Hippocampus RNA Sequencing of Pentylenetetrazole-Kindled Rats and Upon Treatment of Novel Chemical Q808

Abstract

The expression of genes altered in epilepsy remains incomplete, particularly in the hippocampus, which exhibits exquisite vulnerability to epilepsy. Q808 is an innovation chemical compound that has potent anti-convulsant effect. Exploring its mechanism can not only explore the pathogenesis of epilepsy but also provide a theoretical basis for its clinical application. The present study aimed to use RNA sequencing (RNA-seq) to reveal the gene transcriptomic profile of chronic pentylenetetrazole (PTZ)-kindled seizure rats and the difference of the PTZ model rat before and after treatment with Q808. Quantitative real-time PCR (qRT-PCR) was performed to validate the RNA-seq results. The protein level was estimated with Western blot. Hippocampal transcriptomic analysis showed that 289 differentially expressed genes (DEGs) were confirmed in the PTZ-kindled seizure group compared with the vehicle control. Gene cluster analysis identified most of the DEGs linked to neuronal apoptosis, neurogenesis, neuronal projections, and neurotransmitter regulation. After analysis across the three groups, 23 hub genes and 21 pathways were identified, and qRT-PCR analysis confirmed that most of the mRNA levels of hub genes were consistent with the RNA-seq results. Q808 treatment increased the level of ACE, a GABA-related protein. Our analysis showed the comprehensive compendium of genes and pathways differentially expressed for PTZ-kindled seizure rats and upon Q808 treatment in PTZ-kindled seizure, which may provide a theoretical basis to explore the mechanism and unique efficacy of Q808 and the pathophysiology of epilepsy in the future.

Keywords: PTZ-kindled seizure; Q808; RNA sequencing; hippocampus; novel anti-convulsant chemical.

FIGURE 1

Chemical structure of Q808.

FIGURE 2

(A) Seizure score and (B) latency to seizures of PTZ-kindled rats before and after treatment with Q808. Data are shown as mean ± SD. Statistical analysis was carried out by the Mann–Whitney U test and unpaired t-test, respectively. n = 5, *p < 0.05, and **p < 0.01.

FIGURE 3

Volcano plot and heatmaps of hub genes in the hippocampus. (A, B) Volcano plot showing up-regulated (red dots) and down-regulated (green dots) DEGs and normal expressed genes (gray dots). (C) Heatmap showing the expression level of 23 hub genes across the three groups. Blue and red colors represent low and high levels of expression, respectively. Corresponding hierarchical clustering analysis of these hub genes is shown in black bars.

FIGURE 4

(A) GO classification of DEGs between PTZ and Con. groups. (B) GO classification of DEGs between Q808 and PTZ groups. The GO term of the most genes was cellular process, cell, and binding for the biological process, cellular component, and molecular function, respectively.

FIGURE 5

Altered gene expression in the hippocampus. Differentially expressed mRNAs which were identified in the RNA-seq data were further validated by qRT-PCR according to the fold-changes of qRT-PCR (2^−ΔΔCt). *p < 0.05, **p < 0.01, and ***p < 0.001. Data are expressed as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s test (n = 5).

FIGURE 6

The protein levels of ACE in the hippocampus of rats were determined by Western blot analysis. (A) Representative Western blot bands of ACE. (B) Quantitative analysis of ACE. β-Tubulin was used as the internal control. *p < 0.05. Data are expressed as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s test (n = 3).