1. Ghrelin Attenuates Sepsis-induced Acute Lung Injury and Mortality in Rats R2 조용덕 Am J Respir Crit Care Med Vol 176.pp805-813,2007 Rongqian Wu1, Weifeng Dong1, Mian Zhou1, Fangming Zhang1, Corrado P. Marini1, Thanjavur S. Ravikumar1, and Ping Wang1

2. Background Despite significant advances in the understanding of the pathogenesis of sepsis and its management, the mortality rate of severe sepsis remains unacceptably high The ultimate cause of death in patients with severe sepsis is multiple organ failure. The lung is frequently the first failing organ during the sequential development of multiple organ dysfunction in sepsis Acute lung injury (ALI) that clinically manifests as acute respiratory distress syndrome is the primary cause of death under these conditions

3. Background The pathophysiological sequelae of sepsis are caused by an overreaction of the immune system to microorganisms and their products. Excessive cytokine-mediated inflammation plays a fundamental role in the pathogenesis of sepsis-induced ALI. Immunosuppression is also evident at the onset of sepsis. Therefore, direct antiinflammatory strategies have produced modest clinical effects in critically ill patients

4. Background The activation of nuclear factor (NF)-kB has been implicated as an important step in the development of ALI. Increased activation of NF-kB is found in alveolar macrophages, peripheral blood mononuclear cells, and neutrophils from patients with sepsis A greater or more persistent nuclear accumulation of NF-kB is associated with higher mortality and more persistent organ dysfunction, including pulmonary injury Therefore, NF-kB activation is likely to be a logical therapeutic target fo ALI

5. Background Ghrelin -> a novel endogenous ligand for the growth hormone secretagogue receptor (GHSR)-1a -> a 28–amino acid acylated peptide produced predominantly by the stomach -> possess other endocrine and nonendocrine activities reflecting central and peripheral GHSR-1a distribution Li and coworkers reported -> ghrelin mitigates proinflammatory cytokine production and mononuclear cell binding through inhibition of NF-kB in human endothelial cells

6. Background Our studies -> circulating levels of ghrelin decreased significantly in a rat model of polymicrobial sepsis induced by cecal ligation and puncture (CLP) -> ghrelin administration increases cardiac output, reduces total peripheral resistance, and improves organ blood flow under such conditions The improvement of tissue perfusion by ghrelin in severe sepsis appears to be mediated by down-regulation of endothelin-1 in endothelial cells, involving an NF-kB–dependent pathway

7. Background The present study was conducted to test the hypothesis that ghrelin attenuates severe sepsis–induced ALI and mortality through inhibition of NF-kB.

8. Materials and Methods Animal Model of Sepsis -> Male Sprague-Dawley rats (275–325 g) were housed in a temperaturecontrolled room on a 12-h light:dark cycle and fed a standard Purina rat chow diet -> CLP was performed as we previously described, and the animals were randomly assigned to various groups. -> Sham-operated animals (i.e., control animals) underwent the same procedure with the exception that the cecum was neither ligated nor punctured

9. Materials and Methods Administration of Ghrelin -> After a slow intravenous bolus injection of 2 nmol of ghrelin (or 200 ml of vehicle) 5 hours after CLP, -> the minipump was then connected to a jugular venous catheter and implanted subcutaneously. -> Twenty hours after CLP (i.e., 15 h after implantation of the minipump), the rats were killed and blood and tissue samples were collected. -> The total dose of ghrelin each rat received was 45 nmol/kg body weight.

10. Materials and Methods Water Content Determination and Histologic Examination Determination of Pulmonary Blood Flow Bacterial Load Survival Study Determination of Pulmonary Ghrelin, Tumor Necrosis Factor-a, and IL-6 Western Blotting Analysis of NF-kB Nuclear Translocation Evaluation of NF-kB Activity Administration of Ghrelin Receptor Antagonist Statistical Analysis

11. Result Figure 1. Alterations in pulmonary levels of ghrelin in sham-operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after cecal ligation and puncture (CLP). Data are presented as means ±SE (n =6) and compared by one-way analysis of variance (ANOVA) and the Student-Newman-Keuls method: *P, 0.05 versus sham group; #P, 0.05 versus vehicle group.

12. Result Figure 2. Alterations in lung water content in sham-operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after cecal ligation and puncture (CLP). Data are presented as means 6 SE (n 5 6) and compared by one-way analysis of variance and the Student-Newman-Keuls method: *P, 0.05 versus sham group; #P, 0.05 versus vehicle group.

13. Result (A) Photomicrograph of a pulmonary section from a sham-operated rat. (B) Photomicrograph of a lung section from a septic rat 20 hours after cecal ligation and puncture (CLP) treated with vehicle.

14. Result (C ) Photomicrograph of a lung section from a septic rat 20 hours after CLP treated with ghrelin. (D) Histopathologic scoring of lung injury in sham-operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after CLP.

15. Result Figure 4. Alterations in pulmonary blood flow in sham-operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after cecal ligation and puncture (CLP). Data are presented as means ±SE (n=6 or 7) and compared by one-way analysis of variance and the Student-Newman-Keuls method: *P, 0.05 versus sham group; #P, 0.05 versus vehicle group.

16. Result Figure 5. Alterations in bacterial load in the peritoneal fluid (A), blood(B), and lungs (C) in sham- operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after cecal ligation and puncture (CLP). Data are presented as means ± SE (n = 5– 8) and compared by one-way analysis of variance and the Student-Newman-Keuls method: *P, 0.05 versus sham group; #P, 0.05 versus vehicle group.

17. Result Figure 6. Alterations in the survival rate (A) and body weight (B) 10 days after cecal ligation and puncture (CLP) and cecal excision with normal saline treatment (solid triangles) and CLP with ghrelin treatment (open circles). There were 20 animals in each group. The survival rate was estimated by the Kaplan-Meier method and compared by log-rank test. *P, 0.05 versus CLP plus vehicle.

18. Result Figure 7. Alterations in pulmonary levels of tumor necrosis factor (TNF)-a (A) and IL-6 (B) in sham-operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after cecal ligation and puncture (CLP). Data are presented as means 6 SE (n 5 6) and compared by one-way analysis of variance and the Student- Newman-Keuls method: *P, 0.05 versus sham group; #P, 0.05 versus vehicle group.

19. Result Figure 8. Alterations in nuclear levels of nuclear factor (NF)-kB p65 (A), cytoplasm levels of IkBa (B), and NF-kB activities (C ) in the lungs in sham-operated animals and septic animals treated with normal saline (vehicle) or ghrelin 20 hours after cecal ligation and puncture (CLP). Total p44/42 MAPK and b-actin were served as loading control for nuclear and cytoplasmic proteins, respectively. Representative blots are also presented. Data are expressed as means ± SE (n =4–6 per group) and compared by one-way analysis of variance and the Student- Newman-Keuls method: *P, 0.05 versus sham-operated animals; #P, 0.05 versus CLP animals treated with vehicle.

20. Result Figure 9. Alterations in pulmonary levels of tumor necrosis factor (TNF)-a (A) and IL-6 (B) in normal animals 1 hour after normal saline (vehicle) or [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P (D-SP) treatment. Data are expressed as means 6 SE (n 5 6 per group) and compared by Student t test: *P, 0.05 versus vehicle group.

21. Result Figure 10. Alterations in the survival rate (A) and body weight (B) 10 days after cecal ligation and puncture (CLP) and cecal excision with normal saline treatment (solid triangles, derived from Figure 6) and CLP with [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P treatment (open triangles). There were 20 animals in each group. The survival rate was estimated by the Kaplan-Meier method and compared by log-rank test. *P, 0.05 versus CLP plus vehicle.

22. discussion Ghrelin -> an acylated peptide produced predominantly in the stomach, -> discovered to be a natural ligand of the GHSR-1a in 1999 Studies have indicated -> ghrelin appears to play a determinant role in fetal lung development. ->Ghrelin receptor–binding sites have been demonstrated in the adult lung parenchyma and pulmonary artery wall. -> ghrelin administration has been shown to attenuate monocrotaline- induced pulmonary hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy in rats. Therefore, it seems reasonable to propose that ghrelin plays a prevailing role in physiological and pathophysiological functions of the lungs.

23. discussion Our present study -> lung injuries, characterized by increased lung water content, disruption of lung architecture, extravasation of red blood cells, accumulation of inflammatory cells, decreased pulmonary blood flow, are present 20 hours after CLP in vehicle-treated animals. -> These injuries are associated with decreased levels of ghrelin in the pulmonary tissues. -> administration of ghrelin may be a useful adjunct in the treatment of severe sepsis–induced lung injury.

24. discussion -> pulmonary levels of ghrelin 20 hours after CLP in ghrelin-treated animals were 86% higher than those in sham-operated animals despite a 52% decrease after CLP. -> The increase in pulmonary levels of ghrelin is associated with mitigated lung injury, decreased bacterial load, increased pulmonary blood flow, improved survival after CLP. ->administration of ghrelin receptor antagonist decreased the 10-day survival rate after CLP and cecal excision confirms the notion that the beneficial effect of ghrelin in sepsis is mediated through its receptors. Thus, ghrelin may be an effective treatment for severe sepsis– induced ALI.

25. discussion ALI in the setting of sepsis is the result of the actions of an integrated network of soluble inflammatory mediators and a variety of inflammatory cells The formation of proinflammatory mediators such as TNF-a and IL-6 plays an important role in the pathophysiology of ALI. TNF-a is present in the bronchoalveolar lavage fluid patients at risk for acute respiratory distress syndrome (ARDS) and with established ARDS The highest concentrations of TNF-a are found in the bronchoalveolar lavage fluid from patients with sustained ARDS.

26. discussion Raised levels of IL-6 have been described in a number of acute conditions such as burns, major surgery, and sepsis Circulating levels of IL-6 have been shown to be excellent predictors of the severity of ARDS of various etiologies, including sepsis and acute pancreatitis

27. discussion In patients with ARDS, nonsurvivors have significantly higher ratios of bronchoalveolar lavage fluid to plasma cytokine concentrations than survivors -> This suggests the critical role of lung inflammation in mortality. In this regard, the fact that ghrelin administration down-regulated pulmonary levels of TNF-a and IL-6 suggests another possible mechanism by which ghrelin exerts a beneficial effect in severe sepsis–induced ALI.

28. discussion NF-kB is one critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of acute lung injury. Our previous study -> ghrelin inhibits TNF-a–induced NF-kB nuclear translocation in cultured endothelial cells -> ghrelin inhibits the degradation of IkBa and prevents the translocation of NF-kB into the nucleus in the lungs.

29. discussion Activation of NF-kB -> play a central role in the development of pulmonary inflammation and acute lung injury -> In vitro studies have shown that NF-kB regulates gene expression of cytokines, chemokines, and adhesion molecules

30. discussion Our results -> ghrelin does not directly inhibit TNF-a and IL-6 release from endotoxin-stimulated Kupffer cells or peritoneal macrophages. -> The intact vagus nerve is essential for the inhibitory effect of ghrelin on the release of proinflammatory cytokines -> Acetylcholine released by stimulation of the vagus nerve binds to the a7 subunit of the nicotinic acetylcholine receptor expressed on macrophages, suppressing proinflammatory cytokine production by an NF-kB–dependent pathway

31. conclusion pulmonary levels of ghrelin decreased significantly 20 hours after CLP. Intravenous administration of ghrelin after the onset of sepsis -> increased pulmonary levels of ghrelin -> ameliorated lung histology -> reduced lung water content -> elevated lung blood flow, improved survival -> decreased pulmonary levels of proinflammatory cytokines Thus, ghrelin can be developed as a novel treatment for severe sepsis–induced ALI. This protective effect appears to be mediated through inhibition of NF-kB.