Epilepsy is one of the most common neurological disorders worldwide, with pathogenesis involving abnormal neuronal networks and oxidative damage. Effective interventions remain limited, highlighting the importance of exploring novel targets. This study aimed to investigate how downregulation of profilin-1 (PFN1) exerts neuroprotective effects by stabilizing calcium homeostasis and suppressing oxidative stress in hippocampal neurons via the RhoA/ROCK2 signaling pathway in epilepsy.

Bioinformatics analysis was performed using the Gene Expression Omnibus (GEO) and the Human Protein Atlas (THPA) databases to identify key differentially expressed genes in temporal lobe epilepsy. For in vivo experiments, 82 male C57 BL/6 mice (aged 8 to 10 weeks, weighting 20 to 25 g) were divided into control, epileptic model, vector control, and PFN1-interfered groups. The vector control and PFN1-interfered groups received stereotactic injections of siCon or siPFN1 complexes, respectively, followed by kainic acid (KA)-induced epilepsy modeling 72 h later; the other 2 groups received saline and KA only. RT-qPCR and Western blotting validated PFN1 expression in hippocampal neurons. Electroencephalogram and Racine scores were used to evaluate seizure characteristics and electroencephalographic features. Open field tests assessed spontaneous locomotion and exploratory behavior. RT-qPCR, Western blotting, immunohistochemistry, and histological staining analyzed PFN1's protective effects against hippocampal neuronal injury. For in vitro experiments, HT22 cells were pretreated with PFN1 interference before establishing the epileptic cell models using Mg²⁺-free extracellular fluid under identical conditions, with calcium imaging, reactive oxygen species(ROS) probes, and flow cytometry assessing calcium homeostasis and oxidative stress. Co-immunoprecipitation and Western blotting verified PFN1's interaction with the RhoA/ROCK2 pathway.

Bioinformatic analyses revealed significant upregulation of PFN1 expression in epileptic models. Results from characterization demonstrated elevated PFN1 transcription and protein expression (P<0.01) in epileptic model, with PFN1 co-localized with NeuN in the cytoplasm of hippocampal CA1/CA3 neurons. PFN1 knockdown significantly reduced Racine scores (P<0.001), prolonged seizure latency (P<0.001), improved epileptiform discharges and neuronal morphology, and increased NeuN-positive cells and Nissl bodies (P<0.01). Mechanistically, PFN1 formed a complex with RhoA and ROCK2; Its knockdown suppressed pathway protein expression (P<0.05), downregulated CaMK2 levels (P<0.05), and inhibited calcium influx (P<0.001) and ROS generation (P<0.001).

Downregulation of PFN1 may protect neurons in epilepsy by stabilizing calcium homeostasis and suppressing oxidative stress through the RhoA/ROCK2 signaling pathway.

To investigate and validate the expression profiles of Ppp3cb and Ppm1g through differential proteomic analysis of hippocampal tissue in NHE1 gene knockout mice with proteomic analysis.

① Six 2-week-old NHE1 knockout mice were selected as the model group, and 6 wild-type mice of the same age served as the control group, and their genotypes were detected by agar-gel electrophoresis. Open field test and forced swimming test were used to evaluate the behaviors of mice in the model group and control group, and epileptic seizure was graded according to Racine scoring. ②Tandem mass spectrometry was employed to screen the differential proteins in the hippocampus tissues from the model group and the control group. Then the obtained differential proteins were annotated and enriched in the Gene Ontology (GO) database. Search tool for the retrieval of interesting genes (STRING) database was used to analyze protein-protein interaction (PPI) among different proteins. ③The transcriptional and translational levels of Ppp3cb and Ppm1g were detected by qPCR and Western blotting, respectively, and their expression levels in the tissues were observed with immunohistochemistry.

① NHE1 was not expressed in the model group. The mice of the model group had shorter total movement distance (P=0.007 3) and less crossing cells (P<0.000 1) in open field test, and longer period of immobility in forced swimming test (P<0.000 1) when compared with those from the control group. ②When fold change ≥1.2 times and P<0.05 were set as the significant threshold for differential expression, 845 differentially expressed protein sites were detected in the hippocampus, among which 9 proteins (including Ppm1g) were up-regulated and 7 ones (including Ppp3cb) were down-regulated. Gene Ontology (GO) functional analysis showed that after NHE1 knockout, the most significant differences were observed in the concentration of molecular function (MF) related to protein serine/threonine phosphatase activity, concentration of cellular component (CC) related to the plasma membrane, and concentration of biological process (BP) related to negative regulation of biological processes and immune system processes. STRING analysis indicated that the differential proteins Ppp3cb and Slc9a1 directly acted, Ppm1g indirectly acted through Ppp3cb and Slc9a1, and Ppp3cb and Ppm1g interacted. ③The transcriptional and translational levels of Ppp3cb were decreased, and its expression level was reduced in the tissues, while those of Ppm1g were increased, and its expression was elevated in the tissues (P<0.05).

In the hippocampus of NHE1 gene knockout mice, the expression of differential protein Ppp3cb is down-regulated and that of Ppm1g is up-regulated, which provide a basis for further study on their involvement in the pathogenesis of epilepsy.