Abstract
Objective:
To test the hypothesis that administration of ghrelin attenuates inflammatory responses in sepsis through vagal nerve stimulation.
Summary Background Data:
Ghrelin has been demonstrated to possess multiple functions, including stimulation of the vagus nerve. Our recent study has shown that plasma levels of ghrelin were significantly reduced in sepsis; and ghrelin administration improved organ perfusion and function. However, it remained unknown whether ghrelin also decreases proinflammatory cytokines in sepsis and, if so, whether the down-regulatory effect of ghrelin is mediated by activation of the vagus nerve.
Methods:
Male rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 hours after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed 200-μL Alzet mini-pump for 15 hours. At 20 hours after CLP, plasma and peritoneal fluid levels of TNF-α and IL-6 were determined. The direct effect of ghrelin on cytokine production was studied using cultured normal rat Kupffer cells or peritoneal macrophages stimulated by lipopolysaccharide (LPS). In additional animals, vagotomy or sham vagotomy was performed in sham and septic animals immediately prior to ghrelin administration and cytokine levels were then measured.
Results:
Ghrelin significantly reduced TNF-α and IL-6 levels in sepsis. In contrast, ghrelin did not inhibit TNF-α and IL-6 release from LPS-stimulated Kupffer cells or peritoneal macrophages. However, vagotomy, but not sham vagotomy, prevented ghrelin's down-regulatory effect on TNF-α and IL-6 production.
Conclusions:
Ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve. Pharmacologic stimulation of the vagus nerve may offer a novel approach of anti-sepsis therapy.
Administration of ghrelin significantly reduced TNF-α and IL-6 in sepsis. However, ghrelin did not inhibit TNF-α and IL-6 release from endotoxin-stimulated Kupffer cells and peritoneal macrophages. Vagotomy, but not sham vagotomy, prevented ghrelin's down-regulatory effect on TNF-α and IL-6 production. Thus, ghrelin down-regulates cytokine release through the vagus nerve stimulation.
Despite improvement in the management of septic patients with systemic antibiotics, surgical intervention, aggressive fluid resuscitation, and careful monitoring, sepsis continues to be one of the leading causes of death in intensive care units, and a large number of septic patients die of ensuing septic shock and multiple organ failure.1–4 The pathophysiologic sequelae of sepsis are caused by an overreaction of the immune system to microorganisms and their products. During sepsis, bacterial toxins activate macrophages/Kupffer cells to release proinflammatory cytokines and other mediators that initiate specific immune responses. A growing collection of experimental and clinical data has indicated that proinflammatory cytokines play a prominent role in sepsis-induced tissue injury.5,6 The kinetics and magnitude of cytokine release influence the development of sepsis.5,7
Ghrelin is a gastric hormone first identified in the rat stomach in 1999 as an endogenous ligand for the growth hormone secretagogue receptor (GHSR).8 Human ghrelin is a 28-amino acid peptide with an n-octanoyl group at Ser3, a modification essential for its activity.9,10 Rat ghrelin differs from human ghrelin by only 2 amino acids.10 The biologic effects of ghrelin are thought to be mediated through GHSR. Although a group of synthetic molecules featuring growth hormone secretagogue can bind to GHSR, ghrelin is the only identified endogenous ligand for this receptor. Ghrelin was originally reported to induce growth hormone release through pituitary GHSR stimulation. It has a strong stimulatory effect on growth hormone secretion.11–13 However, a large body of evidence has indicated physiologic functions of ghrelin mediated by the central and peripheral GHSR distribution.14 The wide distribution of GHSR suggests multiple paracrine, autocrine and endocrine roles of ghrelin.15–18 One recent study demonstrates the presence of GHSR in afferent neurons of the nodose ganglion, suggesting that ghrelin signals are transmitted to the brain via vagal afferent nerves.19 Moreover, central administration of ghrelin stimulates the vagal efferent nerve in anesthetized rats.20
The vagus nerve is an important link between the involuntary nervous system and proinflammation, which has been suggested for more than 70 years.21 This “parasympathetic” nerve emanates from the cranium and innervates all major organs in a subconscious way. It is finely branched and is composed of both sensory (input) and motor (output) fibers, suggesting that the vagus nerve can sense continuing inflammation and subsequently suppresses it. This mechanism is more efficient and rapid than other anti-inflammatory pathways. In addition, the vagus nerve has been shown to convey the immunologic state of the gastrointestinal tract to the hypothalamus. Thus, the vagus nerve provides the endogenous mechanism to regulate the magnitude of innate immune responses and attenuate inflammation. Activation of parasympathetic efferent nerves during systemic stress confers an additional protective advantage to the host by restraining a potentially adverse peripheral immune response. Tracey et al found that after administration of LPS in the rat, electrical stimulation of the vagus nerve via the activation of nicotinic acetylcholine receptors (α7 receptors), prevented both the release of TNF from macrophages and death.22–25 However, it remains unknown whether the novel peptide ghrelin plays any role in such an anti-inflammatory pathway.26
Circulating levels of ghrelin have been shown to decrease significantly in endotoxemia.27 Our recent studies also demonstrated that plasma levels of ghrelin decrease significantly in a rat model of polymicrobial sepsis induced by cecal ligation of puncture (CLP), raising the possibility of treating sepsis with ghrelin.28 In addition, recent studies have shown that treatment with ghrelin significantly down-regulates circulating levels of cytokines in a rat model of endotoxemia.29,30 However, it remained unknown whether ghrelin also decreases cytokine levels in polymicrobial sepsis and, if so, whether the down-regulatory effect of ghrelin is mediated by vagal nerve activation. The present study was conducted to test the hypothesis that administration of exogenous ghrelin attenuates the inflammatory response in sepsis through vagal nerve stimulation.
MATERIALS AND METHODS
Animal Model of Sepsis
Male Sprague-Dawley rats (275–325 g) were housed in a temperature-controlled room on a 12-hour light/dark cycle and fed a standard Purina rat chow diet. Prior to the induction of sepsis, rats were fasted overnight but allowed water ad libitum. Rats were anesthetized with isoflurane inhalation and the ventral neck, abdomen, and groin were shaved and washed with 10% povidone iodine. CLP was performed as we previously described.31 Briefly, a 2-cm midline abdominal incision was performed. The cecum was exposed, ligated just distal to the ileocecal valve to avoid intestinal obstruction, punctured twice with an 18-gauge needle, squeezed slightly to allow a small amount of fecal matter to flow from the holes, and then returned to the abdominal cavity, following which the abdominal incision was closed in 2 layers by a surgical suture. Sham-operated animals (ie, control animals) underwent the same procedure with the exception that the cecum was neither ligated nor punctured. The animals were resuscitated with 3 mL/100 g body weight normal saline subcutaneously immediately after surgery. The animals were then returned to their cages. The rats were singly housed in a temperature-controlled room after the operation. The animals recovered quickly (within 10 minutes) after the discontinuation of isoflurane inhalation. They moved around and drank water. The cages we used for our study are conventional polycarbonate ones. All experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. This project was approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research.
Administration of Ghrelin
Rat ghrelin (Phoenix Pharmaceuticals, Belmont, CA) was dissolved in normal saline to a final concentration of 100 μmol/L; 200-μL mini-pumps (Alzet, infusion rate 8 μL/hr) were primed with ghrelin solution or vehicle (normal saline) for 3 hours prior to implantation. At 5 hours after CLP, the rats were reanesthetized with isoflurane inhalation. After a slow intravenous bolus injection of 2 nmol ghrelin (or 200 μL vehicle), the mini-pump was then connected to a jugular venouscatheter and implanted subcutaneously. Twenty hours after CLP (ie, 15 hours after implantation of the mini-pump), the rats were reanesthetized with isoflurane inhalation. Cardiac puncture was used for collecting blood samples. Peritoneal fluid was collected and its volume was measured by a pipette. No lavage was performed for peritoneal fluid collection for this portion of the experiment. After sample collecting, the animals were euthanized by a CO2 chamber. No animals died before 15 hours postimplantation. The total dose of ghrelin each rat received was around 45 nmol/kg body weight.
Determination of Serum and Peritoneal Fluid Levels of TNF-α and IL-6
The concentrations of TNF-α and IL-6 in the serum and peritoneal fluids were quantified by using commercially obtained enzyme-linked immunosorbent assay (ELISA) kits specific for rat-TNF-α and IL-6 (BioSource International, Camarillo, CA). The assay was carried out according to the instructions provided by the manufacturer. The total amounts of TNF-α and IL-6 in the peritoneal fluids were calculated by multiplying the concentrations of cytokines by the volume of peritoneal fluids.
Cell Culture
Rat Kupffer cells were isolated from normal male Sprague-Dawley rats by collagenase perfusion of the liver, isopycnic sedimentation in a Percoll gradient, and selective adherence.32 Rat peritoneal macrophages were isolated by peritoneal lavage with Hanks balanced salt solution (HBSS).33 The isolated Kupffer cells and peritoneal macrophages from normal animals were cultured in DMEM containing 10% heat-inactivated fetal bovine serum, 10 mmol/L HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin at the concentration of 106 cells/mL and plated at a density of 5 × 105/well in 24-well cell culture plates. After being washed twice with HBSS, the cells were cultured in culture medium containing LPS (10 ng/mL) with or without ghrelin (10−10–106− mol/L) for a period of 4 hours (Kupffer cells) or 24 hours (peritoneal macrophages). The supernatant was collected by transferring the noncell portion from the culture wells to Eppendorf tubes. Kupffer cells/macrophages are adherent cells. No spin-down was needed for collecting supernatant. The supernatant levels of cytokines (TNF-α and IL-6) were measured by ELISA as described above.
Vagotomy and Sham Vagotomy
In additional groups of animals, the trunks of the subdiaphragmatic vagus were transected.34 Briefly, the rats were reanesthetized with isoflurane inhalation at 5 hours after CLP or sham operation. The midline abdominal incision was reopened prior to the administration of ghrelin, and the dorsal and ventral branches of the vagus nerve were dissected from the esophagus. Each branch of the nerve was tied with surgical sutures at 2 points separated by approximately 1 cm, and then severed between the sutures. Sham-vagotomized animals underwent the same surgical procedure with the exception that their vagus nerves were neither tied nor severed. After the surgery, the animals from both vagotomy and sham vagotomy were allowed food and water ad libitum. Rat ghrelin or vehicle was administered immediately following vagotomy as described above in the vagus nerve intact animals. Serum and peritoneal fluid levels of TNF-α and IL-6 were measured by ELISA at 20 hours after CLP as described above. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate were determined by using assay kits according to the manufacturer's instructions (Pointe Scientific, Lincoln Park, MI).
Statistical Analysis
Results are expressed as mean ± SE. One-way analysis of variance and the Student-Newman-Keuls method were used to compare different groups of experimental animals. Differences in values were considered significant if P < 0.05.
RESULTS
Effects of Ghrelin Administration on TNF-α and IL-6 Release After CLP
As shown in Figure 1, serum levels of TNF-α and IL-6 increased by 5- and 84-fold, respectively, at 20 hours after CLP in vehicle-treated animals as compared with sham-operated animals (P < 0.05). Administration of ghrelin significantly attenuated serum levels of TNF-α and IL-6; however, they were still higher as compared with sham-operated animals (P < 0.05, Fig. 1). As indicated in Figure 2A and C, the concentrations of TNF-α and IL-6 in the peritoneal fluid increased by 4- and 3-fold, respectively, at 20 hours after CLP in vehicle-treated animals (P < 0.05). Treatment with ghrelin reduced the TNF-α level in peritoneal fluid by 39%; however, it was still significantly higher than that in sham-operated animals (P < 0.05, Fig. 2A). Administration of ghrelin reduced the peritoneal level of IL-6 by 63% (P < 0.05), which was similar to sham-operated animals (Fig. 2C). The total amount of TNF-α and IL-6 in the peritoneal fluid increased markedly at 20 hours after CLP in vehicle-treated animals (P < 0.05, Fig. 2B, D). Treatment with ghrelin attenuated the levels of TNF-α and IL-6 by 48% and 66%, respectively (P < 0.05, Figs. 2B, D).
FIGURE 1. Alterations in serum levels of TNF-α (A) and IL-6 (B) in sham-operated animals (Sham) and septic animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 20 hours after cecal ligation and puncture (CLP). Data are mean ± SE (n = 8/group): *P < 0.05 versus sham-operated animals; #P < 0.05 versus CLP animals treated with vehicle.
FIGURE 2. Alterations in the levels of TNF-α (A) and IL-6 (C) as well as the total amount of TNF-α (B) and IL-6 (D) in the peritoneal fluid in sham-operated animals (Sham) and septic animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 20 hours after cecal ligation and puncture (CLP). Data are mean ± SE (n = 8/group): *P < 0.05 versus sham-operated animals; #P < 0.05 versus CLP animals treated with vehicle.
Effects of Ghrelin on TNF-α and IL-6 Release From Kupffer Cells and Peritoneal Macrophages
As shown in Figure 3A and B, TNF-α and IL-6 levels in the supernatant of Kupffer cell culture increased dramatically after the stimulation by LPS (10 ng/mL) for 4 hours. In contrast to its effect in the in vivo animal model, ghrelin did not significantly affect either LPS-induced levels or basal levels of TNF-α and IL-6 released from the cultured Kupffer cells. Similarly, LPS significantly increased TNF-α and IL-6 release from the cultured peritoneal macrophages, and ghrelin did not alter either LPS-induced levels or basal levels of those cytokines (Fig. 3C, D).
FIGURE 3. Alterations in the levels of TNF-α (A, C) and IL-6 (B, D) in Kupffer cell (A, B) and peritoneal macrophage (C, D) culture supernatant. Data are mean ± SE (n = 8/group): *P < 0.05 versus medium group.
Effects of Vagotomy on Ghrelin's Inhibition of TNF-α and IL-6 Releases After CLP
To determine whether ghrelin's down-regulatory effect on proinflammatory cytokines in sepsis is mediated via activation of the vagus nerve, vagotomy or sham-vagotomy was performed in sham and septic animals immediately prior to the administration of ghrelin. As indicated in Figure 4A and B, vagotomy immediately prior to the administration of ghrelin completely prevented the inhibitory effects of this agent on the circulating levels of TNF-α and IL-6. In contrast, sham-vagotomy did not alter the anti-inflammatory effect of ghrelin at 20 hours after CLP (Fig. 4C, D). The volumes of the peritoneal fluid in vagotomized animals were 0.07 ± 0.02, 6.6 ± 0.5, and 6.7 ± 0.6 mL/animal in sham, vehicle-, and ghrelin-treated animals, respectively, which were higher than those in the corresponding animals with sham vagotomy (0.008 ± 0.001, 3.1 ± 0.2, and 2.6 ± 0.2 mL/animal, respectively). Although peritoneal concentrations of IL-6 in vagotomized septic animals were similar to those in sham vagotomized septic animals, the total amount of TNF-α and IL-6 in the peritoneal fluid of vagotomized animals was much higher (Table 1). Most importantly, vagotomy completely prevented the inhibitory effect of ghrelin on the peritoneal fluid levels of TNF-α and IL-6 at 20 hours after the onset of sepsis (Table 1), whereas sham-vagotomy did not alter ghrelin's effects on peritoneal fluid levels of TNF-α and IL-6 (Table 1).
FIGURE 4. Effects of vagotomy or sham vagotomy on serum levels of TNF-α (A, C) and IL-6 (B, D) in sham-operated animals (Sham) and septic animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 20 hours after cecal ligation and puncture (CLP). Data are mean ± SE (n = 6/group): *P < 0.05 versus sham-operated animals; #P < 0.05 versus CLP animals treated with vehicle.
TABLE 1. Effects of Vagotomy or Sham Vagotomy on the Levels of TNF-α and IL-6 as Well as the Total Amount of TNF-α and IL-6 in the Peritoneal Fluid in Sham-Operated Animals (Sham) and Septic Animals Treated With Normal Saline (Vehicle) or Ghrelin at 20 Hours After Cecal Ligation and Puncture (CLP)
Effects of Vagotomy on Ghrelin's Beneficial Effects on Organ Injury After CLP
We have previously shown that ghrelin administration improves organ perfusion and function after CLP.35 To determine whether vagotomy also eliminate ghrelin's beneficial effects on organ functions in sepsis, serum levels of ALT, AST, and lactate were measured at 20 hours after CLP in vagotomized or sham vagotomized animals. As shown in Table 2, both vagotomized and sham vagotomized rats subjected to sepsis had a 2- to 3-fold increase in circulating levels of ALT, AST, and lactate compared with sham-operated animals (P < 0.05). These results are similar to the vagus nerve intact animals. However, ghrelin treatment has no effects on serum levels of ALT, AST, and lactate in vagotomized septic animals. Conversely, when sham vagotomized septic animals were treated with ghrelin, the levels of ALT, AST, and lactate were markedly reduced (P < 0.05, Table 2).
TABLE 2. Effects of Vagotomy or Sham Vagotomy on the Serum Levels of ALT, AST, and Lactate in Sham-Operated Animals (Sham) and Septic Animals Treated With Normal Saline (CLP-Vehicle) or Ghrelin (CLP-Ghrelin) at 20 Hours After Cecal Ligation and Puncture (CLP)
DISCUSSION
We recently demonstrated that, although ghrelin levels decrease at both early and late stages of sepsis, its receptor is markedly elevated in early sepsis.28 Moreover, ghrelin-induced relaxation of resistance blood vessels in the isolated small intestine increases significantly at the early stage of sepsis but was not altered at the late stage of sepsis.28 This suggests that the decreased ghrelin production may herald the hypodynamic response of late sepsis. Since ghrelin receptor and vascular responsiveness to ghrelin are not reduced at the late stage of sepsis, administration of ghrelin may be a useful adjunct in the treatment of clinical case of sepsis. However, ghrelin is a small peptide with a relatively short half-life. Our recent study has indicated that ghrelin's half-life is 11 minutes in the normal rat and 17 minutes at the late stage of sepsis.36 Therefore, a continuous infusion of ghrelin is required to maintain its effective concentration in the circulation. In this study, we used an osmotic mini-pump to infuse ghrelin intravenously after a slow bolos injection. Our results indicate that ghrelin treatment significantly decreased serum and peritoneal fluid levels of TNF-α and IL-6. Recently, Li et al showed that ghrelin down-regulates cytokine release from endothelial cells.29 However, the major sources of circulating inflammatory cytokines in sepsis are macrophages/Kupffer cells but not endothelial cells.37 Moreover, depletion of Kupffer cells by liposomal clodronate prevents LPS-induced cytokine production and subsequent liver damage in the rat.38 We, therefore, chose 2 major resident macrophage populations, Kupffer cells and peritoneal macrophages, to study the mechanism of ghrelin's inhibitory effect on cytokine release in sepsis. In contrast to our expectation, ghrelin has no direct beneficial effects on cytokine release from either Kupffer cells or peritoneal macrophages isolated from normal rats. This would suggest that an indirect mechanism may be involved in ghrelin's effects on inflammatory cytokines.
Date et al reported that ghrelin activates the vagus nerve and that blockade of the gastric vagal afferent abolishes ghrelin-induced feeding and growth hormone secretion.19 This would suggest an important role of the vagus nerve in ghrelin's biologic effects. Since intracerebroventricular administration of ghrelin stimulates the vagal efferent nerve and since ghrelin can pass the blood–brain barrier,36 we hypothesized that the anti-inflammatory property of ghrelin is mediated through vagal nerve stimulation. To test this, vagotomy or sham vagotomy was performed in sham and septic animals immediately prior to the administration of ghrelin. Our results show that vagotomy, but not sham vagotomy, prevented the down-regulatory effects of ghrelin on inflammatory cytokines in sepsis. This suggests that the intact vagus nerve is essential for the inhibitory effect of ghrelin on the release of proinflammatory cytokines. Vagus nerve signaling is a critical component of the afferent loop that modulates immune responses to systemic inflammation.23 Stimulation of the vagus nerve can significantly and rapidly inhibit the release of macrophage-derived TNF-α through the release of acetylcholine.22,39 Although this physiologic mechanism termed the “cholinergic anti-inflammatory pathway,” has major implications in the prevention of overt life-threatening infections, the potential target molecule(s) for the development of possible therapeutic agents remains unknown. Our finding that ghrelin down-regulates inflammatory cytokine releases in sepsis through the stimulation of the vagus nerve suggests that ghrelin may be the circulating mediator for the link between the vagus nerve and the immune system.
Previous studies have shown that vagotomy not only prevents the protective effect of the vagus nerve stimulation but also sensitizes the animals to the lethal effects of endotoxin.25,40 However, we could not find any significant increase in the serum levels of TNF-α and IL-6 after vagotomy in the present study. One explanation for the different results might be related with the timing of vagotomy. We collected samples at 15 hours after vagotomy, whereas the blood samples were harvested at less than 3 hours after vagotomy in those studies.40 Another explanation might be related with the site of vagotomy. The vagotomy performed in our laboratories was a typical truncal vagotomy, whereas others performed vagotomy at cervical levels.40 Similar to our findings, Heider et al showed that acute truncal vagotomy did not increase the risk of the systemic inflammatory response in surgical patients with complicated peptic ulcer disease.41
There are a few limitations of this study. Although the rats were singly housed in a temperature-controlled room after the operation, the animals' core body temperature was not monitored. Water and food were available ad libitum after the operation, but consumption was not recorded. Although every caution has been made to guarantee the completeness of vagotomy, and although our results show that vagotomy not sham vagotomy prevented the effect of ghrelin on cytokines in sepsis, the completeness of vagotomy was not assessed by checking the histology of the esophagus. Moreover, although both vagotomized and sham vagotomized animals have the same anesthesia (ie, isoflurane inhalation), the anesthetic agent was not used for the cell culture experiments.
CONCLUSION
Administration of ghrelin reduced proinflammatory cytokines (TNF-α and IL-6) in sepsis. However, ghrelin did not directly inhibit cytokine release from LPS-stimulated Kupffer cells or peritoneal macrophages. The down-regulatory effect of ghrelin on cytokines release requires the intact vagus nerve, since vagotomy not sham vagotomy prevented the effect of ghrelin on cytokines in sepsis. It is concluded that ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve. Thus, pharmacologic stimulation of the vagus nerve may offer a novel approach of antisepsis therapy.
Footnotes
Supported by National Institutes of Health Grant Nos. R01 GM053008-12 and R01 GM057468-09 (to Dr. Wang). Dr. Wu was supported by a Postdoctoral Fellowship from the American Heart Association (the Heritage Affiliate) (No. 0325802T).
Reprints: Ping Wang, MD, Department of Surgery, North Shore University Hospital, 350 Community Drive, Manhasset, NY 11030. E-mail: pwang@nshs.edu.
REFERENCES
- 1.Deitch EA. Animal models of sepsis and shock: a review and lessons learned. Shock. 1998;9:1–11. [DOI] [PubMed] [Google Scholar]
- 2.Angus DC, Linde-Zwirble WT, Lidicker J, et al. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med. 2001;29:1303–1310. [DOI] [PubMed] [Google Scholar]
- 3.Baue AE. Sepsis research: what did we do wrong? what would Semmelweis do today? Shock. 2001;16:1–8. [DOI] [PubMed] [Google Scholar]
- 4.Cohen J, Guyatt G, Bernard GR, et al. New strategies for clinical trials in patients with sepsis and septic shock. Crit Care Med. 2001;29:880–886. [DOI] [PubMed] [Google Scholar]
- 5.Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med. 2003;348:138–150. [DOI] [PubMed] [Google Scholar]
- 6.Cavaillon JM, dib-Conquy M, Fitting C, et al. Cytokine cascade in sepsis. Scand J Infect Dis. 2003;35:535–544. [DOI] [PubMed] [Google Scholar]
- 7.Perl M, Chung CS, Garber M, et al. Contribution of anti-inflammatory/immune suppressive processes to the pathology of sepsis. Front Biosci. 2006;11:272–299. [DOI] [PubMed] [Google Scholar]
- 8.Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 1999;402:656–660. [DOI] [PubMed] [Google Scholar]
- 9.Bednarek MA, Feighner SD, Pong SS, et al. Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem. 2000;43:4370–4376. [DOI] [PubMed] [Google Scholar]
- 10.Wang G, Lee HM, Englander E, et al. Ghrelin—not just another stomach hormone. Regul Pept. 2002;105:75–81. [DOI] [PubMed] [Google Scholar]
- 11.Arvat E, Di Vito L, Broglio F, et al. Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans. J Endocrinol Invest. 2000;23:493–495. [DOI] [PubMed] [Google Scholar]
- 12.Date Y, Murakami N, Kojima M, et al. Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats. Biochem Biophys Res Commun. 2000;275:477–480. [DOI] [PubMed] [Google Scholar]
- 13.Nass R, Toogood AA, Hellmann P, et al. Intracerebroventricular administration of the rat growth hormone (GH) receptor antagonist G118R stimulates GH secretion: evidence for the existence of short loop negative feedback of GH. J Neuroendocrinol. 2000;12:1194–1199. [DOI] [PubMed] [Google Scholar]
- 14.Cowley MA, Grove KL. Ghrelin: satisfying a hunger for the mechanism. Endocrinology. 2004;145:2604–2606. [DOI] [PubMed] [Google Scholar]
- 15.Hattori N, Saito T, Yagyu T, et al. GH, GH receptor, GH secretagogue receptor, and ghrelin expression in human T cells, B cells, and neutrophils. J Clin Endocrinol Metab. 2001;86:4284–4291. [DOI] [PubMed] [Google Scholar]
- 16.Sakata I, Yamazaki M, Inoue K, et al. Growth hormone secretagogue receptor expression in the cells of the stomach-projected afferent nerve in the rat nodose ganglion. Neurosci Lett. 2003;342:183–186. [DOI] [PubMed] [Google Scholar]
- 17.Papotti M, Ghe C, Cassoni P, et al. Growth hormone secretagogue binding sites in peripheral human tissues. J Clin Endocrinol Metab. 2000;85:3803–3807. [DOI] [PubMed] [Google Scholar]
- 18.Shuto Y, Shibasaki T, Wada K, et al. Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats. Life Sci. 2001;68:991–996. [DOI] [PubMed] [Google Scholar]
- 19.Date Y, Murakami N, Toshinai K, et al. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology. 2002;123:1120–1128. [DOI] [PubMed] [Google Scholar]
- 20.Sato N, Kanai S, Takano S, et al. Central administration of ghrelin stimulates pancreatic exocrine secretion via the vagus in conscious rats. Jpn J Physiol. 2003;53:443–449. [DOI] [PubMed] [Google Scholar]
- 21.Floto RA, Smith KG. The vagus nerve, macrophages, and nicotine. Lancet. 2003;361:1069–1070. [DOI] [PubMed] [Google Scholar]
- 22.Wang H, Yu M, Ochani M, et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 2003;421:384–388. [DOI] [PubMed] [Google Scholar]
- 23.Tracey KJ. The inflammatory reflex. Nature. 2002;420:853–859. [DOI] [PubMed] [Google Scholar]
- 24.Borovikova LV, Ivanova S, Nardi D, et al. Role of vagus nerve signaling in CNI-1493-mediated suppression of acute inflammation. Auton Neurosci. 2000;85:141–147. [DOI] [PubMed] [Google Scholar]
- 25.Borovikova LV, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature. 2000;405:458–462. [DOI] [PubMed] [Google Scholar]
- 26.Wu JT, Kral JG. Ghrelin: integrative neuroendocrine peptide in health and disease. Ann Surg. 2004;239:464–474. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Basa NR, Wang L, Arteaga JR, et al. Bacterial lipopolysaccharide shifts fasted plasma ghrelin to postprandial levels in rats. Neurosci Lett. 2003;343:25–28. [DOI] [PubMed] [Google Scholar]
- 28.Wu R, Zhou M, Cui X, et al. Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis. Am J Physiol Heart Circ Physiol. 2004;287:H1296–H1302. [DOI] [PubMed] [Google Scholar]
- 29.Li WG, Gavrila D, Liu X, et al. Ghrelin inhibits proinflammatory responses and nuclear factor-kappaB activation in human endothelial cells. Circulation. 2004;109:2221–2226. [DOI] [PubMed] [Google Scholar]
- 30.Chang L, Zhao J, Yang J, et al. Therapeutic effects of ghrelin on endotoxic shock in rats. Eur J Pharmacol. 2003;473:171–176. [DOI] [PubMed] [Google Scholar]
- 31.Yang S, Lim YP, Zhou M, et al. Administration of human inter-alpha-inhibitors maintains hemodynamic stability and improves survival during sepsis. Crit Care Med. 2002;30:617–622. [DOI] [PubMed] [Google Scholar]
- 32.Wu R, Zhou M, Wang P. Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells. Regul Pept. 2003;112:19–26. [DOI] [PubMed] [Google Scholar]
- 33.Wang P, Tait SM, Ba ZF, et al. ATP-MgCl2 administration normalizes macrophage cyclic AMP and β-adrenergic receptors after hemorrhage and resuscitation. Am J Physiol. 1994;267:G52–G58. [DOI] [PubMed] [Google Scholar]
- 34.Williams DL, Grill HJ, Cummings DE, et al. Vagotomy dissociates short- and long-term controls of circulating ghrelin. Endocrinol. 2003;144:5184–5187. [DOI] [PubMed] [Google Scholar]
- 35.Wu R, Dong W, Zhou M, et al. Ghrelin improves tissue perfusion in severe sepsis via downregulation of endothelin-1. Cardiovasc Res. 2005;68:318–326. [DOI] [PubMed] [Google Scholar]
- 36.Wu R, Zhou M, Cui X, et al. Ghrelin clearance is reduced at the late stage of polymicrobial sepsis. Int J Mol Med. 2003;12:777–781. [PubMed] [Google Scholar]
- 37.Koo DJ, Chaudry IH, Wang P. Kupffer cells are responsible for producing inflammatory cytokines and hepatocellular dysfunction during early sepsis. J Surg Res. 1999;83:151–157. [DOI] [PubMed] [Google Scholar]
- 38.Sturm E, Havinga R, Baller JF, et al. Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats. J Hepatol. 2005;42:102–109. [DOI] [PubMed] [Google Scholar]
- 39.Pavlov VA, Ochani M, Gallowitsch-Puerta M, et al. Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia. Proc Natl Acad Sci USA. 2006;103:5219–5223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Bernik TR, Friedman SG, Ochani M, et al. Pharmacological stimulation of the cholinergic antiinflammatory pathway. J Exp Med. 2002;195:781–788. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Heider TR, Koruda MJ, Farrell TM, et al. Acute vagotomy does not augment the systemic inflammatory response in patients with peptic ulcer disease. Am Surg. 2004;70:342–346. [PubMed] [Google Scholar]