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Abstract

Salusin-α and salusin-β are newly found bioactive peptides of 28 and 20 amino acids, respectively, which are widely distributed in the hematopoietic system, endocrine system, and central nervous system. Salusins exert cardiovascular effects, including hypotension and bradycardia; promote vascular inflammation; and so on. However, little information is available yet on the relationships of salusin-β with sepsis. In this study, we investigated the distribution and content of endogenous salusin-β in septic rats. A total of 72 specified pathogen-free (SPF) male Sprague-Dawley (SD) rats were randomly divided into control group (sham operation, n = 36) and experimental group (n = 36) with sepsis rat model by cecal ligation and puncture (CLP). The model group rats were sacrificed after 6, 12, and 24 h of treatment. The concentration of salusin-β in spleen, stomach, small intestine, hypothalamus, and serum specimens was detected by enzyme-linked immunosorbent assay (ELISA). It showed that salusin-β was endogenously generated in rat tissues, including spleen, stomach, small intestine, hypothalamus, and serum. The content of salusin-β in the spleen was higher than that in other tissues. The content of salusin-β in the spleen, stomach, and small intestine, together with the serum level of salusin-β, increased significantly at 6 h after CLP compared with the control group (P < 0.05). The content of salusin-β in spleen and serum peaked at 12 h, and in small intestine, it reached the summit at 24 h. Meanwhile, no significant fluctuations in salusin-β content were observed in the stomach. The content of salusin-β in hypothalamus began to increase at 6 h, and a significant increase appeared 12 h after CLP (P < 0.05). In conclusion, this study shows that the time-dependent alterations of salusin-β in septic rats suggest that salusin-β might be involved in the pathogenesis of sepsis.

Keywords

enzyme-linked immunosorbent assay rat salusin-β sepsis

Introduction

Sepsis is the systemic response of the host toward invading microorganisms and their toxins.¹ It has been generally accepted that whole-body inflammatory state, named systemic inflammatory response syndrome (SIRS), is the early hallmark sign of sepsis.² This early phase of excessive systemic inflammation compromises the function of distinct organ systems, resulting in multiple organ dysfunction syndrome (MODS). As the population ages, the incidence of sepsis in the United States continues to rise. Recently, Kadri et al.³ identified all patients with International Classification of Diseases, Ninth Revision (ICD-9) codes for septic shock at 27 academic hospitals in the United States from 2005 to 2014; it is estimated that septic shock incidence increased from 6.7 to 19.3 per 1000 hospitalizations, while mortality decreased from 48.3% to 39.3%. The population of 1.3 billion in mainland China accounts for approximately one-fifth of the whole world population. In speculating, we have at least 5.68 million sepsis and 3.51 million severe sepsis cases that need to be treated in 1 year.^4,5 The intensive care unit (ICU) and hospital mortality rates of severe sepsis were 28.7% and 33.5%,⁶ respectively, while another single-centre retrospective study from a teaching hospital also reports a high ICU mortality rate of 43.9%.⁷ Despite rapid advances in medical care over the past decades, sepsis remains a leading cause of mortality in ICU.

The main theory suggests that SIRS is predominantly mediated by cytokines, so it is a hot topic to find new inflammatory mediators or study the activation of the cytokine network. Salusins are endogenous bioactive peptides, including salusin-α and salusin-β consisting of 28 and 20 amino acids, respectively.⁸ Both of them are identified in a human full-length-enriched complementary DNA (cDNA) library using bioinformatics analysis by Shichiri et al.⁸ Salusins have been distributed widely in different kinds of tissues of humans and rats, such as the cardiovascular system, nervous system, macrophages, and monocytes, as well as human body fluids such as plasma and urine.^8–10 Salusins exert multiple cardiovascular effects, including hypotension, bradycardia,⁸ inhibition of myocardial contraction,¹¹ reduction of cardiac ischemic injury,¹² promotion of cardiomyocyte hypertrophy,¹³ and proliferation of vascular smooth muscle cells.⁸ Most of the studies that have investigated the biological activities of salusins demonstrated that they acted on the cardiovascular system.^14–16 Interestingly, it is shown that salusins probably are involved in the pathogenesis of inflammatory diseases.^17,18 Ozgen et al.¹⁷ have reported that serum salusin-α level is increased in rheumatoid arthritis, a chronic inflammatory disease. Salusin-α and salusin-β immunoreactivity has been detected in human coronary atherosclerotic plaques, with dominance of salusin-β in macrophage foam cells, vascular smooth muscle cells, and fibroblasts.¹⁹ Salusin-α suppresses human foam cell formation, while salusin-β promotes the formation of human foam cells.²⁰ Furthermore, another study indicates that salusin-β accelerates inflammatory responses in vascular endothelial cells via nuclear factor-κB (NF-κB) signals in low-density lipoprotein (LDL) receptor–deficient mice in vivo.²¹ Recently, a new study indicated that salusin-β exhibited potent antibacterial activity against Gram-positive microorganisms, such as Bacillus subtilis NBRC 3513, Bacillus megaterium ATCC 19213, Staphylococcus aureus NBRC 12732, and Staphylococcus epidermidis NBRC 12933. Salusin-β had the ability to depolarize the cytoplasmic membrane, and this may be linked to the antibacterial activity of this agent.²²

However, little information is available yet on the relationships of salusin-β with sepsis. Based on the above studies, we hypothesized that salusins might be involved in the pathogenesis of inflammatory disease, including sepsis. This study was designed to observe whether salusin-β was involved in the pathogenesis of sepsis which determined the distribution and content of endogenous salusin-β in septic rats.

Materials and methods

Animals

Adult male Sprague-Dawley (SD) rats, 3 months old, weighing 250–300 g (Laboratory Animal Center of Gansu University of Traditional Chinese Medicine, Lanzhou, China), were used in this study. All animals were acclimatized under controlled temperature (23 ± 2°C), humidity (50 ± 5%), and 12 h light/12 h dark cycle for 1 week before the experiment. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (1985), National Institutes of Health (NIH), Bethesda, or European Guidelines on Laboratory Animal Care.

Cecal ligation and puncture operation

Cecal ligation and puncture (CLP) in rodents has become the most widely used model for experimental sepsis and is considered the gold standard in sepsis research.²³ As the cecum is an endogenous source of bacterial contamination, puncturing the cecum results in mixed enteric bacteria translocating into the abdominal cavity.²⁴ In order to ensure the uniformity of the process, all the procedures were performed by the same person.

In brief, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg; Sigma, USA), a midline laparotomy was made using minimal dissection, and the cecum was ligated just below the ileocecal valve with 2-0 silk so that intestinal continuity was maintained. The antimesenteric surface of the cecum was perforated with an 18-gauge needle at two locations 1 cm apart and the cecum was gently compressed until the fecal matter was extruded. The bowel was then returned to the abdomen and the incision was closed. At the end of the operation, all rats were resuscitated with saline, 3 mL/100 g body weight, given subcutaneously. The sham-operated groups were given a laparotomy, and the cecum was manipulated but not ligated or perforated. All animals were returned to their cages with free access to food and water. The experimental protocols were approved by Animal Care and Use Committee of First Hospital of Lanzhou University.

Detection of salusin-β in serum and tissues

Blood samples were collected via the internal carotid from septic or sham-operated rats at 6, 12, and 24 h after surgery by a serum separator tube, and the blood samples were clotted for 2 h at room temperature before centrifugation for 20 min at approximately 1000g (Hettich EBA 12R, Germany). The spleen, stomach, small intestine, and hypothalamus tissue samples (100 mg per tissue) were washed in ice-cold phosphate buffered saline (PBS) (0.01 mol/L, pH 7.0–7.2) to remove excess blood thoroughly and weighed before homogenization; then, they were homogenized in 5–10 mL of PBS with a glass homogenizer on ice. After that, the homogenates were centrifugated for 5 min at 5000g (Hettich EBA 12R, Germany). All samples were stored at ⩽−20°C for less than 1 month, and repeat freeze/thaw cycles were avoided.

Salusin-β measurements were conducted in the Biochemistry Department using specific commercial kits (ELISA Kit for Rat Salusin-β, Catalog No: CEC026Ra; Uscn Life Science Inc., PR China). The competitive inhibition enzyme immunoassay technique was employed by this ELISA Kit, and the competitive inhibition reaction was launched between biotin-labeled salusin-β and unlabeled salusin-β (standards or samples) with the pre-coated antibody specific to salusin-β. Briefly, a monoclonal antibody specific to salusin-β was pre-coated onto a microplate, and serum, tissue homogenates, and standards were added to specific wells respectively and incubated for 1 h at 37°C. The plate was washed with PBS three times, and 100 µL of Horseradish Peroxidase (HRP) which conjugated with avidin was added to each microplate well and incubated for 30 min at 37°C. The plate was washed again, and 90 µL of tetramethylbenzidine (TMB) substrate solution was added to each microplate well and incubated for 15–20 min at 37°C in the dark. The reaction was stopped by the addition of 50 µL of 0.5 M H₂SO₄. A microplate reader (Rayto RT-2100C, USA) was used to determine the absorbance values of each well at 450 nm; the intensity of color developed was reversed proportional to the concentration of salusin-β in the sample. The spleen, stomach, small intestine, hypothalamus, and serum specimens were measured using the same kits, the same method, and in the same experiment series.

Statistical analysis

All values are presented as mean ± standard deviation (SD). The content of endogenous salusin-β in serum and tissues was compared by analysis of unpaired Student’s t-test. A one-way analysis of variance (ANOVA) followed by the Newman–Keuls test was used when multiple comparisons were made. Values of P < 0.05 were considered to be statistically significant.

Results

Content of salusin-β in sham-operated rats

Salusin-β was endogenously generated in rat tissues, including spleen, stomach, small intestine, hypothalamus, and serum. The content of salusin-β in serum was maximum in the sham-operated rat and significantly higher than in spleen, hypothalamus, small intestine, and stomach (P < 0.05). The content of salusin-β in the spleen was significantly higher than in hypothalamus, small intestine, and stomach (P < 0.05). The difference was signifi-cant between the hypothalamus and stomach (P < 0.05). No significant difference was observed between the stomach and small intestine (P > 0.05). The content of salusin-β in the spleen, hypothalamus, small intestine, stomach, and serum was not significantly different between 0, 6, 12, and 24 h after sham operation (P > 0.05). The distribution of salusin-β in sham-operated rat tissues is summarized in Figure 1. The content of salusin-β in the spleen, small intestine, stomach, hypothalamus, and serum is summarized in Table 1.

Figure 1.

Content of salusin-β in spleen, hypothalamus, small intestine, and stomach after sham operation. Compared with spleen, *P < 0.05; compared with small intestine, ^ΔP < 0.05; compared with stomach, ^☆P < 0.05.

Table 1.

Content of salusin-β in sham-operated rat ( $\bar{x} \pm s$ ) (n = 9).

Groups	Salusin-β (pg/mg) (pg/mL)
	Spleen	Small intestine	Stomach	Hypothalamus	Serum
Sham 0 h	8.28 ± 0.88	2.93 ± 0.66*	2.70 ± 0.49*	6.36 ± 0.73*^{Δ ☆}	15.00 ± 3.72*^{Δ ☆ #}
Sham 6 h	6.97 ± 0.91	2.69 ± 0.37*	2.13 ± 0.32*	6.33 ± 0.69*^{Δ ☆}	14.64 ± 8.77*^{Δ ☆ #}
Sham 12 h	7.90 ± 0.88	2.56 ± 0.22*	2.76 ± 0.25*	6.78 ± 0.66*^{Δ ☆}	15.84 ± 4.35*^{Δ ☆ #}
Sham 24 h	7.99 ± 0.84	2.79 ± 0.38*	2.51 ± 0.30*	5.41 ± 0.74*^{Δ ☆}	13.55 ± 5.55*^{Δ ☆ #}

Compared with spleen,*P < 0.05; compared with small intestine, ^ΔP < 0.05; compared with stomach, ^☆P < 0.05, compared with hypothalamus, ^#P < 0.05.

Content of salusin-β in rat serum after operation

Salusin-β in rat serum had no significant fluctuations after sham operation. The serum level of salusin-β increased gradually with time after CLP; this was significant at 6 h (26.17 ± 5.64 pg/mL) after CLP, peaked at 12 h (42.31 ± 7.84 pg/mL), and slightly decreased at 24 h (22.53 ± 4.72 pg/mL). The serum level of salusin-β in CLP 0-h group (15.00 ± 3.72 pg/mL) was significantly lower than in CLP 6-, 12-, and 24-h group (P < 0.05). The difference was significant between CLP 12 h and 6 h (P < 0.05). No significant difference was observed between CLP 6 h and CLP 24 h (P > 0.05). The serum level of salusin-β in CLP 6-, 12-, and 24-h group was significantly higher than in sham 6-, 12-, and 24-h group (P < 0.05). The time changes of salusin-β in rat serum after the operation are summarized in Figure 2.

Figure 2.

Time changes of salusin-β in rat serum after sham operation or cecal ligation and puncture (CLP) operation. Compared with sham operation group, ^#P < 0.05; compared with CLP 0 h group, *P < 0.05; compared with CLP 6 h group, ^ΔP < 0.05; compared with CLP 12 h group, ^☆P < 0.05.

Content of salusin-β in rat tissues after CLP

The content of salusin-β in the spleen, small intestine, and stomach increased significantly at 6 h after CLP compared with the CLP 0-h group (P < 0.05), and salusin-β in spleen peaked at 12 h and slightly decreased at 24 h; salusin-β in small intestine increased gradually with time, reaching the summit at 24 h; there was no significant fluctuations in salusin-β in stomach after CLP 6 h. The content of salusin-β in hypothalamus began to increase at 6 h, and a significant increase appeared 12 h after CLP (P < 0.05). The time changes of salusin-β in rat tissues after CLP are summarized in Figure 3 and Table 2.

Figure 3.

Time changes of salusin-β in spleen, small intestine, stomach and hypothalamus after cecal ligation and puncture (CLP) operation. Compared with CLP 0 h group, *P < 0.05; compared with CLP 6 h group, ^ΔP < 0.05; compared with CLP 12 h group, ^☆P < 0.05.

Table 2.

Content of salusin-β in rat after cecal ligation and puncture (CLP) operation ( $\bar{x} \pm s$ ) (n = 9).

Groups	Salusin-β (pg/mg) (pg/mL)
	Spleen	Small intestine	Stomach	Hypothalamus	Serum
CLP 0 h	8.28 ± 0.88	2.93 ± 0.66	2.70 ± 0.49	6.36 ± 0.73	15.00 ± 3.72
CLP 6 h	9.36 ± 0.75*	4.25 ± 0.80*	3.78 ± 0.70*	6.55 ± 0.82	26.17 ± 5.64*
CLP 12 h	11.67 ± 0.93*^Δ	6.09 ± 0.90*^Δ	3.59 ± 0.89*	7.72 ± 0.87*^Δ	42.31 ± 7.84*^Δ
CLP 24 h	10.75 ± 0.92*^{Δ ☆}	7.03 ± 0.90*^{Δ ☆}	3.84 ± 0.85*	7.09 ± 0.78^☆	22.53 ± 4.72*^☆

Compared with CLP 0 h group, *P < 0.05; compared with CLP 6 h group, ^ΔP < 0.05; compared with CLP 12 h group, ^☆P < 0.05.

Discussion

The most important findings of this study are as follows: (1) the distribution of salusin-β in rat was different—spleen > hypothalamus > intestine > stomach—and (2) the time-dependent alterations of salusin-β in septic rats suggest that salusin-β might be involved in the pathogenesis of sepsis.

The pathogenesis of sepsis is still unclear today. The main theory thinks that SIRS is predominantly mediated by cytokines, and it is now well established that bacterial infection or tissue injury leads to the activation of the cytokine network. In sepsis, tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) are the most important proinflammatory cytokines; they act synergistically in activating target cells and inducing the production of more inflammatory mediators and are largely responsible for the clinical manifestations of sepsis.

Salusins are not only considered as a novel bioactive peptide involved in hypotension, bradycardia, reducing cardiac ischemic injury, mitogenic activities, intracellular signaling pathways, and so on, but they are also characterized as a novel candidate of inflammatory cytokines. Some peptides such as casecidin, isracidin, hepcidin, and lactoferricin, which have been detected in human or cow milk, have been shown to exhibit antibacterial activities.²⁵ Analytical examinations of biological fluids such as plasma, milk, and cheese have recently revealed that there are many bioactive peptides, including salusin peptides, in these liquids.^26,27 Previous studies have shown that serum salusin-α levels are increased in rheumatoid arthritis, a chronic inflammatory disease, which leads to early and accelerated atherosclerosis.¹⁷ Moreover, salusin-α suppresses macrophage foam cell formation, while salusin-β stimulates. Several studies have shown that salusin-α exerts anti-atherosclerotic effects by suppressing serum total cholesterol levels and acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) expression, while salusin-β accelerates the development of atherosclerotic lesions associated with up-regulation of scavenger receptors and ACAT-1 in apolipoprotein E–deficient mice.²⁸ Recently, several studies have indicated that stimulation with the inflammatory cytokine TNF-α and lipopolysaccharide (LPS) resulted in the increased secretion of salusin-β without inducing the expression of the gene for preprosalusin, suggesting that TNF-α and LPS stimulated the release of salusin-β. These data demonstrate that salusin-β, which induces macrophage foam cell formation, is secreted in its authentic form from human monocytes/macrophages.¹⁰ Furthermore, a new study indicates that salusin-β exhibited potent antibacterial activity against Gram-positive microorganisms, such as Bacillus subtilis NBRC 3513, Bacillus megaterium ATCC 19213, Staphylococcus aureus NBRC 12732, and Staphylococcus epidermidis NBRC 12933. Salusin-β had the ability to depolarize the cytoplasmic membrane, and this may be linked to the antibacterial activity of this agent.²² However, the relationship of salusin-β with sepsis or the function of salusin-β in inflammation is still unclear.

This study clearly indicated that the serum and tissues of rat contain a certain amount of salusin-β, and the level of spleen tissue was the highest, followed by the hypothalamus, small intestine, and stomach tissues, which were the lowest. This is different from Suzuki’s study,²⁹ which suggests that salusin-β distribution and preprosalusin distribution are not entirely consistent. Besides, the serum level of salusin-β increased at 6 h after CLP, peaked at 12 h, and slightly decreased at 24 h. The increasing tendency of salusin-β is consistent with the serum inflammatory cytokines TNF-α and interleukin-1β (IL-1β) in septic rats.³⁰ Based on previous studies,^10,17,21 we speculated that salusin-β might be involved in the pathogenesis of sepsis.

In conclusion, this study shows that the time-dependent alterations of salusin-β in sepsis rats suggest that salusin-β might be involved in the pathogenesis of sepsis.

Footnotes

Acknowledgements

The authors thank Ms Jian-Yun YUE for technical support at Department of Clinical Laboratory, San Ai Tang Hospital, and Dr Wei-Zhong WANG, Department of Physiology, Second Military Medical University, for critical reading of this manuscript and for helpful comments.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by Award Number 30700266 from the National Natural Science Foundation of China, GSWSKY2016-20 from the Health industry project of Gansu Province of China, and 1308RJZA269 from the Natural Science Foundation of Gansu Province.

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The time changes of endogenous salusin-β in septic rats

Abstract

Keywords

Introduction

Materials and methods

Animals

Cecal ligation and puncture operation

Detection of salusin-β in serum and tissues

Statistical analysis

Results

Content of salusin-β in sham-operated rats

Content of salusin-β in rat serum after operation

Content of salusin-β in rat tissues after CLP

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

References