Introduction
Sepsis is a systemic inflammatory response syndrome that can occur following burns, trauma, and surgery. Sepsis may develop into septic shock [
1], which is characterised by microcirculation disorders, histiocytic damage, and multi-organ dysfunction [
2]. Septic shock is the most common type of shock and has a mortality rate of 40–80% [
3‐
6]. Cardiovascular dysfunction seriously affects the progression of sepsis, and circulatory dysfunctions, including volume depletion, vasodilation, loss of vascular tone, and myocardial depression, are important features of sepsis [
7]. Hypotension can cause a decrease in oxygen delivery and organ perfusion, resulting in impaired organ function, and decreased organ perfusion often leads to multiple organ dysfunction, including acute injury to the kidneys and lungs and myocardial dysfunction.
Long non-coding RNAs (lncRNAs), which are non-coding RNAs greater than 200 nucleotides in length, are poorly conserved endogenous RNAs that do not encode proteins but instead regulate gene expression [
8,
9]. Several studies have shown that lncRNAs play various roles in inflammatory responses and several diseases, such as cancer and cardiovascular disease [
10,
11]. In mice with LPS-induced sepsis, the lncRNA H19 regulates the expression of aquaporin 1 by sponging miRNA-874 and thus is related to septic myocardial function [
12]. The lncRNA NEAT1 promotes inflammatory responses in sepsis-induced liver injury through the Let-7a/TLR4 axis [
13]. MALAT1 regulates cardiac inflammation and dysfunction caused by sepsis [
14]. However, the roles of all lncRNAs in the vascular injury caused by sepsis remain unclear.
In this study, we identified the differentially expressed (DE) lncRNAs and mRNAs in the aortic tissues of LPS-induced endotoxemic rats using transcriptomic microarray analysis. We then performed functional enrichment analysis and annotation to explore the roles of the DE mRNAs in septic vascular injury.
Materials and methods
Model preparation and sample collection
Male Wistar rats (average weight, 200–250 g; average age, 8 weeks), obtained from Charles River Laboratories (Beijing, China), were allowed free access to standard chow and drinking water. The rats were randomly placed into two groups that were treated as follows: rats in the control group were administered an intraperitoneal injection of 0.9% saline (2 mL/kg,
n = 5), and rats in the LPS group were administered an intraperitoneal injection of LPS (L-2880; Sigma-Aldrich, St. Louis, MI, USA) at 10 mg/kg in 0.9% saline (2 mL of a 5 mg/mL preparation,
n = 5) [
15,
16]. At 24 h after LPS injection, the mean arterial blood pressure (MAP) was calculated by non-invasive measurement of blood pressure. The rats were anaesthetised with an intramuscular injection of ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight) and euthanised via CO
2 inhalation. The aortas were isolated, quickly frozen in liquid nitrogen, and stored at − 80 °C until analysis. All experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996), and the study protocol was approved by the Laboratory Animal Welfare Ethics Branch and Biomedicine Ethics Committee of Peking University (approval no. LA2020343).
Microarray analysis
We analysed five pairs of aortic tissues collected from the LPS and control groups using microarray and detected the DE lncRNAs and mRNAs (
n = 5, each group). The Agilent Gene Expression Hybridisation Kit (Agilent Technology Inc., USA) was used for tissue preparation and microarray hybridisation. An Agilent microarray scanner was used to scan the array, and Agilent feature extraction software was used for the analysis. A fold change > 1.5 and a
P < 0.05, were set as the thresholds for differential expression [
17]. We used the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify the important signalling pathways containing the DE mRNAs. Gene Ontology (GO) analysis was used to explore the biological roles of the DE mRNAs in the categories of molecular function, biological process, and cellular component. Gene Set Enrichment Analysis (GSEA) was used to compensate for the shortcomings of individual genes in the analysis.
Protein–protein interaction analysis
To examine the relationships and functions of the DE mRNAs, a protein–protein interaction (PPI) analysis was performed using the STRING database (
https://string-db.org/). The top 100 up-regulated and down-regulated mRNAs were visualised using Cytoscape (v3.6.0) with high confidence ≥ 0.7 and hiding disconnected nodes in the network. The significant modules in the network were scored > 9 using the MCODE (Molecular Complex Detection) Cytoscape plugin.
Microarray validation using quantitative real-time polymerase chain reaction
Total RNA from rat aortic tissue (50 mg) was extracted using 1 mL of TRIzol reagent (Invitrogen, USA). Approximately, 1000 ng of the extracted RNA was reverse transcribed to cDNA using the SuperScript III Reverse Transcriptase Kit (Invitrogen, USA) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using 2 × PCR master mix (Arraystar, USA) and the ViiA 7 qPCR System (Applied Biosystems; Thermo Fisher Scientific, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and expression was analysed using the 2 − ΔΔCt method.
LncRNA–mRNA correlation and co-expression analysis
We evaluated the correlations between the DE mRNAs and lncRNAs by analysing a coding/non-coding (CNC) co-expression network. LncRNA–mRNA pairings with Pearson’s correlation coefficients (PCCs) greater than or equal to 0.90 were identified, and the lncRNA–mRNA co-expression network was visualised using Cytoscape (v3.6.0).
LncRNA–miRNA–mRNA competitive endogenous RNA regulatory network construction
LncRNAs can sponge miRNAs and prevent them from functioning as negative regulators, thus promoting the expression of the target mRNAs. We used miRanda (
http://cbio.mskcc.org/miRNA2003/miranda.html) to predict miRNA-binding sites and considered overlapping miRNA-binding sites on lncRNAs and mRNAs as evidence of lncRNA–miRNA–mRNA interactions. TargetScan (
http://www.targetscan.org/vert_72/) was used for these analyses. Finally, we constructed a competitive endogenous RNA (ceRNA) network.
Determination of serum C-reactive protein (CRP) level
Serum CRP levels were measured using an agglutination test kit (Omega Diagnostics, LTD., Scotland, UK) according to the manufacturer’s instructions.
Determining in vitro aortic vascular reactivity
The harvested aortic rings were equilibrated under 1 g of resting tension for 60 min. Then, isometric tension, generated by the vascular smooth muscle, was measured using a force–displacement transducer (K30; Hugosachs Elektronik, March, Germany) and was recorded using a PowerLab data acquisition device and Chart v4.2 software (AD Instruments, Ltd., Oxfordshire, UK). After equilibration, arterial ring responsiveness was assessed by measuring the contraction in response to 80 mM KCl. To assess vasorelaxation, the aorta was pre-contracted with 1 μM phenylephrine before measuring the relaxation response to ACh (10–9–10–5 M).
Statistical analysis
GraphPad Prism v5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all statistical analyses. Data are presented as the mean and standard deviation. The statistical significance of differences in the microarray data between the two groups was evaluated using Student’s t-test. Differences in expression were considered statistically significant at P < 0.05. Pearson’s correlation test was used to assess the relationship between lncRNAs and mRNAs.
Discussion
Studies have revealed abnormal expression levels of certain lncRNAs in sepsis-induced organ dysfunction. Therefore, we performed a microarray analysis of the lncRNAs and mRNAs in the aortic tissues of septic model rats to identify the changes in lncRNA and mRNA expression in septic vascular tissue with an aim to provide new targets for improving circulatory failure in sepsis. We identified 503 DE lncRNAs (307 up-regulated lncRNAs and 196 down-regulated lncRNAs) and 2479 DE mRNAs (1304 up-regulated mRNAs and 1175 down-regulated mRNAs).
We first analysed the functions of the DE mRNAs. We found that MTFP1 was the top down-regulated DE mRNA. MTFP1 is a nuclear-encoded protein that promotes mitochondrial fission and is closely related to mitochondrial fission and fusion [
21]. Mitochondrial fragmentation often occurs in dysfunctional mitochondria, resulting in decreased ATP production [
22]. Increasing evidence indicates that mitochondrial dynamics are associated with mitochondrial metabolism and vascular relaxation induced by mitochondrial inhibition [
23,
24]. We observed significant down-regulation of MTFP1 in the aortic tissues of rats with LPS-induced sepsis. This indicates that abnormalities in mitochondrial dynamics occur in sepsis. If these abnormalities cause vascular relaxation, then mitochondrial dynamics could be closely related to the decrease in blood pressure observed in sepsis.
We performed GO and KEGG analyses of the DE mRNAs to determine which signalling pathways and functions may be altered in the aortic tissue in septic shock. KEGG analyses were conducted to identify relevant functional pathways, which showed that among the down-regulated pathways, oxidative phosphorylation was the most enriched pathway, and the citrate cycle (TCA cycle) was also highly enriched. GO analysis was performed to study the functions of the DE mRNAs, which showed that the significantly down-regulated biological processes were generation of precursor metabolites and energy, oxidation–reduction process, cellular respiration, energy derivation by oxidation of organic compounds, mitochondrion organisation, electron transport chain, and oxidative phosphorylation. Nine of the ten most significantly down-regulated cellular components were related to mitochondria (mitochondrion, mitochondrial part, mitochondrial envelope, mitochondrial inner membrane, mitochondrial membrane, mitochondrial protein complex, inner mitochondrial membrane protein complex, and mitochondrial membrane part). The significantly down-regulated molecular functions were oxidoreductase activity, electron transfer activity, NADH dehydrogenase activity, cytochrome-c oxidase activity, NADH dehydrogenase (ubiquinone) activity, oxidoreductase activity, and NADH dehydrogenase (quinone) activity. These results suggest that the most significant changes in the vascular tissue under septic shock are associated with energy metabolism, since many molecules related to energy metabolism and their signalling pathways were significantly down-regulated. Recent studies have indicated that the key pathophysiological mechanism of sepsis is metabolic decline rather than structural damage [
7], and ATP levels and the phosphocreatine/ATP ratio were lower in patients with severe septic shock [
25]. Abnormal energy metabolism occurs in various tissues in patients with sepsis, and mitochondrial dysfunction has been observed in skeletal muscle, platelets, and peripheral blood mononuclear cells [
26,
27]. Mitochondrial dysfunction was also observed in liver cells isolated from healthy humans exposed to endotoxins [
7]. GO analysis indicated that mitochondrial structure proteins were significantly down-regulated, suggesting that mitochondrial structural damage plays an important role in metabolic decline. We suspected that as a result of the serious damage to the mitochondrial structure, the biological processes and molecular functions related to the respiratory chain were also significantly altered; oxidative phosphorylation was also significantly altered, ultimately leading to insufficient energy production and vascular function damage. This was the most prominent change observed in sepsis.
Increasing evidence has shown that lncRNAs play vital roles in sepsis-induced organ dysfunction, including vascular tissue damage. Therefore, we conducted a microarray analysis of the lncRNAs in the aortas of model rats with LPS-induced sepsis to identify the relevant lncRNAs. We performed a qRT-PCR validation of the 20 lncRNAs with the most significantly altered expression and selected 12 of the validated lncRNAs for further analysis. We conducted CNC and ceRNA analyses of these 12 lncRNAs. We then performed GO and KEGG analyses of the predicted target genes to identify the molecules and pathways regulated by these DE lncRNAs.
Next, these 12 DE lncRNAs and mRNAs were subjected to CNC network analysis; we first determined the altered mRNAs related to these 12 lncRNAs. The CNC network analysis revealed potential regulatory relationships between mRNAs and these 12 lncRNAs. Interestingly, the predicted mRNAs were highly consistent with the DE mRNA analysis. The KEGG analysis revealed significant enrichment in oxidative phosphorylation and the citrate cycle (TCA cycle). The biological processes oxidation–reduction process, generation of precursor metabolites and energy, cellular respiration, electron transport chain, and respiratory electron transport chain had high enrichment scores. The top nine enriched cellular components were all related to mitochondrial structure. The top three enriched molecular functions were oxidoreductase, electron transfer, and catalytic activities. These results suggest that the most significant DE lncRNAs were involved in energy metabolism. In other words, the regulation of lncRNA levels might be an important mechanism by which sepsis leads to energy metabolism dysfunction in vascular tissue. This indicates that lncRNAs may be key players in sepsis-related vascular damage.
LncRNAs regulate mRNA expression in several ways. One of the primary mechanisms by which lncRNAs alter the expression of downstream mRNAs is by sponging regulatory miRNAs, thus reducing their levels. Therefore, we performed a ceRNA analysis of the 12 lncRNAs and DE mRNAs in the hope of identifying the mRNAs that are regulated by miRNAs. KEGG and GO analyses of these mRNAs revealed that five of the top ten enriched cellular components were related to mitochondrial structure, and oxidoreductase activity was the most enriched molecular function, suggesting that the altered mitochondrial structure was regulated by lncRNA–miRNA interaction. In addition, the KEGG analysis showed that several inflammation-related pathways were highly enriched, including the NF-κB, chemokine, and TNF signalling pathways. These results suggest that although lncRNAs regulate many mRNAs related to energy metabolism, lncRNA–miRNA–mRNA interaction is not the main way. LncRNAs can regulate the mRNAs involved in energy metabolism in other ways. However, lncRNA–miRNA–mRNA interactions may be involved in the inflammatory response. The mechanisms by which lncRNAs regulate mRNAs related to energy metabolism require further study.
The cardiovascular system is essential for maintaining adequate organ perfusion. Therefore, cardiovascular dysfunction has a direct impact on sepsis. Severe circulatory system dysfunction is closely associated with cell metabolism disorders. Multiple organ failure caused by septicaemia is often accompanied by only limited cell death and frequent recovery of organ function, suggesting that metabolic shutdown, rather than structural damage, is the main cause [
7]. Blood vessels require well-structured, functional mitochondria to ensure sufficient energy supply, normal physiological function, and adequate organ perfusion. However, in an inflammatory microenvironment, mitochondrial oxidative phosphorylation is disrupted, which reduces the supply of ATP [
28‐
30]. Previous studies have shown aberrant energy metabolism in several organs during sepsis. For example, LPS-induced acute myocardial injury is mainly caused by mitochondrial damage [
25]. The decrease in mitochondrial respiration leads to excess ROS generation, causing oxidative damage to cells, which can result in cell death. When ATP is insufficient, a variety of cell death processes can occur, including necrosis, apoptosis, reticulum, pyroptosis, and autophagy-induced cell death, causing organ damage [
31]. This reduction in ATP generation leads to a bioenergy deficit. It has long been viewed as a critical factor in sepsis-induced organ failure. Our high-throughput mRNA sequencing revealed significant energy metabolism damage in the aortic tissue, including mitochondria structural damage and aberrant mitochondrial dynamics. We suspect that insufficient energy production in vascular tissue damages smooth muscle cells, directly leading to vascular relaxation and hypotension. Insufficient energy production can also damage vascular endothelial cells, cause disseminated intravascular coagulation, and eventually lead to multiple organ failure [
32]. Some studies have shown that energy-related treatments such as β-receptor blockers can improve the outcomes of patients with heart failure [
3]. Therefore, drugs that improve energy metabolism may also be helpful in the treatment of septic shock. Our high-throughput lncRNA sequencing showed that lncRNAs are the main regulators of this process and that miRNAs are also involved. Thus, further research is warranted.
This study has some limitations. We tested vascular tissue, but we did not isolate a smooth muscle, endothelial tissues, or other vascular cells. Therefore, we could not state which cell types were altered; thus, more detailed research is needed in the future.
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