Transcriptome analysis of DEGs and pathways between Mock and PND group at different time point
To determine whether mRNA was differentially expressed in the hippocampus of the PND group compared with the Mock group, mice were sacrificed on days 1, 7, and 21 after surgery. The hippocampus samples of 6 mice from per group at each time point were randomly selected for RNA extraction and then were submitted to Illumina sequencing. After deep sequencing, 47,669,156/50,465,448/47,446,952 and 43,852,733/47,073,916/50,487,263 total raw reads (average, n = 6) were obtained from the mRNA library of the Mock and PND groups, respectively, on day 1/7/21. The reference annotation-based assembly method was used to reconstruct the transcripts, and fragments per kilobase of the exon model per 1 million mapped fragments and the coverage threshold were used to reduce background noise. Then, the assembled transcripts were compared with known mRNA reference sequences to obtain all transcripts. The DEGs were determined by comparing the obtained transcripts of the Mock and PND groups. The mRNAs with extremely low expression levels were removed (normalization reads < 1), and if the P values were less than 0.05, the mRNAs were considered altered.
After normalization of the data (Fig. 2a), we found that 352, 395, and 772 genes were altered (Fig. 2b), including 91, 129, and 360 upregulated genes and 261, 266, and 412 downregulated genes at days 1, 7, and 21, respectively (Fig. 2C-E, Online Resource 4, Online Resource 5, and Online Resource 6).
To evaluate the reliability of these results, we performed qRT-PCR to check the expression of DEGs with obvious changes. Alanine and arginine rich domain containing protein (AARD), cilia and flagella associated protein 74 (CFAP74), G protein subunit gamma transducin 2 (GNGT2) and zinc finger protein 119b (ZFP119B) were significantly altered at day 1; CFAP74, G protein-coupled receptor 6 (GPR6), potassium voltage-gated channel subfamily E regulatory subunit 4 (KCNE4) and ciliogenesis and planar polarity effector complex subunit 2 (CPLANE2) were significantly changed at day 7; Ankyrin repeat domain 44 (ANKRD44), G protein nucleolar 3 like (GNL3L), heat shock protein family B (small) member 1 (HSPB1) and solute carrier family 38 member 5 (SLC38A5) were significantly changed at day 21. The results were consistent with the sequencing data, indicating the accuracy of sequencing data.
Subsequently, GO analyzes were performed based on these DEGs. On day 1, DEGs are involved in “response to stress”, “response to other organism”, “response to organic substance”, “response to interferon-beta”, “response to cytokine”, “regulation of immune system process” and other related biological processes (Fig. 3a). On day 7, DEGs are involved in “spinal cord association neuron differentiation”, “sperm displacement”, “response to mineralocorticoid”, “response to glucocorticoid”, “response to corticosteroid”, “regulation of biomineral tissue development”, “organic acid transmembrane transporter activity”, “oligopeptide binding”, “neutral amino acid transport”, “negative regulation of heart induction by canonical”, “insemination”, “G protein-coupled receptor binding”, “glutathione binding”, “copulation”, “carboxylic acid transmembrane transporter activity” and “amino acid transport” (Fig. 3c). On day 21, DEGs are involved in “T cell activation”, “side of membrane”, “regulation of response to external stimulus”, “regulation of immune system process”, “positive regulation of multicellular organismal process”, “negative regulation of metanephros development”, “metanephric epithelium development”, “C-X-C chemokine binding” and “cell surface” (Fig. 3e).
Further KEGG pathway enrichment analysis revealed that the DEGs at day 1 were mainly associated with the “NOD-like receptor signaling pathway”, the “B cell receptor signaling pathway”, the “IL-17 signaling pathway”, the “Toll-like receptor signaling pathway”, the “Calcium signaling pathway” and the “FoxO signaling pathway,” etc (Fig. 3b); the “mTOR signaling pathway”, the “Hippo signaling pathway”, the “Wnt signaling pathway”, the “C-type lectin receptor signaling pathway” and the “Apelin signaling pathway” at day 7 (Fig. 3d); the “T cell receptor signaling pathway”, the “PD-L1 expression and PD-1 checkpoint pathway in cancer” and the “chemokine signaling pathway” at day 21 (Fig. 3f). These results suggest that the biological processes and signaling pathways are different between time points.
Screening of DEGs strikingly associated with p53 signaling pathway
Postoperative changes in cognitive functions of aged mice over time: on day 1 and day 7 after surgery, cognitive functions decreased, whereas on day 21 after surgery, cognitive functions of mice had already partially improved (Fig. 1). Therefore, we plan to investigate different DEGs and signaling pathways between day 1, 7, and 21 to further explain the mechanism of PND. All genes were subjected to GSEA analysis (https://www.gsea-msigdb.org), and KEGG pathway diagrams were drawn. After crossing different signaling pathways at each time point, 8 identical pathways were obtained (Data S1), suggesting that there are 8 pathways involved in PND. Among these pathways, the p53 signaling pathway was identified (Fig. 4a). As p53 signaling is an important regulator of neuronal function(Jazvinscak Jembrek et al. 2018), we speculated that p53 signaling may be involved in the PND process. The results showed that p53 signaling was enriched in PND at day 1 and 7 but not at day 21 (Fig. 4b). To identify the potential DEGs regulating p53 signaling in PND, DEGs from each time point were compared, and 18 identical DEGs were identified (Figs. 4c and 4d). Among these genes, 2 DEGs were upregulated and 9 DEGs were downregulated in the PND group, whereas the change trend of the other 7 DEGs was inconsistent (Figs. 4e and 4f). Moreover, GSEA revealed that among these 11 DEGs, 4 DEGs were associated with p53 signaling, including leucine rich repeat kinase 1 (LRRK1), monooxygenase DBH like 1 (MOXD1), membrane-spanning 4-domains, subfamily A, member 6B (MS4A6B) and piezo type mechanosensitive ion channel component 1 (PIEZO1) (Fig. 4g, Data S2). Due to MS4A6B is a mouse-specific gene, we monitored the expression of 3 other genes and p53 via WB. Consistent with the sequencing data, LRRK1, MOXD1, and PIEZO1 were significantly downregulated in PND at day 1 and day 7 but not at day 21. p53 was upregulated at day 1 and day 7 but remained unchanged at day 21 (Fig. 4h).