Ghrelin Attenuates Acute Pancreatitis-Induced Lung Injury and Inhibits Substance P Expression

BASIC INVESTIGATION

Ghrelin Attenuates Acute Pancreatitis-Induced Lung Injury and Inhibits Substance P Expression Xiaolei Zhou, PhD and Chengrui Xue, PhD

Abstract: Objective: To investigate the effect of ghrelin administration on the severity of acute lung injury and on the production of proinflammatory cytokines and Substance P (SP) in rats with acute pancreatitis (AP). Methods: AP was induced in rats by sodium taurocholate injection through pancreaticobiliary duct. Ghrelin 20 nmol/kg was given before and after the treatment. Tumor necrosis factor-␣, interleukin-1␤, and -6 levels in the serum were measured using the radioimmunoassay method. Morphological signs of lung injury, pulmonary water content, microvascular permeability, and myeloperoxidase activity were measured. Meanwhile, the determination of pulmonary SP mRNA level and its expression were performed by reverse transcriptase polymerase chain reaction and immunohistochemistry. Results: The serum proinflammatory cytokines, pulmonary water content, microvascular permeability, and myeloperoxidase activity were increased, and morphological damages were observed in the lung of AP rats. SP mRNA level and its expression were significantly higher in shamoperated rats (P ⬍ 0.05). Morphological damages were attenuated and serum cytokines and pulmonary parameters were reduced by pre- and posttreatment with ghrelin. Pulmonary SP expression was also significantly down-regulated by ghrelin (P ⬍ 0.05). Conclusions: Ghrelin attenuates the severity of acute lung injury induced by AP. The reduction of neutrophil sequestration, limitation of proinflammatory cytokines release, and inhibition of pulmonary SP expression may be the mechanisms involved in the therapeutic effect of ghrelin. Key Indexing Terms: Ghrelin; acute pancreatitis; acute lung injury; substance P. [Am J Med Sci 2010;339(1):49–54.]

A

cute pancreatitis (AP) is a pathological process that depends on premature activation of inactive zymogens into active digestive enzymes and autodigestion of pancreatic tissue.1 Once AP is initiated, the inflammatory events within the acinar cells progress to a generalized systemic inflammatory response syndrome.2 Among the systemic complications, pulmonary complications are the most frequent and potentially the most serious.3 Approximately one third of the patients will develop acute lung injury (ALI),4 which is characterized by an increase in pulmonary microvascular permeability with protein-rich transudate spilling into the alveolar spaces, resulting in decreased lung compliance.5 Recent studies have shown that cytokines and adhesion molecules such as tumor necrosis factor (TNF)-␣, interleukin (IL)-1, and IL-6, and intercellular adhesion molecule-1 as well as neutrophil activation and adhesion contribute to the development and severity of AP and associated ALI.6,7 Substance P (SP), which is released from airway sensory nerves, contributes to the event of neurogenic inflammation in

From the Surgery of Integrated Traditional and Western Medicine Department, Tianjin Medical University General Hospital, Tianjin, China. Submitted March 24, 2009; accepted in revised form July 29, 2009. Correspondence: Chengrui Xue, Ph.D., Surgery of Integrated Traditional and Western Medicine Department, Tianjin Medical University General Hospital, No. 154 AnShan Road, HePing District, Tianjin, 300052, China (E-mail: [email protected]).

the respiratory tract.8 Activation of the neurokinin-1 receptor by SP elicits local vasodilatation and increases microvascular permeability and plasma extravasation.9 SP has also been implicated in inducing the release of proinflammatory mediators and stimulating the chemotaxis of neutrophils.8,9 Thereby, it has shown that SP acts as an important proinflammatory mediator via activation of the neurokinin-1 receptor in the course of AP and associated ALI. On the other hand, both blockage of the neurokinin-1 receptor and genetic deletion of SP greatly attenuate inflammation and damage in the lung.10 –12 Ghrelin, an acylated 28-amino acid protein, was initially purified from rat stomach13 and localizes in endocrine X/A-like cells of the gastric mucosa.14 It acts as an endogenous ligand for the growth hormone secretagogue receptor15,16 and stimulates growth hormone release.17–19 Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis in rats, an effect related to the inhibition of the inflammatory process by reduction in liberation of proinflammatory IL-1␤.20 Moreover, some studies have reported that ghrelin administration also attenuated sepsis-associated ALI induced by cecal ligation and puncture in rats.21 However, it remains unknown whether ghrelin has a therapeutic effect on ALI induced by AP and, if so, whether inhibition of SP plays any role in it. This study was performed to examine the effect of ghrelin administration on the severity of ALI in AP rats and investigate the mechanisms related to the therapeutic effects.

MATERIALS AND METHODS Animals and Treatment Male Wistar rats (250 –350 g) were used for all experiments. The experimental protocols were reviewed and approved by the Committee for Research and Animal Ethics of Tianjin Medical University. The Wistar rats were housed at constant ambient temperature with alternating 12 hours of light-dark cycles. Before the experiment, the rats were fasted overnight with continued access to water. Rat ghrelin (Phoenix Pharmaceuticals, Belmont, CA) was dissolved in normal saline at a final concentration of 100 ␮mol/L. The experiments were performed on 4 groups (10 animals each): AP model group (APG) was treated with normal saline through intraperitoneal injection (3 hours after the injection of sodium taurocholate solution) at 200 ␮L/kg; pre- and posttreated groups (GT1G and GT2G) were treated with ghrelin through intraperitoneal injection (30 minutes before the injection of sodium taurocholate solution or 3 hours later, respectively) at 20 nmol/kg20; shamoperated rats (SOG) were treated with normal saline through intraperitoneal injection (3 hours after the laparotomy) at 200 ␮L/kg. The rats were anesthetized with ketamine (50 mg/kg intraperitoneally; Bioketan, Biowet, Gorzo´w, Poland), and a laparotomy was performed. The duodenum and pancreaticobiliary duct were found, and the hepatic end of the duct was clipped with a noninvasive vascular clip, and then pancreaticobiliary duct retrograde centesis was conducted with an obtuse needle through duodenum seromuscular layer. In APG, GT1G

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and GT2G, 5% of sodium taurocholate (0.1 mL/100 g, taurocholic acid and sodium salt; Sigma Chemical Co., St. Louis, MO) was injected retrograde toward the pancreaticobiliary duct with a microsyringe at an injection rate of 0.20 mL/min.22,23 After the injection, the part of pancreaticobiliary duct entering the duodenum was clipped with a noninvasive vascular clip for 10 minutes. SOG was treated with anesthesia, laparotomy, and duodenal manipulation, but cannulation was not performed. The abdomen wound was closed with 2 layers, and the rats recovered at 37°C. Determination of Serum TNF-␣, IL-1␤, and IL-6 Concentration After sodium taurocholate solution was injected to the rats for 24 hours, the rats were anesthetized with ketamine (50 mg/kg intraperitoneally) and the abdomen wound was opened again. The abdominal aorta was exposed and blood was collected for determination of some serum parameters. Serum TNF-␣, IL-1␤, and IL-6 levels were measured in duplicate by radioimmunoassay using the rat RIA kits (Institute of North Biotechnology, Beijing, China). Concentrations were expressed as nanograms per milliliter or picograms per milliliter. Histological Examination Lung specimens of the rats were harvested and fixed in 10% formalin for histological examination. The tissues were dehydrated and embedded in paraffin and then cut into 5 ␮m sections. The sections were stained with hematoxylin and eosin. The slides were examined by an experienced pathologist without knowing the treatment given. Histopathologic analysis of the lung was performed according to this scoring system: absent, 0; mild, 1; moderate, 2; severe, 3. Three parameters were used as criteria for lung injury, manifested as hemorrhage, edema, and leukocyte infiltration, and the total scores of these parameters were calculated according to the system mentioned above. Measurement of Water Content in the Lung of Rats Lung specimens were resected, dried, and weighed (wet weight). Thereafter, the specimens were desiccated for 24 hours at 80°C and weighed again (dry weight). Water content in the lung (%) ⫽ (wet weight ⫺ dry weight)/wet weight ⫻ 100%. Measurement of Pulmonary Microvascular Permeability After the infusion of sodium taurocholate solution, 1% of Evans Blue dye (Sigma Chemical Co.) in saline (20 mg/kg) was injected into the right femoral vein. The rats were killed 45 minutes later. The pulmonary vasculature was perfused with 5 mL of phosphate-buffered saline via the right ventricle, and the lungs were removed. Thereafter, 100 mg of lung tissue was homogenized in 3 mL of dimethyl formamide and incubated at 54°C overnight. The amount of dye in the lungs was determined spectrophotometrically and extrapolated from a standard curve.24 The results were expressed as micrograms of Evans Blue dye per gram of tissue. Measurement of Myeloperoxidase Activity Sequestration of neutrophils within the lung was evaluated by quantifying tissue myeloperoxidase activity.25,26 The lung tissue samples were homogenized in phosphate buffer (20 mmol/L, pH 7.4), centrifuged at 10,000g for 10 minutes at 4°C, and the resultant pellet was resuspended in phosphate buffer (50 mmol/L, pH 6.0) containing 0.5% of hexadecyltrimethylammonium bromide (Sigma Chemical Co.). The suspension

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was subjected to 4 cycles of freezing and thawing and further disrupted by sonication for 40 seconds. The sample was then centrifuged at 10,000g for 5 minutes at 4°C, and the supernatant was used for the myeloperoxidase assay. Myeloperoxidase activity was determined as described previously by using tetramethylbenzidine (Sigma Chemical Co.) as the substrate. The results were expressed as units per gram of tissue. Measurement of Pulmonary SP mRNA Level by Reverse Transcriptase Polymerase Chain Reaction Total RNA of the lung was isolated by TRIzol (Invitrogen Co., Carlsbad, CA) and treated with RQ1-DNase (Promega Co., Madison, WI) to remove residual contaminations of DNA. Then, the integrity of the total RNA was examined by agarose gel electrophoresis, and the concentration and purity of the total RNA were measured by a UV spectrophotometer. After the total RNA concentration of the sample was calculated, 1 ␮g of the total RNA was used for first-strand cDNA synthesis. Reverse transcriptase reaction was completed by oligo (deoxythymidine) priming and reverse transcriptase (M-MLV; Promega Co.). Polymerase chain reaction was performed by using a kit (TaKaRa; TakaraShuzo Co., Otsu, Japan), including 2 ␮L of cDNA, 2.5 ␮L of 10⫻ buffer, 2.5 ␮L of dNTPs (2.5mmol/L/ each), 1 ␮L of upstream primer (2.5 ␮mol/L), 1 ␮L of downstream primer (2.5 ␮mol/L), 0.4 ␮L of Taq enzyme (5 ug/ul), and 15.6 ␮L of deionized double-distilled water. Upstream and downstream primers for the rat SP gene transcripts were 5⬘AGAGG AAATC GGTGC CAACG-3⬘ and 5⬘-TAATC CAAAG AACTG CTGAG G-3⬘, respectively (145bp); upstream and downstream primers for the rat ␤-actin gene transcripts were 5⬘-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3⬘ and 5⬘-CTA GAA GCA TTG CGG TGG ACG ATG GAG GG-3⬘, respectively (666bp). Thermal cycle conditions for SP were as follows: predegeneration at 94°C for 10 minutes, degeneration at 94°C for 40 seconds, annealing at 60°C for 40 seconds, and extension at 72°C for 50 seconds. After 35 cycles, amplification was completed with an additional step at 72°C for 5 minutes. The amplification was performed in an automatic thermal cycler (Perkin Elmer Inc., Boston, MA). Different cDNA samples were normalized using primer sets to the housekeeping gene ␤-actin. Equal aliquots were taken from each sample and analyzed by agarose gel electrophoresis, and then the video images of ethidium bromide-stained gels were quantified by ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The ratios of SP to ␤-actin signals were used for quantification. Measurement of SP Expression in Lung by Immunohistochemistry Samples of lung tissue for immunohistochemistry examination were fixed in 4% paraformaldehyde and then embedded in paraffin. Sections (4 ␮m) of specimens were cut into polyL-lysin-coated slides. After deparaffinization with dimethyl benzene and gradient hydration with alcohol, endogenous peroxidase activity was blocked by incubating the slides with 3% hydrogen peroxide for 10 minutes. The sections were treated with 10% normal goat serum to block nonspecific sites. After that treatment, SP rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (diluted in 1:50) was added at 4°C. Thereafter, the sections were incubated with biotinylated mouse antirabbit antibody (Santa Cruz Biotechnology) (diluted in 1:400) and peroxidase-conjugated avidin (Santa Cruz Biotechnology) (diluted in 1:200) at 37°C for 30 minutes, respectively. Finally, the brown color solution was developed with the addition of diaminobenzidine for 5 minutes. For the negative Volume 339, Number 1, January 2010

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TABLE 2. Serum TNF-␣, IL-1␤, and IL-6 concentration in each group (n ⫽ 10)

TABLE 1. Histological scoring, pulmonary microvascular permeability, and MPO activity of the lung in each group (n ⫽ 10) Groups

Histological Scores

The Amount of EB Dye (␮g/g)

MPO Activity (U/g)

SOG APG GT1G GT2G

0.5 ⫾ 0.6 4.8 ⫾ 1.3b 3.0 ⫾ 0.9a,b 3.6 ⫾ 0.8a,b,c

13.09 ⫾ 9.48 107.68 ⫾ 48.76b 63.32 ⫾ 28.22a,b 70.48 ⫾ 24.69a,b

0.64 ⫾ 0.35 1.75 ⫾ 0.32b 1.27 ⫾ 0.43a,b 1.33 ⫾ 0.52a,b

a

a

a

All the values are given in mean ⫾ SEM. a P ⬍ 0.05 vs. APG. b P ⬍ 0.05 vs. SOG. c P ⬍ 0.05 vs. GT1G. SOG, sham-operated rat; APG, AP model group; SEM, standard error of mean; EB, Evans Blue; MPO, myeloperoxidase.

control sections, phosphate-buffered saline was substituted for primary SP rabbit polyclonal antibody. Other steps were the same with those above. The ratios of positive stain area to visual field area were used for quantification. Statistical Analysis Results were expressed as mean ⫾ standard error of mean. Statistical analysis was carried out by one-way analysis of variance. Subsequent comparison was made by the StudentNewman-Keuls test; statistical significance was inferred when P ⬍ 0.05.

RESULTS Histological Examination of Lung Tissue In APG, histological examination of the sections confirmed lung injury with significant alveolar edema and thickening, hemorrhage, vasocongestion, and infiltration with leukocytes observed. In GT1G and GT2G, the histological injury in lung was improved significantly. Compared with GT2G, GT1G showed less lung injury (P ⬍ 0.05). The scores of histological evaluation of lung injury were summarized in Table 1.

Groups SOG APG GT1G GT2G

TNF-␣ (ng/mL)

IL-1␤ (pg/mL)

IL-6 (pg/mL)

4.13 ⫾ 0.14a 6.52 ⫾ 0.41b 4.42 ⫾ 0.21a 4.50 ⫾ 0.36a

127.9 ⫾ 5.7a 242.1 ⫾ 24.6b 152.8 ⫾ 16.7a,b 173.5 ⫾ 21.5a,b,c

187.6 ⫾ 32.5a 516.3 ⫾ 39.4b 322.0 ⫾ 36.2a,b 339.4 ⫾ 30.8a,b

All the values are given in mean ⫾ SEM. a P ⬍ 0.05 vs. APG. b P ⬍ 0.05 vs. SOG. c P ⬍ 0.05 vs. GT1G. SOG, sham-operated rat; APG, AP model group; SEM, standard error of mean; TNF, tumor necrosis factor; IL, interleukin.

Serum Cytokines All the measured serum inflammatory cytokines in APG, including TNF-␣, IL-1␤, and IL-6, were significantly higher than that in SOG (P ⬍ 0.05), but pre- and posttreatment with ghrelin led to a significant reduction in the increase caused by AP (P ⬍ 0.05). Moreover, the IL-1␤ concentration in GT1G was significantly lower than that in GT2G (P ⬍ 0.05). The results were shown in Table 2. Pulmonary SP mRNA Level Pulmonary SP mRNA level was significantly higher in APG than that in SOG (P ⬍ 0.05) (Fig. 1). When ghrelin was given to the rats before or after the induction of AP, the increase in SP mRNA levels was significantly attenuated (P ⬍ 0.05) (Fig. 1). Moreover, SP mRNA level in GT1G showed no significant difference from GT2G. The ratios of SP to ␤-actin signals are shown in Table 3. SP Expression in Pulmonary Tissue There was no background staining when SP antibody was omitted. In SOG, bland staining was observed in a few alveolar epithelial cells and vascular endothelial cells (Fig. 2A). More staining was observed in vascular endothelial cells, alveolar epithelial cells, and tracheal mucous membranes in APG

Water Content in Lung In SOG, water content in lung was 68.0% ⫾ 5.2%, and it was significantly increased to 77.7% ⫾ 1.5% in APG (P ⬍ 0.05). Water content was significantly reduced to 73.5% ⫾ 4.8% and 74.2% ⫾ 3.6% in GT1G and GT2G, respectively (P ⬍ 0.05 compared with APG). Pulmonary Microvascular Permeability Microvascular permeability was significantly higher in APG than in SOG (P ⬍ 0.05), and it was significantly reduced by pre- and posttreatment with ghrelin. Moreover, the permeability in GT1G was lower than that in GT2G, but there was no significant difference between them. The results of microvascular permeability were shown in Table 1. Myeloperoxidase Activity As an additional quantitative assessment of the severity of the inflammatory response, myeloperoxidase activity was measured, which was an indicator of neutrophil sequestration in lung. Myeloperoxidase activity in APG was higher than that in SOG (P ⬍ 0.05), and the increase was significantly attenuated in GT1G and GT2G (P ⬍ 0.05). The results of 4 groups were summarized in Table 1. © 2010 Lippincott Williams & Wilkins

FIGURE 1. SP mRNA levels in lung in each group. SP (145 bp); ␤-actin (666 bp). 1: Maker, 2: SOG, 3: APG, 4: GT1G, 5: GT2G.

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TABLE 3. SP mRNA levels and expression in pulmonary tissues of each group (n ⫽ 10) Groups

mRNA Level

Expression

SOG APG GT1G GT2G

0.93 ⫾ 0.15 1.29 ⫾ 0.09b 1.14 ⫾ 0.16a,b 1.16 ⫾ 0.11a,b

0.03 ⫾ 0.02a 0.13 ⫾ 0.05b 0.09 ⫾ 0.04a,b 0.09 ⫾ 0.03a,b

a

All the values are given in mean ⫾ SEM. a P ⬍ 0.05 vs. APG. b P ⬍ 0.05 vs. SOG. SOG, sham-operated rat; APG, AP model group; SEM, standard error of mean.

(Fig. 2B). In addition, SP-positive staining was significantly reduced in pulmonary tissues of GT1G and GT2G (P ⬍ 0.05) (Fig. 2C and D). The ratios of positive stain area to visual field are shown in Table 3.

DISCUSSION Ghrelin has received considerable attention for its growth hormone-releasing properties, in addition to its effects on food intake and adiposity. Moreover, the administration of ghrelin attenuated pancreatic injury in caerulein-induced pancreatitis rats20 and sepsis-associated ALI in rats subjected to cecal ligation and puncture.21 The therapeutic effects of ghrelin may be mediated through down-regulation of proinflammatory cytokines and inhibition of NF-kB. However, the effect of ghrelin on AP-associated ALI and the mechanism still need be studied further. Our study demonstrates for the first time that ghrelin inhibits the expression of SP in the lung in the course of AP, suggesting a potential mechanism for the observed therapeutic effects on AP-associated ALI. AP is a noninfectious inflammatory reaction of the pancreas, associated with autodigestion of the organ, and it involves 3 phases. The initial phase is characterized by intrapancreatic digestive enzyme activation and acinar cell injury; the second phase is characterized by an intrapancreatic inflammatory reaction and varying degrees of acinar cell ne-

FIGURE 2. Immunohistochemistry for pulmonary SP expression. (A) Bland staining was present in a few alveolar epithelial cells and vascular endothelial cells from SOG. (B) But seen in vascular endothelial cells, alveolar epithelial cells, and tracheal mucous membranes from APG as a brownish-yellow colour. (C and D) Pre- and posttreatment with ghrelin at the dose 20 nmol/kg significantly reduced its expression in pulmonary tissue of GT1G and GT2G (counterstaining by eosin, 200⫻).

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crosis; and the third phase is characterized by further progression of the pancreatic injury and the appearance of extrapancreatic changes. ALI is the most important manifestation of extrapancreatic organ dysfunction in AP. In this study, histological examination of the lung from APG confirmed that ALI presented significant alveolar edema and thickening, hemorrhage, vasocongestion, and infiltration with leukocytes observed. Meanwhile, the increases in pulmonary water content and microvascular permeability in APG provided an additional evidence for ALI. Pre-ghrelin injection in GT1G significantly reduced the histological scores of lung injury and attenuated the increases of pulmonary water content and microvascular permeability. In addition, the histological scores of lung injury in GT1G were lower than those in GT2G. Thereby, these results suggest that pretreatment with ghrelin could be a prophylactic treatment for AP patients at high risk for ALI. However, 3 hours of postghrelin injection in GT2G also showed therapeutical effects on AP-associated ALI. The histological scores of lung injury, pulmonary water content, and microvascular permeability in GT2G were significantly lower than those in APG. As a result, the above results support the hypothesis that ghrelin treatment could have a promising role for both prevention and therapy of ALI in any clinical phases of AP. During the process of AP, ALI is associated with the accumulation of neutrophils within the interstitial and alveolar spaces.27 Neutrophil sequestration within an inflamed area is a multistep process that begins with neutrophil activation followed by the rolling of inflammatory cells and the adhesion of circulating activated inflammatory cells to the endothelium via adhesion molecules. In this study, myeloperoxidase activity was measured to assess the sequestration of neutrophils within the lung. The results showed that myeloperoxidase activity was significantly increased after the induction of AP, whereas ghrelin administration significantly reduced myeloperoxidase activity and improved the histological changes. Released cytokines by inflammatory cells can also aggravate ALI.28 Therefore, we studied the production of major cytokines TNF-␣, IL-1␤, and IL-6 in serum. The data show that the release of TNF-␣, IL-1␤, and IL-6 in AP rats was attenuated by ghrelin treatment. These findings indicate that the therapeutic effects of ghrelin on ALI are mediated through the inhibition of neutrophil sequestration and the reduction in liberation of proinflammatory cytokines. The data also show that the concentrations of TNF-␣, IL-1␤, and IL-6 in GT1G are lower than those in GT2G, although there was no significant difference between them in concentrations of TNF-␣ and IL-6. It seems that the liberation of proinflammatory cytokines was more significantly inhibited by pretreatment with ghrelin, which might contribute to the different therapeutic effects between GT1G and GT2G. SP, a preprotachykinin-A gene product, is an immunoregulatory neuropeptide produced at various inflammation sites. Increased SP immunoreactivity has been found in bronchoalveolar lavage samples from patients suffering from lung diseases.29 Moreover, SP has been proposed to be a key mediator in regulating the severity of ALI.30 –33 In this study, there was a significant increase in the pulmonary SP mRNA levels of AP rats. In addition, it was found that the changes in mRNA levels were reflected by parallel changes in SP expression in pulmonary tissue. The above findings are consistent with previous observations in AP-associated ALI in mice,10,34 and indicate that up-regulation of SP may contribute to lung © 2010 Lippincott Williams & Wilkins

inflammation and injury in the development of extrapancreatic organ dysfunction induced by AP. On the other hand, our results also showed that both preand posttreatment with ghrelin could significantly attenuate the increases of pulmonary SP mRNA levels and its expression induced by AP. The inhibition of the action of SP by blockage of the neurokinin-1 receptor with CP96345 has been shown to protect mice against AP and associated ALI.34 These findings are further substantiated by the fact that the deletion of the precursor gene for SP can lead to remarkable reduction in the severity of AP-associated lung injury.25 In view of these previous findings, we believed that the inhibition of pulmonary SP expression may be a potential mechanism for the observed protective effects of ghrelin on AP-induced ALI. Moreover, the neurokinin-1 receptor activation by SP can enhance inflammation by decreasing vascular tone and increasing endothelial microvascular permeability and neutrophil sequestration.34 SP has also been implicated in inducing the release of proinflammatory cytokines, such as TNF-␣, IL-1␤, and IL-6.35,36 Our data show that ghrelin treatment not only decreased lung microvascular permeability but also reduced the level of these serum proinflammatory cytokines in AP rats. This is consistent with the reduced levels of pulmonary SP mRNA levels and provides more evidence for our hypothesis. However, ALI is multifactorial, and numerous mediators other than SP are involved. Therefore, the precise mechanism by which ghrelin elicits its protective effect on AP-associated ALI remains to be investigated in the future study. In summary, the results of this study show that ghrelin treatment attenuates ALI in the sodium taurocholate-induced rat AP model. Pulmonary histological score, water content microvascular permeability, myeloperoxidase activity, and serum proinflammatory cytokines were significantly reduced by ghrelin injection either 30 minutes before or 3 hours after AP induction. Ghrelin also attenuated the increase of pulmonary SP mRNA levels and its expression induced by AP. Therefore, the reduction in neutrophil sequestration, limitation of proinflammatory cytokines release, and the inhibition of pulmonary SP expression may be the mechanisms involved in therapeutic effects of ghrelin on AP-associated ALI. However, further clinical studies are needed to confirm the benefits of ghrelin treatment. REFERENCES 1. Klo¨ppel G. Acute pancreatitis. Semin Diagn Pathol 2004;21:221– 6. 2. Bhatia M. Acute pancreatitis as a model of SIRS. Front Biosci 2009;14:2042–50. 3. Liu XM, Xu J, Wang ZF. Pathogenesis of acute lung injury in rats with severe acute pancreatitis. Hepatobiliary Pancreat Dis Int 2005;4: 614 –7. 4. Bhatia M. Novel therapeutic targets for acute pancreatitis and associated multiple organ dysfunction syndrome. Curr Drug Targets Inflamm Allergy 2002;1:343–51. 5. Gu¨nther A, Walmrath D, Grimminger F. Pathophysiology of acute lung injury. Semin Respir Crit Care Med 2001;22:247–58. 6. Malleo G, Mazzon E, Siriwardena AK, et al. TNF-alpha as a therapeutic target in acute pancreatitis—lessons from experimental models. ScientificWorldJournal 2007;7:431– 48. 7. Frossard JL, Saluja A, Bhagat L, et al. The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury. Gastroenterology 1999;116:694 –701. 8. Groneberg DA, Quarcoo D, Frossard N, et al. Neurogenic mechanisms in bronchial inflammatory disease. Allergy 2004;59:1139 –52.

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Zhou and Xue

9. O’Connor TM, O’Connell J, O’Brien DI. The role of substance P in inflammatory disease. J Cell Physiol 2004;201:167– 80.

injury in experimental pancreatitis in rats: pulmonary protective effects of crotapotin and N-acetylcysteine. Shock 2002;18:428 –33.

10. Lau HY, Bhatia M. The effect of CP96, 345 on the expression of tachykinins and neurokinin receptors in acute pancreatitis. J Pathol 2006;208:364 –71.

24. Klinger JR, Murray JD, Casserly B, et al. Rottlerin causes pulmonary edema in vivo: a possible role for PKCdelta. J Appl Physiol 2007;103:2084 –94.

11. Sun J, Bhatia M. Blockade of neurokinin-1 receptor attenuates CC and CXC chemokine production in experimental acute pancreatitis and associated lung injury. Am J Physiol Gastrointest Liver Physiol 2007; 292:G143–53.

25. Bhatia M, Slavin J, Cao Y, et al. Preprotachykinin-A gene deletion protects mice against acute pancreatitis and associated lung injury. Am J Physiol Gastrointest Liver Physiol 2003;284:G830 – 6.

12. Hegde A, Zhang H, Moochhala SM, et al. Neurokinin-1 receptor antagonist treatment protects mice against lung injury in polymicrobial sepsis. J Leukoc Biol 2007;82:678 – 85.

26. Bhatia M, Brady M, Zagorski J, et al. Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury. Gut 2000;47:838 – 44.

13. Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone releasing acylated peptide from stomach. Nature 1999;402:656 – 60.

27. de Campos T, Deree J, Martins JO. Pentoxifylline attenuates pulmonary inflammation and neutrophil activation in experimental acute pancreatitis. Pancreas 2008;37:42–9.

14. Hayashida T, Nakahara K, Mondal MS, et al. Ghrelin in neonatal rats: distribution in stomach and its possible role. J Endocrinol 2002; 173:239 – 45.

28. Martín Alonso MA, Santamaría A, Saracíbar E, et al. Cytokines and other immunological parameters as markers of distant organ involvement in acute pancreatitis. Med Clin (Barc) 2007;128:401– 6.

15. Sun Y, Wang P, Zheng H, et al. Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 2004;101:4679 – 84. 16. Dixit VD, Weeraratna AT, Yang H, et al. Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility. J Biol Chem 2006;281:16681–90. 17. Malago´n MM, Luque RM, Ruiz-Guerrero E, et al. Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes. Endocrinology 2003;144:5372– 80. 18. Maier C, Schaller G, Buranyi B, et al. The cholinergic system controls ghrelin release and ghrelin-induced growth hormone release in humans. J Clin Endocrinol Metab 2004;89:4729 –33. 19. Zizzari P, Halem H, Taylor J, et al. Endogenous ghrelin regulates episodic growth hormone (GH) secretion by amplifying GH pulse amplitude: evidence from antagonism of the GH secretagogue-R1a receptor. Endocrinology 2005;146:3836 – 42. 20. Dembinski A, Warzecha Z, Ceranowicz P, et al. Ghrelin attenuates the development of acute pancreatitis in rats. J Physiol Pharmacol 2003;54:561–73. 21. Wu R, Dong W, Zhou M, et al. Ghrelin attenuates sepsis-induced acute lung injury and mortality in rats. Am J Respir Crit Care Med 2007;176:805–13. 22. Xia SH, Fang DC, Hu CX, et al. Effect of BN52021 on NF␬-Bp65 expression in pancreatic tissues of rats with severe acute pancreatitis. World J Gastroenterol 2007;13:882– 888. 23. Leme AS, Lichtenstein A, Arantes-Costa FM, et al. Acute lung

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29. Nieber K, Baumgarten CR, Rathsack R, et al. Substance P and beta-endorphin-like immunoreactivity in lavage fluids of subjects with and without allergic asthma. J Allergy Clin Immunol 1992;90(4 Pt 1):646 –52. 30. Puneet P, Hegde A, Ng SW, et al. Preprotachykinin-A gene products are key mediators of lung injury in polymicrobial sepsis. J Immunol 2006;176:3813–20. 31. Beer S, Weighardt H, Emmanuilidis K, et al. Systemic neuropeptide levels as predictive indicators for lethal outcome in patients with postoperative sepsis. Crit Care Med 2002;30:1794 – 8. 32. Wong SS, Sun NN, Lantz RC, et al. Substance P and neutral endopeptidase in development of acute respiratory distress syndrome following fire smoke inhalation. Am J Physiol Lung Cell Mol Physiol 2004;287:L859 – 66. 33. Zhang H, Hegde A, Ng SW, et al. Hydrogen sulfide up-regulates substance P in polymicrobial sepsis-associated lung injury. J Immunol 2007;179:4153– 60. 34. Bhatia M, Saluja AK, Hofbauer B, et al. Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis associated lung injury. Proc Natl Acad Sci USA 1998;95:4760 –5. 35. Yamaguchi M, Ozawa Y, Mishima H, et al. Substance P increases production of proinflammatory cytokines and formation of osteoclasts in dental pulp fibroblasts in patients with severe orthodontic root resorption. Am J Orthod Dentofacial Orthop 2008;133:690 – 8. 36. Tokuda M, Miyamoto R, Sakuta T, et al. Substance P activates p38 mitogen-activated protein kinase to promote IL-6 induction in human dental pulp fibroblasts. Connect Tissue Res 2005;46:153– 8.

Volume 339, Number 1, January 2010