Sepsis is a severe clinical syndrome resulting from systemic host response to infection. The mortality rate with severe bacterial sepsis, especially that associated with shock, still approaches 50% despite appropriate antimicrobial therapy and optimal care. In severe sepsis, endotoxin (lipopolysaccharide [LPS]) released by bacteria results in aggregation of monocytes/macrophages and release of multiple inflammatory mediators such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), which play a prominent role in sepsis-induced tissue injury. The kinetics and magnitude of cytokine release influence the development of sepsis.^{[1] and [2]}

The peptide apelin, isolated from bovine stomach tissue extracts, was newly identified as the endogenous ligand of the human orphan putative G-protein–coupled receptor protein related to the angiotensin receptor AT₁, also named angiotensin II receptor like-1 (AGTRL1) by reverse pharmacology.³ Apelin is secreted as a 77 amino acid prepropeptide and processes 12, 13, 17, and 36 amino acid moieties produced in different tissues with varying activity.⁴ Apelin and AGTRL1 mRNA is abundantly expressed in rats and human brain, which suggests its central regulatory role. As a multiple functional neuropeptide, apelin regulates body fluid homeostasis, food intake, respiratory rhythm, and biological rhythm.^{[5] and [6]} Apelin receptor is localized in rat and human myocardium, as well as the medial layer of human coronary artery, aorta, and saphenous vein grafts.^{[7], [8] and [9]} Apelin is an important adipokine because plasma apelin is mainly derived from the lining of vascular endothelial cells; blood vessels in the human heart, kidney, adrenal gland, lung; and endothelial cells of large conduit vessels and adipocytes.^{[10] and [11]}

Apelin and AGTRL1 are abundant in cardiovascular tissues. Similar to the activity of the receptor, apelin administration causes a significant decrease in mean arterial blood pressure (MABP), and it has a strong inotropic action, which suggests its important regulatory role in cardiovascular homeostasis in a paracrine/autocrine manner.^{[12] and [13]} In patients with heart failure and dilated cardiomyopathy, plasma apelin concentration was increased in the early stage and decreased in the late stage.^{[14] and [15]} Interestingly, apelin-knockout mice display aging-associated reduced cardiac contractility, evident at 6 months of age. Furthermore, in a pressure overload–induced heart-failure model, apelin-knockout mice showed severely impaired heart contractility.¹⁶ Apelin has been considered the most potent endogenous positive-inotropic peptide, which suggests that decrease in endogenous apelin plays a pivotal role in heart failure. Exogenous administration of apelin exerts in vivo inotropic effects on normal and failing hearts.¹⁷

In addition, apelin was shown to have anti-inflammatory, anti-infection, and inhibitory effects on inflammatory mediator release, with expression of AGTRL1 mRNA in several human T- and B-cell lines.^{[18] and [19]} Apelin inhibited the entry of HIV-1 and HIV-2 into NP-2/CD4 cells expressing AGTRL1.²⁴ Considering the unusual combination of inotropy and anti-inflammatory effects of apelin, we speculated that apelin could be an endogenous cardioprotective substance and antagonize myocardial impairment in sepsis by attenuating inflammatory responses.

In the current study, we investigated rats with severe septic shock induced by cecal ligation and puncture (CLP) showing low plasma and myocardial content of apelin and low myocardial protein level of AGTRL1. Furthermore, exogenous administration of apelin ameliorated disordered hemodynamics and metabolism in these rats. Apelin attenuated the plasma release of inflammatory factors and could directly inhibit secretion of MCP-1 and IL-8 in cultured rat peritoneal macrophages. Thus, apelin might be a promising therapeutic target for severe sepsis and septic shock.

Methods

Animals and Reagents

All animal care and experimental protocols were in compliance with the Animal Management Rule of the People's Republic of China (Ministry of Health, P.R. China, document no. 55, 2001). Male Sprague-Dawley rats (220 to 250 g) were provided by the Animal Department, Health Science Center, Peking University. Apelin (human apelin1-36) peptide was synthesized by Phoenix Pharmaceuticals (Belmont, CA), and apelin enzyme immunoassay (EIA) kit and anti-rat AGTRL1 antibody were supplied by Phoenix Pharmaceuticals. Enzyme-linked immunosorbent assay (ELISA) kits for MCP-1 and IL-8 were from R&D Systems (Minneapolis, MN). Primary antibodies for AGTRL1 (sc-33838) were from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibody for β-actin (sc-1616) and secondary antibodies rabbit anti-goat and goat anti-rabbit immunoglobulin G were from Santa Cruz Biotechnology. Nitrocellulose membrane was from Hybond-C (Amersham Life Science, UK), and ECL was from Beijing Applygen Technologies (Beijing, China). Trizol agent was from Gibco (Gaithersburg, MD); dNTP was from Clontech (Palo Alto, CA); and M-MuLV reverse transcriptase, Taq DNA polymerase, RNasin and oligo(dT)15 primer were from Promega (Madison, WI). Sequences of oligonucleotide primers are in Table 1. All the sequences of oligonucleotide primers were synthesized by Sai Bai Sheng (Beijing, China). Other chemicals and reagents were of analytical grade.

Animal Model of Polymicrobial Sepsis

Male Sprague-Dawley rats were housed under standard conditions (room temperature 20 ± 1°C, humidity 60 ± 10%, lights from 6 AM to 6 PM) and given standard rodent chow and water freely. All experiment procedures were performed in accordance with the Guidelines of Animal Experiments from the Committee of Medical Ethics, National Health Department of China. The polymicrobial septic model of rats with CLP was as described.²⁰ A 3-cm ventral midline incision was made in rats anesthetized with methoxyflurane inhalation. The cecum was exposed and ligated just below the ileocecal valve with use of a 3-0 silk suture to avoid intestinal obstruction. The cecum was punctured twice with use of an 18-gauge needle and then returned to the abdominal cavity, and the incision was closed. Each rat received normal saline (4 mL/100 g body weight) by subcutaneous injection immediately after CLP. The area of incision was bathed with 1% lidocaine to provide analgesia during the study. Sham-operated animals underwent the same surgical procedure, except the cecum was neither ligated nor punctured.

Rats were divided into 5 groups for treatment: (1) control; (2) sepsis; (3) sepsis+vehicle; (4) sepsis+low-dose apelin; and (5) sepsis+high-dose apelin (Fig. 1). At 20 hours after CLP or sham operation (the late, hypodynamic stage of sepsis), rats were deeply anesthetized with pentobarbital sodium (45 mg/kg, intraperitoneal administration), and 2 catheters filled with heparin saline (500 U/mL) were inserted into the right femoral and right carotid arteries, the latter further inserted into the left ventricle, to measure MABP and left ventricular (LV) pressure. Heart rate, maximal LV pressure development (LVdp/dt_max), LV end-systolic pressure, and LV end-diastolic pressure (LVEDP) were recorded on a Powerlab (4S, Australia) as described.²¹ Then blood was collected in heparinized syringes from the left ventricle and transferred to tubes for glucose, lactic acid, MCP-1, and IL-8 determination and lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity assay in plasma. All animals were killed by exsanguination; hearts were removed and placed in cold normal saline (4°C).

Enzyme Immunoassay for Apelin Determination

Myocardial tissue (10 mg) was boiled in 0.1 mol/L acetic acid for 10 minutes, homogenized, and centrifuged at 12,000 rpm for 20 minutes; the supernatant was used to quantify total protein concentration via the Bradford assay. Equal amounts of total protein were used in the apelin-36 EIA assay kit following the manufacturer's instructions. Plasma was used directly for the assay. The ED₅₀ for rat apelin was 8.62 pg/tube, and cross-reactivity with rat apelin-36 was 100% and 0% with apelin-16 and apelin-13, respectively.

mRNA Levels of Apelin, AGTRL1, and Brain Natriuretic Peptide

Total myocardial RNA was prepared by in situ lysis with Trizol reagent.²² One microgram of total tissue RNA was reverse-transcribed into a single-strand cDNA with use of M-MuLV reverse transcriptase and oligo (dT) 15 primers. Polymerase chain reaction (PCR) was performed in a 0.2-mL tube containing 2 μL tissue cDNA, 5 pmol/L per each apelin-S and apelin-A primer (Table 1), a mixture of 1 μL, 2.5 mmol/L per each dNTP mixture of 1 μL, 1.5 mmol/L MgCl₂ of 1.5 μL, 10× PCR buffer of 2.5 μL, and 1.25 unit Taq DNA polymerase, in a total volume of 25 μL. After a denaturing at 95°C for 5 minutes, the solution underwent PCR at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds for 30 cycles, then 72°C for 5 minutes. Then 8 μL of PCR product was separated in a 1.5% agarose gel and stained with ethidium bromide. The optical density of the 190-bp band was measured by use of the Gel Documentation System (Bio-Rad, Hercules, CA). Two microliters of PCR product was amplified again with the 2 rat β-actin primers at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds for 24 cycles, then 72°C for 5 minutes, and the optical density of the β-actin band (291 bp) was measured. The mRNA ratio of apelin to β-actin was considered the relative amount of apelin mRNA. The relative mRNA amounts of AGTRL1 and brain natriuretic peptide (BNP) were determined according to the above method.

Western Blot Analysis

Myocardial tissue was homogenized in a lysis buffer (1% NP-40, 20 mmol/L Tris/HCl [pH 8.0], 137.5 mmol/L NaCl, l mmol/L Na₃VO₄, l mmol/L PMSF, and 10 μg/mL aprotinin). Protein concentration of the lysate was determined by the Bradford method. An equal volume of 2× Sodium dodecyl sulfate (SDS)-sample buffer (0.125 mol/L Tris/HCl [pH 7.4], 4% SDS, and 20% glycerol) was added, and the samples were boiled for 5 minutes. Samples of 100 μg protein underwent 10% SDS-polyacrylamide gel electrophoresis (PAGE) for 3 hours at 60 mA. The proteins were then transferred electrophoretically onto a nitrocellulose membrane and incubated for 1 hour in tris-buffered saline containing 5% nonfat powdered milk. The membranes were then incubated with goat polyclonal antibody against rat AGTRL1 (1:500 dilutions) at 4°C overnight. After being washed 3 times for 10 minutes each in TBST, the membranes were incubated with rabbit anti-goat immunoglobulin G antibody for 1 hour. The membranes were then washed 3 times for 10 minutes each in TBST, then underwent enhanced chemiluminescence detection. Autoradiographs were scanned and relative densities quantified.

The AGTRL1 expression in macrophages was also detected. Peritoneal macrophages were isolated and lysed in buffer (1% NP-40, 20 mmol/L Tris/HCl [pH 8.0], 137.5 mmol/L NaCl, l mmol/L Na₃VO₄, l mmol/L PMSF, and 10 μg/mL aprotinin). Samples of 100 μg protein underwent 10% SDS-PAGE and subsequent procedures as described previously.

MCP-1 and IL-8 Protein Secretion in Plasma

MCP-1 and IL-8 levels in plasma were determined by use of a commercially available sandwich-type ELISA. ELISA plates were coated with a specific murine monoclonal antibody against rat MCP-1 and IL-8. Dilutions of cell-free supernatants were added in duplicate, then a second horseradish peroxidase-conjugated goat polyclonal Ab against MCP-1 and IL-8. After a washing to remove any unbound Ab-enzyme reagent, a substrate solution (a 1:1 solution of hydrogen peroxide and tetramethylbenzidine) was added to the wells. The color development was stopped with 2 N sulfuric acid, and the intensity of the color was measured at 540 nm on a spectrophotometer. The sensitivity of the ELISA for both MCP-1 and IL-8 was 3 pg/mL. The intra-assay coefficient of variation was 0.5% and the interassay coefficient of variation 10%.

Rat Peritoneal Macrophage Culture and MCP-1 and IL-8 Protein Secretion in Medium

Peritoneal macrophages were isolated as described with modification.²³ Briefly, rats were killed by decapitation and placed with the abdomen facing up, which was cleaned with 70% ethanol. The skin of the abdominal region was dissected to expose the abdominal wall, which was then soaked with 70% ethanol. About 20 mL of cold phosphate-buffered saline was injected. The needle was removed and the abdomen was gently massaged. The phosphate-buffered saline was drawn back and the peritoneal fluid was dispensed into 50 mL polypropylene tubes. The cells were centrifuged and resuspended in DMEM supplemented with 15% fetal bovine serum. Peritoneal macrophages were seeded (10⁵ cell per well) onto 96-well plates for MCP-1 and IL-8 assay.

Macrophages were divided into groups for culture: (1) control (DMEM alone); (2) LPS (DMEM with 50 ng LPS/well for 6 hours); (3)-(6) LPS + apelin (10⁻⁹ to 10⁻⁶ mol/L) (DMEM with 50 ng LPS/well plus 10⁻⁹ to 10⁻⁶ mol/L, respectively, for 6 hours). Then, cells were collected and IL-8 and MCP-1 levels were determined according to the above methods.

Statistical Analysis

Some of the results were normalized to total protein, with all data expressed as mean ± SD. Comparisons among groups were evaluated by 1-way analysis of variance and then Student-Newman-Keuls test; comparisons between 2 groups involved the Student t-test. Linear regression analysis was used to assess correlation between variables. P < .05 was considered statistically significant.

Results

Characteristics of Rats with Sepsis

Compared with controls, rats with sepsis showed a severe disturbance of hemodynamic features, with decreased MABP by 33.3%, tachycardia (heart rate increased by 26.9%), inhibited cardiac function (+LVdp/dt_max and −LVdp/dt_max decreased by 38.2 and 53.9%, respectively), and LVEDP elevated 3-fold (Table 2). Rats showed significant hypoglycemia (blood glucose decreased by 48.1%) and hyperlactatemia (plasma lactic acid concentration increased by 325%) (Fig. 2). Plasma LDH and CPK activities were higher than those in control rats (Fig. 2).

Decreased mRNA and Protein Levels of Apelin and AGTRL1 and Increased mRNA Level of BNP in Rats with Sepsis

Sequence analysis with the relative primers ensured that the semiquantitative reverse transcription PCR products of apelin, AGTRL1, and BNP were correct. The mRNA relative level of apelin and AGTRL1 in the myocardia of rats with sepsis was decreased, by 71% and 50%, respectively, but that of BNP was increased, by 56% (Fig. 3A, C).

In rats with sepsis, EIA detected lower levels of apelin in plasma (Fig. 3E) and myocardial (Fig. 3F), by 42.3% and 58%, respectively, than that in control rats. Plasma and myocardial apelin contents were positively correlated with LVdp/dt_max values (r = 0.68, P < .05; and r = 0.68, P < .05, respectively).

Western blot analysis revealed the myocardial AGTRL1 protein level in rats with sepsis 55% lower than that in control rats (Fig. 3B, D).

Apelin Ameliorated Disordered Cardiac Function and Hemodynamics in Rats with Sepsis

Compared with the sepsis-alone group, rats under low or high apelin treatment (300 ng·kg·day or 1.5 μg·kg·day, respectively) showed higher MABP, by 31% and 46% and lower heart rate, by 18.2% and 23.7%, respectively. Low- or high-dose apelin increased heart contractility; elevated values of +LVdp/dt_max by 18% and 47%; and −LVdp/dt_max by 45% and 68%; and lowered LVEDP by 30.7% and 59.3%, respectively (Table 2).

Also compared with the sepsis-alone group, rats under low- or high-dose apelin showed alleviated myocardial injury, such as reduced myocardial LDH and CPK leakage and attenuated plasma LDH activity, by 29% and 47%, respectively (Fig. 2C), and CPK activity, by 34% and 37%, respectively (Fig. 2D). Apelin alleviated severe hypoglycemia and hyperlactacidemia; high-dose apelin increased plasma glucose content by 45% (Fig. 2A), and both low- and high-dose apelin attenuated plasma lactic acid content, by 32% and 38.5%, respectively (Fig. 2B).

Apelin Treatment Attenuated Plasma MCP-1 and IL-8 Levels in Rats with Septic Shock

Plasma MCP-1 and IL-8 levels were increased, by 4.8- and 30-fold, respectively (both P < .05), in rats with sepsis as compared with controls and were negatively correlated with LVdp/dt_max (r = 0.98, P < .05). Compared with the sepsis-alone group, rats under low- and high-dose apelin showed lower plasma level of MCP-1, by 38% and 33%, and IL-8, by 21% and 26%, respectively (Fig. 4A, B).

Apelin Inhibits Production of MCP-1 and IL-8 in LPS-stimulated Cultured Rat Peritoneal Macrophages in vitro

ELISA was used to measure the content of inflammatory factors in medium released from cultured peritoneal macrophages stimulated by LPS (50 mg/L) for 6 hours. MCP-1 and IL-8 contents were higher by 3.4- and 12-fold (all P < .05), respectively, than without LPS stimulation, and apelin directly inhibited the production of MCP-1 and IL-8 with LPS stimulation. Apelin incubation (10⁻⁸ and 10⁻⁷ mol/L) reduced MCP-1 production by 29.3% and 58.8%, respectively, as compared with LPS treatment alone; apelin (10⁻⁷ mol/L) reduced the production of IL-8 by 21.3% (Fig. 5A, B).

Western blot analysis confirmed the protein expression of AGTRL1 in rat macrophages (Fig. 5C).

Discussion

Apelin is an endogenous peptide ligand that binds to AGTRL1, a G-protein–coupled receptor that shares significant homology with the angiotensin II type 1 receptor. The apelin gene encodes a 77-amino acid (aa) prepro-apelin polypeptide that is processed into the C-terminal fragments apelin-36 (aa 42 to 77), apelin-17 (aa 61 to 77), and apelin 13 (aa 65 to 77).⁴ Apelin and its receptor are localized in the rat and human cardiovascular system. Accumulating evidence has implicated apelin in multiple organ functions, such as diuresis, fluid intake, obesity, food intake, body temperature control, and blood vessel formation. In mammals, gene targeting of AGTRL1 did not reveal any overt phenotypes in mice. AGTRL1-knockout mice were viable and fertile and, besides a significantly lower body weight than wild-type littermates, showed no obviously visible phenotype.⁴⁴ However, AGTRL1^−/− mice were unable to concentrate their urine to the same extent as wild-type mice in response to the arginine vasopressin V2 receptor agonist desmopressin.⁴⁴ In another AGTRL1 knockout model, AGTRL1- deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects.⁴⁵ In contrast, Apelin-deficient mice were viable, fertile, appeared healthy, and exhibited normal body weight, water and food intake, heart rates, and heart morphology.^{[16] and [45]} However, both apelin and AGTRL1 null mice that survived to adulthood manifested decrements in contractile function.^{[16] and [45]} In rodents, apelin treatment lowered blood pressure and exerted a potent positive inotropic action in the heart.^{[12] and [13]} In addition, clinical studies of heart failure patients suggested a possible role of apelin in heart failure.^{[14] and [15]} Administration of apelin-16 induced a dose-dependent increase in developed tension in isolated rat heart preparations, with a significant increase in contractility 2 minutes after the start of apelin infusion and maximal increase after 24 minutes.⁴ Apelin shows inotropic effects in normal rat hearts and failing hearts induced by myocardial infarction.¹⁷ In addition, apelin was shown to have immunoregulatory effects.²⁴ So we speculated that apelin might play an important regulatory role in the pathogenesis of septic shock.

The present study showed that rats with septic shock had typical severe hemodynamic disturbances, with decreased MABP, bradycardia, inhibited cardiac function, elevated LVEDP, severe hypoglycemia and hyperlactacidemia, and increased plasma LDH activity, as was previously reported.^{[25] and [26]} Rats with severe sepsis showed decreased mRNA and protein levels of apelin and AGTRL1 in myocardial tissues. EIA analysis revealed decreased plasma and myocardial content of apelin. These results are accordance with those in late-stage heart failure.¹⁴ Atrial and plasma levels of apelin were significantly lower in patients with heart failure from coronary heart disease than that in normal subjects; plasma apelin level was significantly correlated with atrial apelin level,²⁷ and plasma apelin level was increased in early stages of heart failure and lowered in later stages, which suggests that decreased plasma and myocardial apelin levels could be a key factor in the progression of heart failure.²¹ In this present study, plasma and myocardial apelin contents were correlated positively with LVdp/dt_max values in rats with sepsis, so the decrease in plasma and myocardial apelin level might be an important factor in the progression of severe septic shock.

The regulation mechanism of apelin/AGTRL1 expression and secretion is still unclear. Administration of insulin (1 to 100 nmol/L) increased and that of dexamethasone (0.1 to 100 nmol/L) decreased apelin mRNA level in 3T3-L1 adipocytes in a dose-dependent manner, which suggests that insulin and glucocorticoids regulate apelin gene expression in adipocytes.²⁸ As well, acute stress increased AGTRL1 mRNA expression in the hypothalamic parvocellular paraventricular nucleus, but repeated restraint stress induced a sustained upregulation of parvocellular paraventricular nucleus AGTRL1 mRNA expression in intact rats²⁹; removal of endogenous glucocorticoids by adrenalectomy also resulted in increased expression of AGTRL1 mRNA in the paraventricular nucleus, which suggests a negative regulation of AGTRL1 mRNA expression by glucocorticoids. However, production and regulation of apelin and AGTRL1 in the cardiovascular system are unclear.

In this work, administration of apelin ameliorated cardiac dysfunction and myocardial injury in rats with sepsis. Low- or high-dose apelin (300 ng·kg·day or 1.5 μg·kg·day) significantly increased MABP. Apelin treatment increased +LVdp/dt_max and −LVdp/dt_max and attenuated plasma LDH and CPK activities. In addition, it ameliorated the hypoglycemia and hyperlactacidemia of sepsis. Considerable evidence has shown that hypoglycemia is one of concomitant symptoms in late-stage sepsis, but hyperglycemia was one of the concomitant symptoms in early in sepsis. In chow-fed mice, acute intravenous injection of apelin had a powerful glucose-lowering effect associated with enhanced glucose utilization in skeletal muscle and adipose tissue.³⁹ Apelin exerts direct inhibitory actions in pancreatic β-cells.⁴⁰ As well, apelin-36 inhibited glucose-stimulated insulin secretion both in vivo and in vitro.⁴¹ Apelin has positive inotropic effects in vivo in both normal rat hearts and rat hearts in heart failure after myocardial infarction, so it was considered an acute inotropic agent in patients with ischemic heart failure.¹⁷ Whether apelin restores plasma glucose content by regulating heart function or by regulating insulin effects and glucose utilization in skeletal muscle and adipose tissue needs to be further investigated.

However, the protective mechanism of apelin against cardiac injury has not been fully elucidated. Apelin is one of the most potent, endogenous, positive inotropic substances identified, and the inotropic response to apelin may involve activation of phosphatidolipase C, protein kinase C, and sarcolemmal sodium hydrogen exchange and sodium calcium exchange.¹² Apelin exerts a selective positive inotropic action in the failing myocardium by increasing intracellular [Ca²⁺]i transients rather than changing myofilament calcium responsiveness.³⁰ The direct cardioprotective actions of apelin against myocardial ischemia-reperfusion injury involves the activation of phosphatidylinositol-3-OH kinase-Akt/protein kinase B and p44/42 mitogen-activated protein kinase, components of the reperfusion injury salvage kinase pathway.³¹ The mechanisms of the apelin inotropic effect may result from enhancing the activity of sodium hydrogen exchange with consequent intracellular alkalinization.³² However, the inflammatory response is the key process in septic shock. The bacterial toxin LPS binds with cell-surface Toll-like receptors and results in activation of nuclear factor κB, which induces inflammatory factors such as tumor necrosis factor-α, IL-6, IL-1α, and MCP.^{[33] and [34]} Both MCP-1 and IL-8 are chemokines with a powerful effect on monocytes and cause rolling monocytes to adhere firmly onto monolayers of activated endothelial cells, thus playing an important role in monocyte aggregation. MCP-1 and IL-8 content was elevated in patients with sepsis and was positively correlated with severity of disease.^{[42] and [43]} During sepsis, bacterial toxins activate macrophages to release proinflammatory cytokines and other mediators that initiate specific immune responses.^{[1] and [2]} Growing experimental and clinical data have indicated that proinflammatory cytokines play a prominent role in sepsis-induced tissue injury.^{[1] and [35]} The kinetics and magnitude of cytokine release influence the development of sepsis. In our study, the plasma contents of MCP-1 and IL-8 were increased in rats with severe septic shock and were negatively correlated with LVdp/dt_max; administration of exogenous apelin significantly reduced plasma MCP-1 and IL-8 levels. Furthermore, LPS stimulated the production and release of MCP-1 and IL-8 in cultured rat peritoneal macrophages, with apelin treatment directly inhibiting their LPS-induced generation and thus suggesting its important anti-inflammatory role in septic shock. Despite prior studies showing that macrophages do not express the apelin receptor,^{[36] and [37]} apelin was recently found to diminish the formation of abdominal aortic aneurysm, with a reduction in macrophage infiltrates, and apelin stimulation of cultured macrophages significantly reduced MCP-1 and tumor necrosis factor-α mRNA levels; both murine monocytes and several lines of mouse macrophages expressed AGTRL1 mRNA.³⁸ Furthermore, our results confirmed AGTRL1 protein expressed in rat macrophages. These results demonstrated that apelin directly inhibits macrophage activity through activating AGTRL1.

Conclusion

We reported plasma and myocardial apelin content and AGTRL1 mRNA and protein levels decreased during severe septic shock in rats. Reduced levels of endogenous apelin and its receptor level are associated with severe septic shock. Exogenous administration of apelin ameliorated cardiac dysfunction, hemodynamic and myocardial injury, and cytokine (MCP-1 and IL-8) generation in such rats. Apelin in vitro treatment directly inhibited MCP-1 and IL-8 production and release in cultured peritoneal macrophages stimulated by LPS. This finding suggested that reduced level of endogenous apelin and its receptor is associated with severe septic shock. Apelin might be an endogenous cardio-vaso-protective factor for septic shock and inflammation, apelin and its receptor AGTRL1 might be a new therapeutic target for severe septic shock.

Disclosures

None.