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Integrated transcriptomics and metabolomics analysis of the hippocampus reveals altered neuroinflammation, downregulated metabolism and synapse in sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is an intricated complication of sepsis that brings abnormal emotional and memory dysfunction and increases patients’ mortality. Patients’ alterations and abnormal function seen in SAE occur in the hippocampus, the primary brain region responsible for memory and emotional control, but the underlying pathophysiological mechanisms remain unclear. In the current study, we employed an integrative analysis combining the RNA-seq-based transcriptomics and liquid chromatography/mass spectrometry (LC-MS)-based metabolomics to comprehensively obtain the enriched genes and metabolites and their core network pathways in the endotoxin (LPS)-injected SAE mice model. As a result, SAE mice exhibited behavioral changes, and their hippocampus showed upregulated inflammatory cytokines and morphological alterations. The omics analysis identified 81 differentially expressed metabolites (variable importance in projection [VIP] > 1 and p < 0.05) and 1747 differentially expressed genes (Foldchange >2 and p < 0.05) were detected in SAE-grouped hippocampus. Moreover, 31 compounds and 100 potential target genes were employed for the Kyoto Encyclopedia of Genes and Genomes (KEGG) Markup Language (KGML) network analysis to explore the core signaling pathways for the progression of SAE. The integrative pathway analysis showed that various dysregulated metabolism pathways, including lipids metabolism, amino acids, glucose and nucleotides, inflammation-related pathways, and deregulated synapses, were tightly associated with hippocampus dysfunction at early SAE. These findings provide a landscape for understanding the pathophysiological mechanisms of the hippocampus in the progression of SAE and pave the way to identify therapeutic targets in future studies.


Introduction
Sepsis and septic shock are prevalent and severe symptoms in an intensive care unit (ICU). They account for 25%-80% of mortality worldwide (Dellinger et al., 2013;Fleischmann-Struzek et al., 2020;Evans et al., 2021) and usually present with multiorgan dysfunction syndromes, including the heart, kidney, lung, intestine, spleen, and liver. However, the disturbance in peripheral liquid homeostasis causes further damage to the brain, defined as sepsis-associated encephalopathy (SAE), which contributes to the leading cause of death (Young et al., 1990;Eidelman et al., 1996;Schuler et al., 2018). SAE is a severe complication that occurs in 7%-71% of sepsis cases and vastly increases the mortality due to the severity of sepsis, patient status, and diagnosis criteria (Iacobone et al., 2009;Gofton and Young, 2012). The hallmark of SAE is a brain injury, possibly impacting the long-term cognitive (memory loss, attention deficit, or language issues) (Iwashyna et al., 2010;Gofton and Young, 2012;Pandharipande et al., 2013) and psychological functions (depression, anxiety, or post-traumatic stress disorder) (Eidelman et al., 1996;Gofton and Young, 2012;Heming et al., 2017;Neves et al., 2018;Schuler et al., 2018). Although various studies have focused on the diagnosis, clinical progression, and pathological or pathophysiological mechanisms, there are still large gaps in profoundly understanding the mechanisms and identifying the early brain injury and timely intervention for sepsis-suffering patients (Flierl et al., 2010;Gofton and Young, 2012).
Sepsis initiates a series of systemic inflammatory responses leading to deregulated peripheral inflammation, neuroinflammation, ischemic damage, oxidative stress, and metabolic disorders, disrupting the integrity of the brain-blood barrier (BBB) and, thus, causing the progression of SAE (Gofton and Young, 2012). Furthermore, the histological studies on animal models have shown enlarged ventricles, reduced hippocampus volume, and increased apoptosis of hippocampal neurons in lipopolysaccharide (LPS)-exposure rodents (Wang et al., 2013;Chen et al., 2021). Therefore, the hippocampus is an overly sensitive brain region in LPS-triggered animals. Of note, years of studies on experimental sepsis animal models, including LPS injections and cecal-ligation puncture (CLP), have established the dysregulated phenotype of the hippocampus (Hippensteel et al., 2019;Chao et al., 2020;Talukdar et al., 2020) and have been employed to evaluate further the cognitive decline or impairment in the cortex and hippocampus region (Neves et al., 2018;Mei et al., 2021;Shen et al., 2021).
Multiple omics approaches, such as expression array, transcriptomic, and metabolomic, have been employed to elucidate the biological events from different brain regions or spheres to uncover the etiology and molecular mechanisms of cognitive or neurofunction impairment. However, single approaches have gained fewer insights into the complicated mechanisms (Schilder et al., 2022). In contrast, multi-omics analysis is a powerful approach to elucidate the pathophysiological mechanism of diseases, such as neurodegeneration diseases, depression, and other mental diseases (Zhang et al., 2018;Clark et al., 2021;Schilder et al., 2022). Therefore, integrated multi-omics data analysis is promising to identify potential biological relationships and deepen the understanding of SAE.
In the SAE-related study, single omics, including metabolomics and transcriptomics, have been widely used to decipher the molecular mechanisms in the hippocampus (Hara et al., 2015;Salvesen et al., 2017;Geng et al., 2020). Nevertheless, the literature would benefit from more comprehensive study based on multi-omics analysis. Herein, we adopted an RNA-seq and metabolomics-based integrated analysis to explore the possible pathophysiological mechanisms of the hippocampus when mice developed SAE-like symptoms at early stages by an intraperitoneal (i.p.) injection with LPS. The integrated analysis demonstrated that inflammation, neuroinflammation, and synapse and metabolic pathways were mainly altered in LPSchallenge hippocampus tissues.
Tongren Hospital, Shanghai Jiao Tong University School of Medicine. After 8 h or 24 h post-injection, mice were killed, with the numbers of animals for histology analysis, omics, or biochemical analysis of the hippocampus being described below.

Open-field test
The open-field test (OFT) widely examined rodent locomotor activity and exploratory behaviors. All open-field testing took place inside an arena (50 cm [l] × 50 cm [w] × 40 cm [h]). The arena was divided into the center and peripheral regions by the VisuTrack system (Shanghai XinRuan Information Technology Co., Ltd., Shanghai, China). Mice that were injected with LPS (10 mg/kg body weight) and controls (injected with saline) for 24 h were removed from their home-cage by the tail and placed directly into the open field center. Tracking/recording was initiated upon the first locomotion grid beam break and lasted 5 min. The trajectory of each mouse was analyzed with the VisuTrack system. Total distance, speed, and freezing time were recorded to evaluate the movement ability of mice, and center entries in the central region were measured to detect mice's anxiety. The data were interpreted as mean ± SEM (standard error of the mean) (n = 9 per group) with Student's t-test.
Tissue preparation, nissl staining, and hematoxylin-eosin staining Twenty-four hours post-injection, mice injected with saline or LPS were anesthetized with 10% chloral hydrate, and whole blood was collected by eyeball sampling before being killed by cervical dislocation. Next, the whole brain was dissected, rinsed with precooled saline, and fixed in 4% paraformaldehyde for paraffin-embedded sections or further dissected for the hippocampi. Then, the dissected hippocampi were snap-frozen by liquid nitrogen and stored at −80°C for subsequent RNA (n = 3 per group) and metabolites analysis (n = 6 per group). Finally, entire brain coronal sections (5 µm) were used for the Nissl staining or hematoxylin-eosin staining (n = 3 mice per group). These stainings were performed as described previously (Xie et al., 2010), scanned by the NDP slide scanner (Hamamatsu NanoZoomer S60), and viewed by its viewing software (NDP.View2). The mean optical density of Nissl staining was measured using Image-Pro Plus 6.0 software (Media Cybernetics, CA, United States) and interpreted as the mean ± SEM.

RNA extraction and sequencing library construction
Total RNA of each frozen hippocampi was extracted by Trizol reagent (Life-technology, United States) with RNeasy Mini Kit (Qiagen#74106). RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, United States). RNA integrity was analyzed by Agilent 2100 bioanalyzer (RIN >6.0 and 28S/18S ≥ 0.7); thus, it could qualify for subsequent library construction using NEBNext Ultra RNA Library Prep kit (#E7530; New England BioLabs, Inc., Ipswich, MA, United States) according to the manufacturer's instructions.

RNA sequencing and differentially expressed genes analysis
The libraries were sequenced on an Illumina NovaSeq 5,000 platform, and 150-bp paired-end reads were generated. In total, 45.18-49.38 Mb raw reads for each sample were generated. Raw data (raw reads) of fastq format were firstly processed using Trimmomatic (Bolger et al., 2014), and the lower quality reads were removed to obtain the clean reads. Then about 44.21-48.31 Mb clean reads for each sample were retained for subsequent analyses.
The clean reads were mapped to the mouse genome (GRCm38.p6) using HISAT2 (Kim et al., 2015). We calculated the FPKM (Roberts et al., 2011) of each gene by using Cufflinks (Trapnell et al., 2010) and obtained the read counts by HTSeqcount (Simon et al., 2015). Furthermore, we performed differential expression analysis using the DESeq (2012) R package (Anders and Huber, 2013). A P-value of <0.05 and log2|Foldchange| of >1.0 were set as the threshold for significantly differentially expressed genes (DEGs). Hierarchical cluster analysis of DEGs was performed to demonstrate the expression pattern of genes between the SAE (S) and control (Ctrl) group samples. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al., 2008) pathway enrichment analysis of DEGs were performed using R based on the hypergeometric distribution. Bioinformatic analysis was performed using OECloud tools (https://cloud.oebiotech.cn).
Additionally, gene set enrichment analysis (GSEA, http:// software.broadinstitute.org/gsea/index.jsp) was used to further screen the signaling pathways associated with SAE (Subramanian et al., 2005). GSEA is a computational method for determining whether a priori-defined set of genes shows statistically significant, concordant differences between two biological states. GSEA does not focus on only significantly/highly changed genes but examines all the genes belonging to a specific biological process.

Reverse transcription and quantitative polymerase chain reaction
As previously described (Lu et al., 2017;Lu et al., 2021), RNA isolation and RT-qPCR were performed. Briefly, RNA was extracted by Trizol reagent, and 1 μg total RNA was used for cDNA synthesis and reversed transcribed by ReverTra Ace kit (TOYOBO). Then, the cDNA was used to quantify by the lightcycle480 system (Roche) with 2 × Power SYBRgreen mix (Applied Biosystems, Carlsbad, CA, United States). Finally, quantification of gene expression was calculated by normalization to GAPDH or B-ACTIN using the 2 −ΔCt method. Primer sequences for qPCR are available on request.

Sample preparation for metabolomics
For sample preparations, 20 μL of internal standard (2chloro-l-phenylalanine, 0.3 mg/ml in methanol) and 400 μL of methanol: water (4:1 = vol: vol) were added to each hippocampal sample (30 mg/group sample), and ground with a grinder machine (60 Hz, 2 min). Mixtures of the sample were extracted by ultrasonication for 10 min in the ice water bath and kept at −20°C for 30 min. The extraction was centrifuged at 13,000 rpm and 4°C for 10 min. Next, 300 μL of supernatant was transformed into a liquid chromatography/mass spectrometry (LC-MS) injection vial to evaporate and dissolved again in 200 μL methanol: water (4:1 = vol: vol) by sonification (vortex 30 s and sonification 3 min). The samples were kept at −20°C for 2 h and then centrifuged at 13,000 rpm and 4°C for 10 min. The supernatants (150 μL) from each tube were collected using crystal syringes, filtered through 0.22-μm filters, and transferred to LC vials. The vials were stored at −80°C until LC-MS analysis. Of note, all reagents for extraction were precooled at −20°C.

LC-MS analysis
The liquid chromatography was performed using a Dionex U3000 UHPLC equipped with ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm, Waters). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The analysis was performed with elution gradient as follows: The QE mass spectrometer (ThermoFisher) was used to acquire MS/MS spectra in information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur 4.0.27, Thermofisher). The acquisition software continuously evaluates the full scan MS spectrum in this mode. The ESI source conditions were set as follows: sheath gas flow rate of 35 Arb, Aux gas flow rate of 8 Arb, capillary temperature of 320°C, full MS resolution of 70,000, MS/MS resolution of 17,500, collision energy of 10/20/40 eV in NCE mode, and spray Voltage of 3.8 kV (positive) or −3.0 kV (negative).

Data preprocessing and analysis
The acquired LC-MS raw data were analyzed by Progenesis QI v2.3 software (Nonlinear Dynamics, Newcastle, United Kingdom) with the following parameters: the precursor tolerance of 5 ppm/10 ppm, fragment tolerance of 10 ppm/20 ppm, and retention time (RT) tolerance of 0.02 min. The product ion threshold was 5%. Internal standard detection parameters were deselected for peak RT alignment, isotopic peaks were excluded for analysis, and the noise elimination level was set at 10.00. The minimum intensity was set to 15% of the base peak intensity. The Excel file was obtained with three-dimension datasets including m/z, peak RT, and peak intensities, and RT-m/z pairs were used as the identifier for each ion. The resulting matrix was further reduced by removing any peaks with a missing value (ion intensity = 0) in >50% of samples. The internal standard was used for data quality control (reproducibility).
Metabolites were identified by progenesis QI software based on public databases, such as HMDB (http://www.hmdb.ca/), Lipidmaps (v2.3) (http://www.lipidmaps.org/), and self-built database (Luming Biotech). The resulting positive and negative ion scans were combined into a dataset for further analysis with R ropls package. Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis OPLS-DA were performed to visualize the metabolic alterations among experimental groups. The Hotelling's T2 region, shown as an ellipse in score plots of the models, defined the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranked the overall contribution of each variable to the OPLS-DA model, and those variables with VIP >1 were considered relevant for group discrimination. In this study, the default 7-round crossvalidation was applied, with 1/seventh of the samples being excluded from the mathematical model in each round to guard against overfitting. The criteria for differentially expressed metabolites (DEMs) were VIP of >1.0 obtained from the OPLS-DA model and p of <0.05 from a two-tailed Student's t-test on the normalized peak areas.

Integrative analysis of transcriptome and metabolome
Building the core genes-metabolites regulation network is better to describe the primary clustered genes and metabolites responsible for the SAE. Therefore, as many studies have shown, we filtered the low expression-abundance DEGs before correlations network analysis and KEGG Markup Language Frontiers in Pharmacology frontiersin.org 04

FIGURE 1
Proinflammatory cytokines and morphological alterations in the hippocampus with neurobehavior changes in mice undergoing acute sepsis phase. (A) RT-qPCR analysis of proinflammatory cytokines (TNF, IL6, and IL1B) alteration in the hippocampus from saline-or LPS-injected (5 or 10 mg/kg body weight) mice. The mRNA expression was normalized to GAPDH. Data are presented as mean ± SEM (n = 3). Two-way ANOVA with Tukey's multiple comparisons tests, *p < 0.05, **p < 0.01, ***p < 0.001 (compared with LPS 5 mg/kg body weight at indicated time points). (B) Representative Nissl staining of the hippocampus from saline-or LPS-injected mice. The mean optical intensity was quantified and tested with an unpaired t-test. Data are presented as mean ± SEM (two sections per mouse, n = 6), **p < 0.01. Bar 250 µm. (C) Hematoxylin-eosin staining of the (Continued ) Frontiers in Pharmacology frontiersin.org 05 (KGML) network analysis. Pearson correlation coefficients were calculated for transcriptome and metabolome data integration (Luo and Brouwer, 2013). Briefly, based on the DEGs expression data and DEMs concentration data, the Pearson correlation coefficients were calculated by R, and a cluster analysis heatmap of the correlations between the top 100 EMs and DEGs was drawn. Furthermore, DEGs and DEMs were mapped to the KEGG database. KGML, another format storing pathway information in the KEGG database, contained the relationship between all the essential elements in the KEGG pathway and orthologous genes in the KEGG GENES database. Gene, metabolites interaction network, and inter-pathway relationship network were obtained from the KGML file and visualized and interpreted using Cytoscape (version 3.5.1) with MetScape plug-in (version 3.1.3) (Gao et al., 2010).

Statistical analysis
Statistical analysis was performed on raw data for each group by unpaired Student's t-test, using GraphPad Prism 8. Data were shown as mean ± SEM. P-values referred to the probability of the null hypothesis meaning that the means do not differ. A P of <0.05 was considered significant, and a p of ≥0.05 was not significant.

Results
Proinflammatory cytokines and morphological alterations in the hippocampus with neurobehavior changes in the mice undergo acute sepsis phase As many studies have shown (Lewis et al., 2016;Savi et al., 2021), we initially constructed the SAE model referring to the sepsis model made by peritoneal injection of a moderate dose of LPS in C57BL6J mice. Then, we evaluated the alterations of cytokines, histology, and behaviors prior to the transcriptomics and metabolomics analysis. The time-chase experiment monitored the dynamic changes of proinflammatory cytokines in the hippocampal tissues by RT-qPCR. The results showed that Tnf, IL1β, and Il6 mRNA decreased at 24 h LPS post-injection compared to 8 h ( Figure 1A). Furthermore, the cytokine genes in the hippocampus of the LPS-injected group (5 or 10 mg/kg body weight) were more highly expressed than in the saline group. Of note, the high dose of LPS (10 mg/kg body weight) triggered a higher expression of proinflammatory genes than the low dosage. Thus, we determined to use the high dosage for subsequent studies. Furthermore, the histology examination showed a decreased staining in the hippocampal regions, especially the dentate gyrus (DG) region, as showed by Nissl staining ( Figure 1B), which indicated hippocampal neurons injury. Hematoxylin and eosin staining showed normal hippocampus morphology in the saline group, whereas minor cell staining showed shrunk and enlarged cells in the adjacent distance among hippocampus regions, especially in the DG region in the LPS group ( Figure 1C).
We examined the mice at 24 h post-injection with LPS (10 mg/kg body weight) or saline by open-field test. The results showed behavioral changes, i.e., a decrease in total distance travel, speed, and center entries, but an increase in freezing time ( Figure 1D−H) in LPS-injected mice, which was consistent with previous studies with the LPS-sepsis mice model and indicated a potential strike on their cognitive performance (Savi et al., 2021). Most mice demonstrated LPS-manifested back piloerection, noticeably slowed but still ambulant, eyes not fully open, and some were covered with secretions. Furthermore, most mice were stationary with occasional investigative movements and decreased overall activity. The mice also exhibited no response to the auditory stimulus, while they demonstrated strong responses and moved a few steps to escape when touched. Therefore, the animal model was suitable for SAE studies, and we determined to use this model to detect the omics alterations in the hippocampus.

Non-targeting metabolomic analysis of the hippocampus in acute sepsis phase
We performed LC-MS-based metabolomics profiling of the hippocampus in the SAE model and control groups (n = 6 mice per group) to determine the alterations of metabolites. A total of 5,109 metabolites (2133 and 2976 identified in positive and negative ion mode, respectively) were detected by LC-MS and subsequently used for multivariate analysis. The PCA and OPLS-DA score plot showed that the SAE group was discriminated from the saline group (Figures 2A,B). Furthermore, the permutation tests showed no overfitting of data ( Figure 2C), indicating that the OPLS-DA model was valid for the analyses. FIGURE 1 (Continued) hippocampus from saline-or LPS-injected mice (n = 3). (D) Representative trajectory of the total distance (E), speed (F), freezing time (G), and center entries (H) in the LPS mice and saline groups. Comparisons were performed with an unpaired t-test. Data are presented as mean ± SEM (n = 9), **p < 0.01, ***p < 0.001.

Transcriptome and DEGs analysis of the hippocampus in the early stage of SAE mice
We next performed RNA-seq-based profiles analysis using the same batch of hippocampus tissues as in LC-MS metabolomics analysis to uncover the molecular basis of the hippocampus from SAE mice. A total of 41703102-4411552 new reads were sequenced, mapped into the mouse genome, and deposited in the NCBI sequence read archive (SRA, BioProject accession number PRJNA827615).
The PCA analysis of the hippocampus transcriptome signature showed clear segregation between SAE (S1−S3, n = 3) and control (Ctrl 1-3, n = 3) ( Figure 4A). Next, we made a DESeq analysis to determine the significant DEGs with a cutoff |Log2FC| of > 1 and p of < 0.05 ( Figure 4B) and identified 662 upregulated and 1085 downregulated DEGs in the SAE of interest ( Figure 4C; Supplementary Table S1). Moreover, a pool of 32 DEGs related to inflammatory cytokines and chemokines, inflammatory response, and metabolic enzyme genes was confirmed by RT-qPCR analysis ( Figure 4D). These data showed consistency in the expression pattern between control and SAE hippocampus tissues.
KEGG analysis was performed to obtain the top 20 significant pathway terms with the upregulated or downregulated DEGs. For the upregulated DEGs, the KEGG term included most of the inflammatory response, cytokine signaling pathways, necroptosis, and apoptosis in the hippocampus ( Figure 4E; Supplementary Figure S1). In addition to the downregulated DEGs, the metabolism (nitrogen, sphingolipid, ether lipid, glutathione, alanine, aspartate and glutamate, glycine, serine and threonine, purine, sulfur, arachidonic acid, and lysine degradation), synapse function (glutamatergic synapse and GABAergic synapse), and signaling pathways (e.g., neuroactive ligand-receptor interaction, calcium signaling pathway, ECM-receptor interaction, PI3K-Akt signaling pathway, and phospholipase D signaling pathway) were enriched ( Figure 4F and Supplementary Figure S2), indicating that the metabolism was suppressed in SAE-grouped hippocampus. Furthermore, GSEA analysis (Subramanian et al., 2005) of the biological event ( Figure 5 and Supplementary Table S2) further corroborated these top-ranked inflammatory response-related pathways, lipid metabolism, cell death, and synapse.

Integrative analysis of transcriptome and metabolome
A core genes-metabolites regulation network must decipher the significant clustered genes and metabolites responsible for the SAE progression. According to the KEGG enrichment analysis with the RNA-seq and metabolomics data, we observed that 392 DEGs and    Figure 6A and Supplementary Table S3). Additionally, the KGML network was built based on the 100 DEGs and 31DEMs datasets to gain the global network information during SAE progression. The results showed that the critical nodes and metabolism pathways were highly overpresented ( Figure 6B).

Discussion
The development of acute sepsis in patients can cause septic shock and further develop as SAE (Iacobone et al., 2009; Gofton and Young, 2012). In this study, we examined the enriched expressed genes and metabolites of the hippocampus to decipher the possible mechanisms of SAE in the early phase with an LPS-induced experimental sepsis model. The results showed that acute sepsis mice developed inflammation (dramatic upregulation of proinflammatory cytokines), hippocampal neuron injury, and behavior changes, which indicated that a reproducible SAE-like-symptom occurred in hippocampal tissues as in previous findings (Wang et al., 2013;Chen et al., 2021). Metabolomics has been vigorously used to explore the enriched metabolites and related metabolic pathways in the development of SAE (Hara et al., 2015;Salvesen et al., 2017;Geng et al., 2020), which leads to the discovery of biomarkers or sensitive indicators to detect their pathophysiological activity. However, the metabolomic profiles only illuminate a landscape of metabolic alterations during the progression of SAE. Additionally, the gene expression profiles most uniquely reflect the protein levels that directly regulate the metabolic process. Therefore, a multiomics analysis could offer a more comprehensive understanding of the molecular regulatory network. According to the literature, no reports on the hippocampus in SAE were documented with multi-omics analysis. Interestingly, this study integrated two different omics data to identify functional associations between them and pinpointed pathways that were perturbed among the lipid metabolic, amino acid, and carbohydrate pathways of the hippocampus of the SAE model.

Lipids metabolism alterations in the hippocampus of SAE
The lipids altered in sepsis have been documented in many studies (Amunugama et al., 2021;Preau et al., 2021). In the present study, the lipids metabolism-related pathways were highly enriched in the hippocampus of SAE by integrative analysis of metabolomics data and RNA-seq data. Furthermore, the lipid metabolism-related pathways were enriched by using the DEMs, which were further corroborated by top-ranked pathways, including ether lipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, phospholipase D signaling pathway, and sphingolipid signaling pathway as enriched by DEGs (Figures 4, 5). Steroids (corticosterone [CORT], 11dehydrocorticosterone [11-DHC], 3β, 7α-dihydroxy-5cholestenoate, and 11β, 21-dihydroxy-3, 20-oxo-5βpregnan-18-al) were accumulated in the hippocampus of SAE. Moreover, lipids and steroids metabolism-related genes were detected to be upregulated (ALOX5, CYP1B1, CYP27B1, CYP3A13, PLA2G4E, SOAT2, UGT1A6A, and UGT1A7C) or downregulated (DHCR24, DHCR7, CYP11A1, PLA2G4B, SOAT1, and SRD5A1) in the hippocampus tissues of SAE (Supplementary Table S1, Figure 3C). Those results indicated that steroid synthesisrelated pathways were significantly affected and consistent with previous findings that the CORT was increased in murine sepsis survivors and exhibited a behavioral neuroendocrine syndrome (Spencer-Segal et al., 2020). Furthermore, CORT was reported to induce neuroinflammation and bring depression-like behaviors or other neuron dysfunction syndromes in the hippocampus (Komoltsev et al., 2021;Bras et al., 2022), which indicated that the increase of CORT is a critical factor in the progression of SAE. Additionally, other enriched steroids were precursors of CORT, but their functions are still unknown.
Apart from the upregulated steroids, 5,6-epoxy-8,11,14eicosatrienoic acids (5, 6-EET) are tightly related to arachidonic acid metabolism and isovaleric acid, which is associated with cognitive impairment and systemic inflammation (Zheng et al., 2022), and were decreased in the SAE-grouped hippocampus (Table 1 and Figure 3C). This result indicated that their anti-inflammatory effects were blocked, which might cause further cognitive impairment in the hippocampus during the progression of SAE (Dalli et al., 2017;Wang et al., 2021). The mentioned steroids induced inflammation, further corroborated with the enriched inflammation-related pathways, including the tumor necrosis factor (TNF) signaling pathway, nuclear factor kappa B (NF-κB) signaling pathway, nucleotide oligomerization domain (NOD)-like receptor signaling pathway, toll-like receptor signaling pathway, and chemokine signaling pathway as top-ranked pathways by RNA-seq analysis. RT-qPCR confirmed that several genes were a part of the inflammatory mediators driving inflammation. PLA2G3, CH25H, NLRC5, and PLAUR (Murase et al., 2017;Wang et al., 2019;Gonias, 2021;Izumi et al., 2021;Rasmussen et al., 2021) were upregulated, while CLEC7A (Deerhake and Shinohara, 2021) was downregulated in the hippocampus of SAE ( Figure 4D; Supplementary Table S1), and they can also affect the neurological function (Rasmussen et al., 2021). The PLA2 family members of inflammation mediators participate in glycerophospholipid metabolism (Sun et al., 2005) and affect choline production. The sphingolipid signaling pathway was another enriched lipid metabolismrelated pathway in our studies. Consistent with previous findings, it regulates the BBB in the LPS-injected mice model for SAE (Vutukuri et al., 2018). Therefore, the altered lipids mediate inflammation or neuroinflammation in the hippocampus during the progression of SAE.
Frontiers in Pharmacology frontiersin.org Amino acid metabolism alterations in the hippocampus of SAE Amino acid metabolism was disturbed in the hippocampus of SAE in gas chromatography (GC)-MSbased metabolomics analyses (Geng et al., 2020). In light of this, more altered amino acids and derivatives, including the increase in L-glutamine, taurine, L-valine, L-histidine, L-threonine, D-lysine, glutaric acid, 5-hydroxykynurenine, nicotinuric acid, and saccharopine and the reduction in creatine, glutathione, oxidized glutathione, L-proline, pantetheine 4′-phosphate, 2-keto-glutaramic acid, and 5′methylthioadenosine, in the hippocampus of SAE was detected in our study, which belong to several metabolisms of amino acids, and this difference among them was likely to explain by diverse methods of metabolomics analysis.
The increase in L-glutamine, taurine, L-valine, L-histidine, L-threonine, and D-lysine has been found in the plasma of sepsis patients and correlated to the severity of sepsis (Chiarla et al., 2011), indicating that the hippocampus has similar metabolic adaptations with peripheral tissues. The saccharopine degradation pathway is the primary route of lysine degradation, and accumulated saccharopine impairs normal neuron development (Guo et al., 2022). The increase in glutamine leads to decreased 2-ketoglutaramic acid, indicating that glutamine metabolism is deregulated in SAE conditions. Glutaric acid accumulation is linked to neurodegeneration (Rodrigues et al., 2013;Rodrigues et al., 2019). 5-hydroxykynurenine, as the precursor of serotonin, is involved in the serotonin pathway, which produces the neurotransmitter serotonin and affects emotional status (Palacios-Filardo and Mellor, 2019). Creatine and choline were decreased in the hippocampus of patients with mild cognitive impairment (Tumati et al., 2013), indicating their associations with cognitive impairment in SAE. It is reported that taurine has versatile effects in modulating neuroinflammatory activity, cellular redox homeostasis, Ca 2+ homeostasis, and neurogenesis (Jakaria et al., 2019). Therefore, the increased taurine in SAE indicates a feedback metabolism regulation to the neurological dysfunction. Because 5′methylthioadenosine is a metabolite that has antiinflammatory effects in preventing endotoxin-induced lethality and inflammatory response (Hevia et al., 2004), its decreased level would deteriorate the inflammation of the hippocampus in SAE.
Glutathione metabolism and the GSH: GSSG redox homeostasis is critical to maintaining neuron physiology (Aoyama, 2021). Interestingly, both glutathione (GSH) and oxidized glutathione (GSSG) were decreased in the hippocampus of SAE mice. However, we observed that the ratio of GSH/GSSG was 1:1.85 ( Figure 3F), which indicated that GSH: GSSG redox homeostasis was disturbed in the hippocampus due to sepsis. Collectively, the amino acid metabolism-related pathway, as mentioned above, would disrupt the redox homeostasis, drive inflammatory response, and dampen the antiinflammatory effects in the hippocampus during the early stage of SAE progression.
Glucose metabolism and nucleotide metabolism alterations in the hippocampus of SAE D-glycerate 3-phosphate (G3P) decreased and subsequently increased L-lactic acid production, indicating that glycolysis was reinforced for a metabolic adaptation to the inflammatory response in the SAE-grouped hippocampus. However, several metabolites (fumaric acid and ascorbic acid) holding anti-inflammatory and/or neuroprotective effects were decreased, and the oxidative stress metabolite gluconic acid was increased in the SAE group (Linker et al., 2011;Ament et al., 2021).
Regarding the nucleotides, the level of purine nucleobases (guanosine and adenine) and pyridine nucleobases (uridine, cytidine, and cytosine) decreased in the SAE hippocampus, indicating that their pyrimidine pathway was impaired and in shortage of energy. Uracil has been shown as the intermediate metabolite to neuroprotection and neuroinflammation (Lecca and Ceruti, 2008). Adenine and pseudouridine could cause inflammation; however, other nucleotides and derivatives (inosine, guanosine, pyridoxamine, and cyclic AMP) have anti-inflammatory effects (Gerbatin et al., 2017;Itokawa et al., 2018;Veremeyko et al., 2018;Lovaszi et al., 2021) that decrease as SAE progresses. This result suggested that the metabolites could not affect the anti-inflammatory response. Adenosine 3′-monophosphate (AMP, for ATP synthesis), cyclic AMP (cAMP, second messenger), and dephospho-CoA (for CoA synthesis) were downregulated in the SAE hippocampus. Uracil, guanosine, pyridoxamine, and cAMP could be pivotal to neuron repair and neuroprotection (Lecca and Ceruti, 2008;Gerbatin et al., 2017;Itokawa et al., 2018;Veremeyko et al., 2018), indicating that their supplementation might be a therapeutic strategy in the treatment of SAE. Additionally, the metabolisms mentioned above corroborated by the enrichment with downregulated DEGs in the hippocampus of SAE ( Figure 4F) and suggested that the glycolysis was enhanced. The nucleotides metabolism also decreased, which indicated an insufficient energy supply disturbing the balance between anabolic and catabolic processes to fuel the neurons and minimize the metabolic brain injury, i.e., the metabolisms of purine, amino acids, and lipids turned into the catabolic process.  Frontiers in Pharmacology frontiersin.org 13 Deregulated synapse and behaviors in the hippocampus of SAE Behavior abnormality and cognitive dysfunction are common characteristics of SAE progression (Eidelman et al., 1996;Gofton and Young, 2012;Heming et al., 2017;Neves et al., 2018;Schuler et al., 2018). Synaptic plasticity relies on the hippocampus synapses normal functions, thus, forming the long-term potentiation (LTP) to improve learning and memory (Magee and Grienberger, 2020). In the present study, we noted several altered metabolites, such as L-glutamine, choline, and cAMP, that were enriched in the synapses-related biological events including glutamatergic synapse, GABAergic synapse, cholinergic synapse, and the calcium signaling pathways (Figure 3), were most affected and deregulated in SAE-grouped hippocampus, which possibly Integrative analysis of transcriptome and metabolome. (A) Correlation heatmap of links between top 100 expressed genes and metabolites based on the Pearson correlation algorithm. The color bar denotes the value of correlation coefficiency, *p < 0.05, **p < 0.01, ***p < 0.001. (B) KGML network building based on the DEGs and DEMs to obtain the critical nodes and metabolism pathways. The triangle with red or green denotes the upor downregulated DEGs, respectively. The circle with red or green denotes the up-or downregulated DEMs, respectively, and the square denotes the pathway. The key metabolic or signaling pathways were shown with pink fonts.
Frontiers in Pharmacology frontiersin.org 15 explained the deterioration of locomotion, exploration, and anxiety-like behaviors that likely implicated learning and memory during the progression of SAE. Additionally, other metabolites, such as CORT, steroids (5,6-EET and isovaleric acid), amino acids (glutaric acid, creatine, and choline), and nucleotides, may directly or indirectly affect behavior (Lecca and Ceruti, 2008;Rodrigues et al., 2013;Tumati et al., 2013;Palacios-Filardo and Mellor, 2019;Rodrigues et al., 2019;Spencer-Segal et al., 2020;Komoltsev et al., 2021;Bras et al., 2022), for example, CORT induce a depressive-or seize-like behaviors via neuroinflammation dysfunction (Komoltsev et al., 2021;Bras et al., 2022). Furthermore, several signaling pathways affected neuron plasticity, including the PI3K-Akt signaling, calcium signaling pathway, and cAMP signaling pathways, and were enriched as the top-ranked pathways in the SAE-grouped hippocampus by RNA-seq analysis (Figure 4). These findings indicated that the dysregulated synapse functions were linked to the behavioral alterations in early SAE. However, further studies need to monitor the long periods of SAE behaviors to corroborate the early phase changes.
Although our study depicted that dysregulated neuroinflammation, metabolism, and synapse pathways were tightly associated with the hippocampus at the early SAE, several key predictions, such as the lipid mediator, and amino acids, need further functional validation. Furthermore, it is still a challenge to directly monitor the cognitive behaviors of SAE rodents at the early stages (Savi et al., 2021) because they move infrequently and are manifested by other alterations in peripheral organ systems or other brain regions at this stage. Additionally, the maturated LPS-sepsis model advanced our studies, but the different sepsis models vary in their mortality and clinic associations (Lewis et al., 2016). Besides, the gender difference was closely correlated with prognosis of sepsis, as the estrogen protective role in inflammation (Kondo et al., 2021). Therefore, future work needs to consider other SAE models including in female rodents and monitor their behavior changes, genes, and metabolites in the middle or late stage of SAE.
In summary, RNA-seq-based transcriptomics and LC-MS-based metabolomics were gathered for the first time to obtain a comprehensive landscape of the hippocampus of SAE. Our integrated studies showed that various dysregulated metabolism pathways, including lipids metabolism, amino acid, glucose, and nucleotide metabolism, inflammation-related pathways, and deregulated synapses were possible mechanisms of SAE progression in the hippocampus. This study provided comprehensive omics data analysis to understand the possible pathophysiological mechanism of the hippocampus in the early phase of SAE. Furthermore, it might pioneer the SAE field to identify future therapeutic targets for brain protection.

Data availability statement
The datasets of RNA-seq have been deposited in sequence read archive (SRA) and assigned a BioProject accession number PRJNA827615. Further reasonable requests for raw data should be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the ethics committee of Tongren Hospital, Shanghai Jiao Tong University School of Medicine (No. A2022-012-01).