陆军军医大学学报 (Jun 2024)
Expression of Ppp3cb and Ppm1g in the hippocampus of NHE1 gene knockout rats based on proteomics
Abstract
Objective 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. Method 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. ② Tandem 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. ③ The 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. Results 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. ② When 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). Conclusion 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.
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