Maresin1 (MaR1), a new anti-inflammatory and proresolving lipid mediator, has been proven to exert organ-protective effects in septic animal models. However, the potential mechanisms are still not fully elucidated. In this study, we sought to explore the impact of MaR1 on metabolic dysfunction in cecal ligation and puncture- (CLP-) induced septic mice. We found that MaR1 significantly increased the overall survival rate and attenuated lung and liver injuries in septic mice. In addition, MaR1 markedly reduced the levels of proinflammatory cytokines (TNF-
Sepsis is a life-threatening clinical syndrome characterized by multiple organ dysfunctions due to uncontrolled host inflammatory responses towards infection or injury [
Maresin1 (MaR1), derived from docosahexaenoic acid (DHA) by macrophages via 12-lipoxygenase [
Metabolomics is an “omics” technique that attempts to analyze all low-molecular-weight metabolites in biological samples and explore metabolic mechanisms underlying disease development or drug treatment [
Maresin1 (7,14-dihydroxydocosa-4Z,8Z,10,12,16Z,19Z-hexaenoic acid, MaR1) was from Cayman Chemical Company (Ann Arbor, MI, USA). The chemical structure of MaR1 is shown in Figure
MaR1 improved the survival rate of mice in cecal ligation and puncture- (CLP-) induced septic model (a); the chemical structure of MaR1 (b); experimental schedule (c). MaR1 (100 ng/mice, i.p.) was administered to C57BL/6 mice 1 h after surgery, and an equal volume of saline was given in both the sham and CLP groups. The survival rate was observed for 8 days. Survival of the two subgroups was estimated by Kaplan–Meier survival curves; comparisons were performed by the log-rank test (
Eight-week-old male C57B6/L mice weighing 20–25 g (Shanghai Experimental Animal Centre, China) were housed four per cage and maintained in a specific pathogen-free room with controlled temperature (22–24°C) and humidity (60–65%) under a 12 h light/dark cycle. The mice were given standard laboratory chow and water ad libitum. Principles of laboratory animal care were followed, and all procedures were conducted according to the guidelines established by the National Institutes of Health (NIH Publication No. 85-23, revised 1996), and every effort was made to minimize suffering. This study was approved by the Animal Care and Use Committee of Wenzhou Medical University.
Mice were anesthetized with 2% sodium pentobarbital (80 mg/kg, intraperitoneally) and randomly assigned to three groups: sham group, CLP group, and CLP + MaR1 group (MaR1: 100 ng/mice, intraperitoneally). Sepsis was induced surgically by CLP as described previously [
The mice which had undergone CLP surgery were randomly administered MaR1 (100 ng) or saline in the sham group (intraperitoneal injection;
The lung and liver samples were obtained immediately after exsanguination (24 h after CLP) then embedded in paraffin and stained with haematoxylin and eosin (H&E) for light microscope analysis. Organ injury scores were performed by two independent investigators blinded to the research assignment following the applicable criteria [
For electron microscopy experiments, the lung tissues were harvested 24 h after CLP and the effect of MaR1 was assessed by transmission electron microscopy analysis. Mice were euthanized and then perfused with cold PBS, followed by 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and processed for transmission electron microscopy (TEM), as described previously [
Lung and liver samples were collected, frozen in liquid nitrogen rapidly, and stored at −80°C until use. The extraction was performed as previously described [
The 1H NMR spectra were measured by using a Bruker Avance III 600 spectrometer equipped with a triple resonance probe (Bruker BioSpin, Rheinstetten, Germany) at 128 K. A standard single-pulse sequence (ZGPR) with water signal presaturation was employed to acquire NMR spectra of tissue extracts. For serum sample, the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with a fixed receiver-gain value was performed in order to minimize broad NMR peaks from proteins and lipids. The main acquisition parameters were set as follows: spectral
The 1H NMR spectra were corrected manually for phase and baseline using TopSpin 3.0 software (Bruker BioSpin, Rheinstetten, Germany). The spectra of serum samples were referenced to the methyl peak of lactate at 1.33 ppm, while the spectra of tissue samples were referenced to the TSP peak at 0 ppm. The “icoshift” procedure was used to align NMR spectra in MATLAB software (R2012a, The MathWorks Inc., Natick, MA, USA) [
In this study, all mice were randomly assigned to experimental procedures including housing and feeding, animal grouping, CLP surgery, and MaR1 treatment. Principal component analysis (PCA) was used to obtain an overview of the metabolic pattern of animal models using the SIMCA software (v. 12.0, Umetrics, Umeå, Sweden). Prior to PCA, NMR data were Pareto-scaled for improving data comparability. All data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s LSD test using SPSS software (version 13.0, SPSS). Survival of the two subgroups was estimated by Kaplan–Meier survival curves and compared by the log-rank test. In this study, a statistically significant difference was considered when
As shown in Figure
MaR1 attenuated lung and liver injury in CLP-induced septic mice. The lung and liver tissues were obtained immediately after exsanguination (24 h after CLP), and the effect of MaR1 was assessed histologically in H&E-stained sections (original magnification ×100). Inset shows 40x magnification of the slides. (a-c) Lung tissues, (d-f) liver tissues, (g) lung injury scores (
To assess the effect of MaR1 on inflammatory response in CLP-induced septic mice, the concentrations of proinflammatory cytokines in serum were detected at 24 h after CLP. The levels of TNF-
MaR1 reduced TNF-
As shown in Figures
MaR1 mitigated mitochondrial damage in CLP-induced septic mice. For electron microscopy experiments, the lung tissues were harvested 24 h after CLP and the effect of MaR1 was assessed by transmission electron microscopy analysis (original magnification ×15,000). The mitochondrial ultrastructure changes in three experimental groups were indicated by the white arrows.
Typical 1H NMR spectra in serum, liver, and lung samples are illustrated in Figures
1H-NMR-based metabolic profiles patterns in septic mouse treatment with MaR1. Representative 1H NMR spectra of serum (a), lung (c), and liver (e) samples obtained from the mice, respectively. The PCA score plot based on the 1H NMR spectra of serum (b), lung (d), and liver (f) samples obtained from mice of three experimental groups.
In this study, PCA was applied to examine changes in metabolic patterns among sham, CLP, and MaR1 groups. PCA can clearly distinguish among these three groups based on serum metabolome (Figure
Furthermore, metabolites were quantified and illustrated in a heat map (Figure
Heat map visualization of the identified differential metabolites in septic mouse treatment with MaR1. Heat maps of metabolites in serum (a), lungs (b), and liver (c) of septic mouse treatment with MaR1.
In the lung, we also identified several metabolites that show a recovery trend after MaR1 treatment; for example, acetate and isoleucine levels were apparently increased in the CLP group relative to the sham group but decreased in the CLP + MaR1 group (Figure
Most metabolites in the liver did not show a recovery trend, but increased levels of valine and acetate in the CLP group relative to the sham group were mildly reduced after MaR1 administration (Figure
Furthermore, metabolic pathway analysis was performed on the basis of metabolites that show a recovery trend after MaR1 treatment. Figure
Metabolic pathway analysis in serum of septic mouse treatment with MaR1. Pathway analysis carried out by the pathway topology analysis among three groups (a) and the pathway flowchart of pyruvate metabolism (b). The key metabolites are shown in (c)~(e).
In lung tissue, we identified alanine, aspartate, and glutamate metabolism as a key metabolic pathway (Figures
Metabolic pathway analysis in lung of septic mouse treatment with MaR1. Pathway analysis carried out by the pathway topology analysis among three groups (a) and the pathway flowchart of alanine, aspartate, and glutamate metabolism (b). The key metabolites are shown in (c)~(f).
Maresin1 (MaR1) is a new family of special proresolving mediators (SPMs) that are derived from endogenous DHA. SPMs have been shown to exert potent protective effects in humans’ biology and health [
Although MaR1 has demonstrated a potential protective effect on sepsis, its clinical application is still in its infancy [
Pyruvate metabolism has been closely associated with human disease by maintaining energy metabolism homeostasis [
Metabolomics results also reveal that MaR1 treatment recovered abnormal metabolism of alanine, aspartate, and glutamate in CLP-induced septic mice. Glutamate plays a vital role in the biosynthesis of proteins in living beings. The reduction in glutamate level in plasma has been linked to poor outcomes in septic shock patients [
Alanine has a close association with multiple metabolic pathways such as glycolysis, gluconeogenesis, and the TCA cycle. In this study, the alanine level in lung tissue was significantly reduced in CLP-induced septic mice as compared with normal control mice. This finding may be due to a significant increase in ALT level in CLP-induced septic mice, since ALT can catalyse the transfer of an amino group from alanine to
Aspartate is synthesized from an intermediate of the TCA cycle, oxaloacetate, by transamination. Our results show that MaR1 treatment mildly recovered the CLP-induced reduction in lung aspartate level. The same trend was also obtained in the level of lung fumarate, which is an important intermediate of the TCA cycle. Therefore, MaR1 could maintain energy metabolism homeostasis in the lung of CLP-induced septic mice. Consistent with this finding, we also found that MaR1 ameliorated the CLP-induced ultrastructural damage of mitochondria in lung tissue by transmission electron microscopy analysis.
Oxidative stress has been implicated in the pathogenesis of sepsis [
The present study suggested that MaR1 treatment improved the survival rate of CLP-induced septic mice in multiple ways, including the recovery of lung and liver injuries, the reduction of inflammatory cytokines, and the attenuation of mitochondrial damage. Furthermore, using a metabolomics analysis, we also found that MaR1 could alleviate metabolic disorders in CLP-induced septic mice, mainly involving pyruvate metabolism, alanine, aspartate, and glutamate metabolism, as well as lung taurine level. Yet, it is worth noting that MaR1 significantly regulated the metabolic changes in serum and lung but not liver in septic mice. The potential explanation underlying this phenomenon could be due to the time effect of MaR1 treatment on sepsis. Therefore, the impacts of MaR1 on hepatic metabolic response might occur for an extended period. Our findings reveal a novel metabolic mechanism for the anti-inflammatory and proresolving actions of MaR1 in sepsis. MaR1 may provide new opportunities to design “metabolic targeted” therapies with high accuracy in treating sepsis. However, several limitations or future works should be considered: on the one hand, based on the present preliminary study, cause-effect associations among MaR1, metabolism, and sepsis cannot be proved. Therefore, cellular experiments or other animal models are recommended to discover more evidence on the therapeutic mechanisms of MaR1 in sepsis. On the other hand, metabolomics needs to be coupled with other omics techniques (e.g., genomics and proteomics) for a better understanding of the effect of MaR1 on metabolic regulation and sepsis treatment. We believe that a deeper insight into the MaR1 therapeutic mechanisms will promote its clinical development and application.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors have no potential conflicts of interest to disclose.
Yu Hao and Hong Zheng contributed equally to this work.
We thank Jian-Guang Wang, Qian Wang, and Hong-Xia Mei for their support and advices in our experiment. This work was funded by the grants from the National Natural Science Foundation of China (no. 81570076 and no. 81571862) and the Xinmiao Talents Program of Zhejiang Province (2017R413077).