy mouse models of GSE88992 and GSE73878, as well as in the Glu-induced HT-22 cell model. In these models, Timp1 knockdown increased cell viability, reduced apoptosis, decreased LDH, promoted mitochondrial membrane potential, and lowered ROS, Fe²⁺, and acyl-CoA synthetase long-chain family member 4 (Acsl4) levels. In KA-induced animal models, Timp1 knockdown reduced seizure score, cleaved caspase-3 (C-caspase3)/total caspase-3, and Acsl4 expression. cAMP response element-binding protein 1 (Creb1) transcriptionally activated Timp1. In the Glu-induced HT-22 cell model, Creb1 knockdown decreased Timp1 protein expression, increased cell viability and mitochondrial membrane potential, and reduced apoptosis, LDH, ROS, Fe²⁺, and Acsl4 levels, while Timp1 overexpression partially reversed these effects. Taken together, Creb1 promotes the progression of epilepsy by activating Timp1, providing a new perspective and potential targets for epilepsy treatment.

The online version contains supplementary material available at https://doi.org/10.1007/s10616-026-01084-7.