Sevoflurane upregulates neuron death process-related Ddit4 expression by NMDAR in the hippocampus

Postoperative cognitive dysfunction (POCD) is a serious and common complication induced by anesthesia and surgery. Neuronal apoptosis induced by general anesthetic neurotoxicity is a high-risk factor. However, a comprehensive analysis of general anesthesia-regulated gene expression patterns and further research on molecular mechanisms are lacking. Here, we performed bioinformatics analysis of gene expression in the hippocampus of aged rats that received sevoflurane anesthesia in GSE139220 from the GEO database, found a total of 226 differentially expressed genes (DEGs) and investigated hub genes according to the number of biological processes in which the genes were enriched and performed screening by 12 algorithms with cytoHubba in Cytoscape. Among the screened hub genes, Agt, Cdkn1a, Ddit4, and Rhob are related to the neuronal death process. We further confirmed that these genes, especially Ddit4, were upregulated in the hippocampus of aged mice that received sevoflurane anesthesia. NMDAR, the core target receptor of sevoflurane, rather than GABAAR, mediates the sevoflurane regulation of DDIT4 expression. Our study screened sevoflurane-regulated DEGs and focused on the neuronal death process to reveal DDIT4 as a potential target mediated by NMDAR, which may provide a new target for the treatment of sevoflurane neurotoxicity.


INTRODUCTION
More than 300 million operations are performed worldwide each year, and a continued increase is observed in all economic environments [1]. Postoperative cognitive dysfunction (POCD) is a common cognitive impairment in patients during the perioperative period and is mostly found in elderly patients, with a reported incidence ranging from 15% to 60% [2,3]. POCD is mainly characterized by progressive postoperative memory impairment, cognitive decline, and executive dysfunction. In addition, the effects of POCD may not be temporary and can lead to neurological dysfunction years after surgery [4,5], which is associated with an increased risk of life-threatening illness and death. Neuronal apoptosis, a high-risk factor inducing POCD, leads to decreased neurogenesis, impaired synaptic plasticity, neuroinflammation, and oxidative stress in POCD patients [6][7][8]. Sevoflurane can induce POCD-related behaviors in animal, such as mice [9] or rats [10,11]. However, the neurobiological basis of sevoflurane neurotoxicity remains largely unknown.
General anesthetic neurotoxicity has been extensively examined in recent years [12][13][14]. An increasing number of studies have shown that inhaled anesthetics may cause neurotoxicity, leading to hippocampal neuronal damage and apoptosis, which result in cognitive dysfunction [15][16][17]. Sevoflurane, the most commonly used inhalation anesthetic, induces neuronal apoptosis [18][19][20][21][22]. Sevoflurane enhanced the production of lactate in aged marmoset brains [23]. Lactate accumulation can induce AGING neuronal apoptosis or even acidosis in critically ill patients [24][25][26]. Sevoflurane was shown to activate gamma-aminobutyric acid subtype A receptor (GABAAR) to induce apoptosis of immature dentate granule cells in mice [27]. Apoptosis is regulated by multiple pathways, among which the mechanism of neuronal apoptosis induced by sevoflurane through related signaling pathways has attracted increased attention [28][29][30]. Sevoflurane inhibits the ERK1/2 signaling pathway by antagonizing the N-methyl-Daspartate receptor (NMDAR) and upregulates the expression of the apoptotic proteins caspase-3 and Bax in mitochondria, resulting in apoptosis of hippocampal neurons [31]. Additionally, sevoflurane promotes the expression of the apoptotic factor connexin 43 (Cx43) and leads to neuronal apoptosis by activating the JNK/cJun/AP-1 signaling pathway [32]. However, a comprehensive analysis of the differentially expressed genes (DEGs) regulated by sevoflurane and further investigation of the molecular mechanism is lacking.
Transcriptomic analysis has identified comprehensive gene expression patterns to help reveal potential mechanisms for various neurological diseases [33,34] and has shown that inhaled anesthetic are associated with neurological damage [35]. Here, we applied multistage comprehensive bioinformatics methods to explore the possible pathogenesis of sevoflurane neurotoxicity. We focused on the potential key genes with different expression levels in the hippocampus of aged rats after sevoflurane anesthesia, established the functional annotation of their potential target genes and used gene enrichment analysis to reveal the role of DEGs associated with the cell death process in sevoflurane anesthesia. We further performed an in vivo experiment in aged mice that received sevoflurane exposure to confirm the pattern of upregulation of these genes in the hippocampus and further found that the NMDAR mediates the sevoflurane regulation of DDIT4 expression. This work showed the molecular mechanism of sevoflurane-induced neuronal apoptosis and provided a new potential target for sevoflurane toxicity.

DEG identification
A total of 10032 unique genes were annotated with the SwissProt database. The expression boxplot of all genes for each sample is shown in Figure 1A after normalization with the rma function by using the oligo package. The differential expression analysis identified 194 upregulated and 32 downregulated genes after treatment with 2.5% sevoflurane in 100% oxygen for 4 hours in an anesthetizing chamber with the criterion of a P value less than 0.05. Among all DEGs, 160 DEGs (153 upregulated DEGs and 17 downregulated DEGs) could be annotated in metascape, they were listed in Table 1, and the heatmap of the DEGs between the two groups is displayed in Figure 1B.

Functional enrichment analysis of DEGs
Biological processes and KEGG annotation were applied to explore the function of DEGs. All DEGs significantly played a role in localization, signaling, metabolic process, development process, and positive regulation of biological process (Figure 2A). Twentyfour biological process terms were filtered with P value less than 0.001, and DEGs were significantly enriched in the regulation of neuronal apoptosis ( Figure 2B). Next, we screened the biological processes associated with neuron death with the key words neuron and death, and 5 biological processes (positive regulation of cell  death, regulation of neuron death, negative regulation of neuron death, regulation of neuron apoptotic process and negative regulation of neuron apoptotic process) were dysregulated by sevoflurane ( Figure 3A). Most of the genes enriched in disordered biological processes associated with cell death were upregulated after sevoflurane inhalation ( Figure 3B-3F). A total of 10 KEGG pathways, such as peroxisome, AGE-RAGE signaling pathway in diabetic complications, inositol phosphate metabolism, vascular smooth muscle contraction, rap1 signaling pathway, and glycerophospholipid metabolism, were enriched by DEGs ( Figure 2C).

Protein-protein interaction network construction and hub gene selection
A total of 57 nodes and 97 interactions of the DEGs were identified in STRING and were visualized in Cytoscape ( Figure 4). We calculated the number of genes enriched in biological process terms, and the genes that were enriched in at least 10 terms are listed in Table 2. The cytoHubba application identified 58 hub genes with 12 algorithms, including 29 genes that were identified by at least five different methods as candidate hub genes (Table 3). Six hub genes (Agt, Cdkn1a, Ddit4, Pdgfra, Rapgef3, and Rhob) were both selected with two methods ( Figure 5A). The six hub genes were upregulated after sevoflurane inhalation ( Figure 5B). We further validated the expression of the six hub genes in vivo. We found that 4 h of 3% sevoflurane treatment increased the mRNA levels of Agt, Cdkn1a, Ddit4, Pdgfra, Rapgef3, and Rhob in the mouse hippocampus ( Figure 5C). Among the 6 hub genes, 4 genes (Agt, Cdkn1a, Ddit4, and Rhob) were also enriched in biological processes associated with neuronal death ( Figure 5D).

Sevoflurane upregulated the expression of Ddit4
DDIT4, an encoded protein that regulates development and DNA damage and participates in various pathological processes, was significantly enriched in the regulation of neuron death and positive regulation of cell death ( Figure 6A). NMDAR and GABAAR are considered important targets of sevoflurane [36][37][38]. Therefore, we further explored whether DDIT4 is regulated by NMDAR or GABAAR. We found that activation of GABAAR by injection of the GABAAR agonist muscimol (1.25 μg) into the mouse hippocampus did not cause a significant change in Ddit4 expression ( Figure 6B). However, after injection of the NMDAR antagonist MK-801 (0.25 μg) into the mouse hippocampus (injection coordinates: AP −2.1 mm, ML 1.5 mm, DV −2.1 mm) by brain stereotactic injection, the mRNA expression of Ddit4 was increased ( Figure  6C).
While using sevoflurane for anesthesia treatment, we injected 0.5 μg NMDA into the hippocampus (AP −2.1 mm, ML 1.5 mm, DV −2.1 mm) of mice and found that the increased expression of Ddit4 caused by sevoflurane could be rescued by NMDA, indicating that the effect of sevoflurane on the expression of DDIT4 might occur through the NMDA receptor ( Figure 6D). The western blot results show that DDIT4 level was elevated after sevoflurane-treated, but decrease after NMDA supplementation ( Figure 6E).

DISCUSSION
As a common perioperative neurological impairment in elderly patients, POCD strongly affects rapid recovery and long-term quality of life and places a heavy burden on patients' families and society [39]. Neuronal apoptosis induced by sevoflurane is one of the possible factors leading to POCD [10,40,41]. Sevoflurane may lead to neuronal death or neuroinflammation to induce cognitive impairment [42]. In this study, we comprehensively analyzed a total of 170 DEGs, 153 upregulated genes and 17 downregulated genes, in the hippocampus of aged rats after sevoflurane anesthesia, and 4 hub genes (Agt, Cdkn1a, Ddit4, and Rhob) were critically related to the biological process of cell death. We further confirmed the upregulation of these genes, especially Ddit4, in the hippocampus of the aged mice that received 4 hours of sevoflurane anesthesia. NMDAR, the core target receptor of sevoflurane, rather than GABAAR, mediates the sevoflurane regulation of DDIT4 expression.   We screened the DEGs from the hippocampus of rats, which is closely related to cognitive function [43] and may play an important role in the pathogenesis of POCD [44,45]. Agt encodes angiotensinogen, an angiotensin precursor protein that functions in the reninangiotensin system (RAS). In addition to the liver, Agt is also expressed in the brain. Increasing evidence has shown that the brain RAS plays a key role in Alzheimer's disease, stroke, alcoholism, and depression [46]. Angiotensin regulates iron homeostasis in dopaminergic neurons and microglia through type 1 receptors, thus affecting neurodegenerative diseases such as Parkinson's disease [47]. The interruption of angiotensinogen synthesis in astrocytes in the rat brain affects the function of the locus coeruleus, which may be responsible for cognitive, behavioural, and sleep disorders [48]. In this study, we found that Agt participates in both the positive and negative regulation of neuronal apoptosis. This evidence suggests that the overexpression of Agt in the hippocampus of aged rats after sevoflurane anesthesia may lead to dysfunction of the brain RAS system by affecting neuronal apoptosis.
Cdkn1a encodes cyclin-dependent kinase inhibitor 1A, which is mainly involved in cell cycle regulation. Several studies have shown that cell cycle-related molecules and pathways play a variety of important roles in influencing neuronal function. In some brain diseases, it is thought that cell cycle arrest may increase the susceptibility to cell death [49]. The failure of cell cycle regulation leads to neuronal dysfunction and cell death, which may be the underlying cause of several neurodegenerative diseases and the ultimate common pathway of other neurodegenerative diseases [50,51]. Our study confirmed that the Cdkn1a gene is enriched in the biological process of positive regulation of cell death, and the overexpression of Cdkn1a after sevoflurane treatment may disrupt the normal cell cycle and accelerate neuronal death in the hippocampus.
Similarly, the small molecule GTPase Rhob encoded by Rhob is an important regulator of cytoskeletal tissue and vesicle and membrane receptor transport. Researchers have found that RHOB is highly expressed in the hippocampus and may be essential for synaptic plasticity in the hippocampus [52]. Moreover, Rhob plays a key role in the apoptotic response, and its deletion affects the apoptotic response of tumor cells to DNA damage [53]. Therefore, both Cdkn1a and Rhob may be the possible pathological basis of sevoflurane neurotoxicity.
In this study, we found that Ddit4 is the only key gene enriched in both neuronal death and unidirectional regulation of apoptosis. Ddit4, also known as REDD1 and RTP801, encodes proteins that regulate development and DNA damage and participate in a variety of pathological processes. Suppression of DDIT4 expression decreases cell apoptosis in many kinds of cells [54][55][56].
Overexpression of DDIT4 promoted SUNE1 cell proliferation but inhibited apoptosis [57]. Here, we showed that sevoflurane upregulates DDIT4 expression, which suggests that neuronal apoptosis is induced by sevoflurane neurotoxicity.
The apoptosis-related neuronal death process regulated by sevoflurane leading to cognitive impairment has been recognized. Inhalation of 2% sevoflurane for 5 hours can activate the NF-κB signaling pathway and promote neuronal apoptosis and the production of inflammatory factors, thus affecting learning and memory abilities [58]. Activation of the PI3K/Akt signaling pathway reduces hippocampal neuronal apoptosis and exerts a protective effect against sevoflurane-induced brain injury in aged rats [59]. We also confirmed that 3% sevoflurane treatment increased the mRNA levels of Agt, Cdkn1a, Ddit4, Pdgfra, Rapgef3, and Rhob in the mouse hippocampus. The expression of Ddit4 in the hippocampal CA1 region was significantly altered after chronic cerebral hypoperfusion, indicating that it may play an important role in neuronal injury [60]. Inhibition of DDIT4 could reverse metformin-induced cell cycle arrest and significantly protect against the deleterious effects of the drug on cellular transformation [61]. Inhibition of DDIT4 expression also exerted a neuroprotective effect after ischemia-reperfusion injury [62]. These results suggest that DDIT4 may be a key target for intervention in cell apoptosis induced by sevoflurane.
General anesthetics play an anesthetic role mainly by inhibiting the target receptor NMDAR and activating GABAAR to regulate nerve signal transduction and can further induce a wide range of physiological effects through NMDAR and GABAAR to regulate downstream molecular signal pathways [31,38,63,64]. Inhibition of NMDAR by MK801 leads to apoptosis of neurons [65,66]. MK-801 also inhibits proliferation and increases apoptosis in hippocampal neural stem cells [67]. We found that the upregulation of DDIT4 expression in the hippocampus by sevoflurane can be inhibited through the supplementation of NMDA in the hippocampus. The injection of MK-801 into the hippocampus of mice also significantly promoted the expression of DDIT4. However, GABAAR activation did not significantly affect the regulatory effect of sevoflurane on DDIT4 expression. This finding indicates that sevoflurane regulates the expression of DDIT4 through NMDAR rather than GABAAR.
However, a limitation in our analysis was that we screened 4 hub genes while we only explored the The data shown are the means ± SDs, n = 3. ns P > 0.05, * P < 0.05. AGING mechanism of DDIT4 elevation after sevoflurane inhaling. Mechanisms of the change in the other three genes need to be explored in future experiments. Here we used the antagonist of NMDAR MK-801 to determine the NMDAR mediating sevoflurane regulation, the gain and loss function of NMDAR subunits by RNAi will be performed in the future. In addition, we will further perform the overexpression or knockdown of DDIT4 in the hippocampus to investigate whether Ddit4 is involved in the sevoflurane-induced neuron death.
Our study comprehensively analyzed sevofluraneregulated DEGs to indicate that Ddit4 may be a potential target of sevoflurane-induced neuronal apoptosis and determined that the NMDAR/DDIT4 pathway may be a potential target of sevoflurane neurotoxicity, which provides new possibilities for the prevention and treatment of sevoflurane neurotoxicity.

Microarray data analysis
GSE139220 expression profiles were retrieved and obtained from the NCBI-GEO website (https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE139220) [68]. The whole transcriptomic data of hippocampal tissue from 3 rats that received 100% oxygen at an identical flow rate for 4 h in an identical chamber and 3 rats that received 2.5% sevoflurane in 100% oxygen for 4 hours in an anesthetizing chamber were included. The raw data were normalized with the rma function by using the oligo package on the R version 4.2.2 platform [69]. The expression data were annotated with the SwissProt database. If the target gene was annotated with two or more probes, the mean value was calculated. Then, the Limma package for the R environment was used to detect the differentially expressed genes (DEGs) in hippocampal tissue between the control group rats and the sevofluranetreated rats [70]. DEGs were identified based on a P value less than 0.05.

DEG functional enrichment analysis
Gene enrichment analysis of DEGs was performed on the web-based portal Metascape (http://metascape.org/) [71] using the Gene Ontology biological processes and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [34]. The enrichment terms were visualized using the ggplot2 package in R.

Protein-protein interaction network construction
For all DEGs, a protein-protein interaction (PPI) network was constructed using the STRING database (https://cn.string-db.org/) [72]. Then, the network was visualized on Cytoscape software version 3.9.1, which can be freely downloaded on the website https://cytoscape.org/ and can be used to detect hub genes with the cytoHubba app [73,74].

Hub gene selection
To explore the hub genes, we used two screening methods. One is that this gene is involved in multiple biological processes, and the other is that hub genes were screened by 12 algorithms with cytoHubba in Cytoscape, and the genes that were identified by both methods were considered to play a critical role in sevoflurane neurotoxicity. The hub genes enriched in the neuron death process were considered to be involved in neurotoxicity.

In vivo validation
We performed in vivo tests to validate the results from the microarray data.

Quantitative real-time PCR (qPCR)
Total RNA from the hippocampus was isolated by using RNAiso Plus (TaKaRa, China). cDNA synthesis from mRNA was performed by using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). Then, the cDNA was used for qPCR detection by using Fast qPCR Mix (TaKaRa). Primers for the qPCR analysis of mRNA are shown as follows:

Statistical analysis
Statistical analysis was performed on the R version 4.2.2 platform. The quantitative data are presented as the mean ± SD. The microarray data and in vivo PCR validations are displayed with boxplots. Unpaired twotailed Student's t-test was used to determine significant differences between the two groups. A P value less than 0.05 was considered significant.

Data availability statement
GSE139220 is available from the NCBI-GEO database with the link-https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE139220. The datasets analyzed during this study are available from the corresponding author upon reasonable request.

ACKNOWLEDGMENTS
We are very grateful to the providers who submitted the transcriptomic data to the public databases.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this study.