Abstract

Introduction

Acute pancreatitis is a necrotic and inflammatory process that suddenly occurs in peripheral and internal regions of the pancreas ¹1. Yuan H, Jin X, Sun J, Li F, Feng Q, Zhang C, et al. Protective effect of HMGB1 a box on organ injury of acute pancreatitis in mice. Pancreas 2009; 38: 143-148, doi: 10.1097/MPA.0b013e31818166b4.
https://doi.org/10.1097/MPA.0b013e318181... . Approximately 20% of patients with acute pancreatitis may develop a more severe form with occurrence of organ dysfunction, which is known as severe acute pancreatitis (SAP) ²2. Hayden P, Wyncoll D. Severe acute pancreatitis. Curr Anaesth Crit Care 2008; 19: 1-7.. SAP is characterized by the development of a systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) as well as local pancreatic complications, and is associated with a mortality rate of 15-30%, despite continuing improvement in clinical care ³3. Ammori BJ. Role of the gut in the course of severe acute pancreatitis. Pancreas 2003; 26: 122-129, doi: 10.1097/00006676-200303000-00006.
https://doi.org/10.1097/00006676-2003030... ,⁴4. Lankisch PG, Lerch MM. Pharmacological prevention and treatment of acute pancreatitis: where are we now? Dig Dis 2006; 24: 148-159, doi: 10.1159/000090318.
https://doi.org/10.1159/000090318... . MODS and complications of infection, such as pancreatic necrosis and sepsis, contribute to the increased mortality of SAP ⁵5. Buter A, Imrie CW, Carter CR, Evans S, McKay CJ. Dynamic nature of early organ dysfunction determines outcome in acute pancreatitis. Br J Surg 2002; 89: 298-302.,⁶6. Yasuda T, Ueda T, Takeyama Y, Shinzeki M, Sawa H, Nakajima T, et al. Significant increase of serum high-mobility group box chromosomal protein 1 levels in patients with severe acute pancreatitis. Pancreas 2006; 33: 359-363, doi: 10.1097/01.mpa.0000236741.15477.8b.
https://doi.org/10.1097/01.mpa.000023674... . Meanwhile, the release of inflammatory mediators, including tumor necrosis factor (TNF), interleukin (IL)-1, IL-8, and other inflammatory cytokines, is the primary cause of SIRS and MODS ¹1. Yuan H, Jin X, Sun J, Li F, Feng Q, Zhang C, et al. Protective effect of HMGB1 a box on organ injury of acute pancreatitis in mice. Pancreas 2009; 38: 143-148, doi: 10.1097/MPA.0b013e31818166b4.
https://doi.org/10.1097/MPA.0b013e318181... .

High-mobility group box 1 (HMGB1), a large group of low-molecular-weight (∼30 kDa) nucleoproteins, belongs to the largest and best characterized group of nonhistone chromosomal proteins ⁷7. Bustin M, Reeves R. High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. Prog Nucleic Acid Res Mol Biol 1996; 54: 35-100, doi: 10.1016/S0079-6603(08)60360-8.
https://doi.org/10.1016/S0079-6603(08)60... . It was originally identified as a DNA-binding protein possessing potent proinflammatory properties ⁸8. Park JS, Arcaroli J, Yum HK, Yang H, Wang H, Yang KY, et al. Activation of gene expression in human neutrophils by high mobility group box 1 protein. Am J Physiol Cell Physiol 2003; 284: C870-C879, doi: 10.1152/ajpcell.00322.2002.
https://doi.org/10.1152/ajpcell.00322.20... . Subsequently, it has been demonstrated that HMGB1 is actively secreted by live inflammatory cells, such as stimulated macrophages and monocytes, and is released passively from necrotic or damaged cells ⁹9. Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002; 418: 191-195, doi: 10.1038/nature00858.
https://doi.org/10.1038/nature00858... . In addition, proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and HMGB1, are thought to have a crucial role in the pathogenesis of acute pancreatitis ¹⁰10. Zhang ZW, Zhang QY, Zhou MT, Liu NX, Chen TK, Zhu YF, et al. Antioxidant inhibits HMGB1 expression and reduces pancreas injury in rats with severe acute pancreatitis. Dig Dis Sci 2010; 55: 2529-2536, doi: 10.1007/s10620-009-1073-0.
https://doi.org/10.1007/s10620-009-1073-... ,¹¹11. Luan ZG, Zhang H, Ma XC, Zhang C, Guo RX. Role of high-mobility group box 1 protein in the pathogenesis of intestinal barrier injury in rats with severe acute pancreatitis. Pancreas 2010; 39: 216-223, doi: 10.1097/MPA.0b013e3181bab5c5.
https://doi.org/10.1097/MPA.0b013e3181ba... . In SAP, HMGB1 has been reported to be involved in SIRS as a cytokine downstream of the inflammatory factors TNF-α and IL-1β ¹1. Yuan H, Jin X, Sun J, Li F, Feng Q, Zhang C, et al. Protective effect of HMGB1 a box on organ injury of acute pancreatitis in mice. Pancreas 2009; 38: 143-148, doi: 10.1097/MPA.0b013e31818166b4.
https://doi.org/10.1097/MPA.0b013e318181... . Furthermore, the serum HMGB1 level is significantly increased in patients with SAP, and the increase is correlated with disease severity ⁶6. Yasuda T, Ueda T, Takeyama Y, Shinzeki M, Sawa H, Nakajima T, et al. Significant increase of serum high-mobility group box chromosomal protein 1 levels in patients with severe acute pancreatitis. Pancreas 2006; 33: 359-363, doi: 10.1097/01.mpa.0000236741.15477.8b.
https://doi.org/10.1097/01.mpa.000023674... . Recently, HMGB1 has been shown to be a key mediator of inflammation and organ failure in SAP ¹²12. Luan ZG, Ma XC, Zhang H, Zhang C, Guo RX. Protective effect of ethyl pyruvate on pancreas injury in rats with severe acute pancreatitis. J Surg Res 2013; 181: 76-84..

Although SAP is characteristic of a serious pathogenic condition with high mortality, and numerous studies have emphasized the important role of proinflammatory cytokines in SAP-related conditions, such as SIRS and MODS, and even death, there have still been no breakthroughs in SAP treatment ¹³13. Felderbauer P, Muller C, Bulut K, Belyaev O, Schmitz F, Uhl W, et al. Pathophysiology and treatment of acute pancreatitis: new therapeutic targets - a ray of hope? Basic Clin Pharmacol Toxicol 2005; 97: 342-350, doi: 10.1111/j.1742-7843.2005.pto_274.x.
https://doi.org/10.1111/j.1742-7843.2005... . Growth hormone is produced by the anterior pituitary to stimulate cell proliferation and tissue regeneration in the gastrointestinal tract and pancreas ¹⁴14. Han X, Sosnowska D, Bonkowski EL, Denson LA. Growth hormone inhibits signal transducer and activator of transcription 3 activation and reduces disease activity in murine colitis. Gastroenterology 2005; 129: 185-203, doi: 10.1053/j.gastro.2005.05.018.
https://doi.org/10.1053/j.gastro.2005.05... . It is also able to modulate cytokine production by subpopulations of inflammatory cells ¹⁵15. Andiran N, Yordam N. TNF-alpha levels in children with growth hormone deficiency and the effect of long-term growth hormone replacement therapy. Growth Horm IGF Res 2007; 17: 149-153, doi: 10.1016/j.ghir.2007.01.002.
https://doi.org/10.1016/j.ghir.2007.01.0... . At present, growth hormone is thought to protect intestinal barrier integrity, and to stimulate production of insulin-like growth factor 1 (IGF-1), which can inhibit the development of acute pancreatitis ¹⁶16. Jaworek J, Leja-Szpak A, Dembinski A, Tomaszewska R, Szklarczyk J, Kot M, et al. Involvement of sensory nerves in the protective effect of growth hormone on acute pancreatitis. Growth Horm IGF Res 2009; 19: 517-522, doi: 10.1016/j.ghir.2009.06.001.
https://doi.org/10.1016/j.ghir.2009.06.0... .

Somatostatin, recently discovered as an endogenous neuropeptide, has multiple modulatory effects on the immune system. The distribution of specific somatostatin receptor might partially explain the heterogeneity of effects of somatostatin or its analogs on immunocytes ¹⁷17. Ferone D, van Hagen PM, Semino C, Dalm VA, Barreca A, Colao A, et al. Somatostatin receptor distribution and function in immune system. Dig Liver Dis 2004; 36 (Suppl 1): S68-S77, doi: 10.1016/j.dld.2003.11.020.
https://doi.org/10.1016/j.dld.2003.11.02... , which are responses to autocrine and paracrine modes of action. Moreover, somatostatin (or cortistatin) produced by activated cells may interact with other cells expressing the receptors ¹⁷17. Ferone D, van Hagen PM, Semino C, Dalm VA, Barreca A, Colao A, et al. Somatostatin receptor distribution and function in immune system. Dig Liver Dis 2004; 36 (Suppl 1): S68-S77, doi: 10.1016/j.dld.2003.11.020.
https://doi.org/10.1016/j.dld.2003.11.02... . A randomized study found somatostatin could alleviate the inflammatory response, up-regulate cell immune function, and improve the condition of patients with SAP ¹⁸18. Tang WF, Wan MH, Zhu L, Chen GY, Xia Q, Huang X. [Immuno-modulatory effect of somatostatin combined with traditional Chinese medicine on severe acute pancreatitis at early stage: a randomized control trial]. Zhong Xi Yi Jie He Xue Bao 2005; 3: 103-107, doi: 10.3736/jcim20050206.
https://doi.org/10.3736/jcim20050206... .

Therefore, the objective of this study was to examine whether HMGB1 is differentially expressed in SAP and to investigate the effects of growth hormone and somatostatin on the expression of HMGB1 and the severity of pancreas and ileum injury in an experimental SAP model.

Material and Methods

Chemicals and other reagents

Sodium taurocholate was obtained from Sigma Chemical Co. (USA). Dimethylformamide and paraformaldehyde were obtained from Sino-American Bio-Tech Inc. (China). The primers were synthesized by the Institute of Cell Biology, Shanghai Institutes for Biological Sciences (SIBS), and the Chinese Academy of Science. Growth hormone and somatostatin were purchased from Laboratoires Serono S.A. (Switzerland). Endotoxin detection kits were purchased from Shanghai Institute of Medical Chemistry (China), enzyme-linked immunosorbent assay (ELISA) kits from T&B Co. (USA), and Trizol was obtained from Invitrogen Inc. (USA). We prepared sodium taurocholate (3.5%).

Animal model and grouping

Male Sprague-Dawley rats weighing 280 to 320 g were obtained from the Experimental Animal Center of Chinese Academy of Science. Rats were housed in individual cages, and maintained at 20±2°C and a 12-h light/dark cycle for at least 1 week to acclimate to the surroundings. They were fasted overnight before experiments, but had free access to water. The procedure for animal surgery was performed in accordance with the guidelines of the Animal Care and Use Committee of Chinese Academy of Science, and every effort was made to minimize the number of animals used and their suffering.

The rats were randomly divided into six groups of 20 rats each: i) sham-operated group, not injected with anything; ii) SAP group, 3.5% sodium taurocholate injected into pancreatic duct ¹⁹19. Li ZD, Ma QY, Wang CA. Effect of resveratrol on pancreatic oxygen free radicals in rats with severe acute pancreatitis. World J Gastroenterol 2006; 12: 137-140. (2.5 mL/kg; injection pressure, 3.4-3.9 kPa); iii) SAP+NS group, after injection of 3.5% sodium taurocholate, rats were injected with an equal volume of normal saline (injection pressure, 1.3-1.4 kPa); iv) SAP+G group, 1 h after injection of 3.5% sodium taurocholate, 0.5 μg·kg⁻¹·d⁻¹ growth hormone (G) was injected under the skin; v) SAP+S group, 1 h after injection of 3.5% sodium taurocholate, 84 μg·kg⁻¹·d⁻¹ somatostatin (S) was injected by femoral vein puncture; vi) SAP+G+S group, 1 h after injection of 3.5% sodium taurocholate, 0.5 μg·kg⁻¹·d⁻¹ G was injected under the skin and 84 μg·kg⁻¹·d⁻¹ S was injected by femoral vein puncture. After being treated for 24 h, rats were sacrificed, and the pancreas and ileum were removed. A portion of the tissues was post-fixed in 4% paraformaldehyde for an additional 24 h, and then transferred to 75% alcohol for hematoxylin and eosin (HE) staining. A portion of the tissues was post-fixed in electron microscopy preservation solution for electron microscopic observation. The other tissues were stored at -20°C for reverse transcription-PCR (RT-PCR).

RNA extraction and RT-PCR analysis

Total RNA was extracted from fresh pancreas tissue of male SD rats with TRIZOL reagent (Invitrogen). Following the manufacturer's instructions, 500 U RNase inhibitor (RNAguard; Amersham Phamacia Biotech, USA) was added to the starting material, usually 500 mg of pancreas tissue. RNA was stored at -80°C until use. Reverse transcription reactions were performed as follows: 2 µL total RNA (1 µg/µL), 4 µL 5× buffer, 2 µL Oligo dT, 1 µL, RNasin (40 U/µL), 1 µL dNTP mix, 1 µL reverse transcriptase (200 U/µL). The total volume was 20 µL with the addition of 9 µL diethyl pyrocarbonate (DEPC)-treated distilled water. The procedure included: 42°C, 1 h; 95°C, 10 min. The cDNA was stored at -20°C until use. The primers used in this study were HMGB-1 forward5′-GAGATCCTAAGAAGCCGAGA-3′, reverse5′-CTTCCTCATCCTCTTCATCC-3′; GAPDH, forward5′-CTCAAGATTGTCAGCAATGC-3′, reverse5′-CAGGATGCCCTTTAGTGGGC-3′.

The PCR reaction performed to assay the expression of HMGB1 was carried out as follows: 1 µL cDNA, 2 µL primers, 10 µL 2× Tag PCR Master Mix, and 7 µL 0.1% DEPC-water. The PCR procedure included 95°C for 5 min; 35 cycles of 95°C for 1 min, 58°C for 30 s, 72°C for 30 s and 72°C for 10 min. PCR products were separated by 2% agarose gel electrophoresis and visualized under ultraviolet light by ethidium bromide staining.

Histological score of injured pancreas

To directly observe the histological changes of tissues in each group, small pieces of pancreas tissues were harvested, fixed immediately in 4% phosphate buffered formalin for 24 h, and then immersed in 75% ethanol. After paraffin embedding, 5-μm transverse sections were prepared and stained with HE for morphological observation. The images were recorded using an Olympus microscope (Japan).

Pancreatic tissues of 8 rats from each group were examined histologically under a light microscope; 20 random fields per slide were scored for edema, acinar necrosis, hemorrhage, and inflammation by an experienced pathologist according to Schmidt's standard method ²⁰20. Schmidt J, Rattner DW, Lewandrowski K, Compton CC, Mandavilli U, Knoefel WT, et al. A better model of acute pancreatitis for evaluating therapy. Ann Surg 1992; 215: 44-56, doi: 10.1097/00000658-199201000-00007.
https://doi.org/10.1097/00000658-1992010... . The scoring criteria of pancreatic pathology are shown in Table 1.

Measurement of cytokines and endotoxin

After being treated for 24 h, the blood of SAP rats in each group was collected and stored at -20°C until analysis. The levels of IL-1β, IL-6 and TNF-α were measured by ELISA. Afterwards, endotoxin was measured using a chromogenic tripeptide assay.

Statistical analysis

Results are reported as means±SD. ANOVA was performed with the SPSS Windows 13.0 statistical analysis software (USA). P<0.05 was considered to be significant. All experiments were replicated, with representative data shown.

Results

Histological changes in pancreas tissues

The histological scores of injured pancreas were obtained by blinded observers and indicated no obvious morphological changes (Figure 1C), necrosis, or hemorrhage in the sham-operated group. However, necrosis and hemorrhage were observed in the SAP group (Figure 1A). The interstitial pancreatic tissue was significantly wider and the lobular gap was increased. Infiltration of inflammatory cells and focal or patchy necrosis of pancreatic tissue were obvious by light microscopy, and the histological scores were significantly different from those of tissues from the sham-operated group (P<0.01, Table 2). In the SAP+G+S group (Figure 1D), edema and inflammatory cell infiltration were found occasionally. In the SAP+G and SAP+S groups (Figure 1E and F), inflammatory changes were still visible; the inflammatory response was decreased, and the resulting histological scores were changed significantly compared with those in SAP group (P<0.05, see Table 2 andFigure 1).

Histological changes in ileum tissues

After HE staining, histological changes in ileum tissues as seen by light microscopy are shown in Figure 2. In the SAP group, interstitial congestion and edema, mild inflammatory cell infiltration, and mild interstitial hemorrhage were observed (Figure 2A). Villi were edematous; greatly shortened, disordered, and occasional necrotic villi were seen along with thinning of the mucosa (Figure 2B). In the SAP+G group (Figure 2C), cells in the epithelium of villi appeared to be proliferating actively, the mucosa was thickened, and the inflammatory response was still visible. In the SAP+S group (Figure 2D), inflammatory changes were decreased. Many lymphoid nodules, epithelial hyperplasia and germinal centers were found, but the degree of activity was significantly lower than that in the SAP+G group. In the SAP+G+S group (Figure 2E-H), the intestinal inflammatory response was decreased; villi were distorted, elongated and branched; epithelial cells were highly columnar; goblet cells were increased in number; and nuclei overlapped significantly.

Histological changes in ileum tissues as seen by electron microscopy are shown inFigure 3. In the sham-operated group (Figure 3A), the cells were dense, microvilli were orderly and arranged compactly, and organelles retained their integrity. In the SAP group (Figure 3B), cells were obviously swollen, intracellular particles were dispersed, organelles were damaged, and microvilli were disarranged. There were also many necrotic cells with partial membrane damage. While in the SAP+G group (Figure 3C), the normal form and structure of most cells was maintained, microvilli were slightly enlarged, and intercellular junctions were intact. In the SAP+S group (Figure 3D), intracellular matter was slightly dispersed and organelles were still intact. In the SAP+S+G group (Figure 3E), the arrangement of microvilli was orderly, structural integrity was maintained, and organelle structures were also complete and clearly visible.

Measurements of villus height and mucosal thickness (Table 3) revealed that in the SAP group, the height of villi was significantly reduced, and the intestinal wall had become significantly thinner than in the sham-operated group (both P<0.05). The degree of villus shortening in the SAP+G, SAP+S, and SAP+G+S groups was less than in the SAP group (P<0.05). In particular, the villi in the SAP+G+S group were closer in height to that in the sham-operated group.

HMGB-1 expression in pancreatic tissues

RT-PCR revealed mRNA amplification products of HMGB-1 of 595 bp, and GAPDH mRNA amplification products of 394 bp (Figure 4). In addition, after being treated for 24 h, the mRNA expression of HMGB-1 was detected in every group. Significant differences existed between the SAP and sham-operated groups (P<0.05) as well as the SAP+NS and sham-operated groups (P<0.05). Compared with the SAP group, the expression of HMGB-1 was significantly down-regulated in the SAP+G+S group (P<0.05); but no obvious difference existed between the SAP+G+S and sham-operated groups.

Endotoxin, IL-1β, IL-6, and TNF-α levels

The endotoxin levels in SAP and SAP+NS groups were significantly higher than in the sham-operated group (P<0.05, Figure 5). Additionally, in the SAP+S, SAP+G, and SAP+G+S groups, the endotoxin levels were significantly lower than in the SAP or SAP+NS groups (P<0.05), but no significant difference was observed between the SAP+G+S and sham-operated groups.

The IL-1β, IL-6, and TNF-α levels in these groups are reported inFigure 6. The levels of IL-1β, IL-6, and TNF-α in the SAP and SAP+NS groups were significantly higher than those in the sham-operated group (P<0.05). Furthermore, the IL-1β, IL-6, and TNF-α levels in the SAP+S, SAP+G and SAP+G+S groups were significantly lower than those in the SAP and SAP+NS groups (P<0.05).

Comparison of survival rates in rats

Kaplan-Meier analysis was used to compare the survival rates in each group over a period of 7 days (Figure 7). The 7-day survival rate was 100% in the sham-operated group, while in the SAP group, six rats had died at 24 h, and all rats were dead at 48 h giving a 7-day survival rate of 0%. In the SAP+NS group, the 7-day survival rate was also 0%. The differences in survival rates observed in both SAP and SAP+NS groups compared with that in the sham-operated group were significant (P<0.01). In the SAP+S, SAP+G, and SAP+G+S groups, the 7-day survival rates were 60, 30, and 90%, respectively. The differences in survival rate observed in SAP+S, SAP+G, and SAP+G+S groups compared with that in the SAP group, especially in the SAP+S+G group, were all significant (P<0.05).

Discussion

SAP is a serious, potentially life-threatening type of acute pancreatitis that follows a severe course in approximately 25% of patients, with local and/or systemic complications leading to a high mortality rate ²¹21. Piascik M, Rydzewska G, Milewski J, Olszewski S, Furmanek M, Walecki J, et al. The results of severe acute pancreatitis treatment with continuous regional arterial infusion of protease inhibitor and antibiotic: a randomized controlled study. Pancreas 2010; 39: 863-867, doi: 10.1097/MPA.0b013e3181d37239.
https://doi.org/10.1097/MPA.0b013e3181d3... . The complications occurring in SAP may be both local (pancreatic necrosis and pseudocyst) and systemic (organ dysfunction, shock, and acute respiratory distress syndrome) ²²22. Su MS, Lin MH, Zhao QH, Liu ZW, He L, Jia N. Clinical study of distribution and drug resistance of pathogens in patients with severe acute pancreatitis. Chin Med J 2012; 125: 1772-1776..

During SAP, increasing permeability of gastrointestinal mucosa occurs in its early phase and facilitates bacterial/endotoxin translocation ²³23. Ammori BJ, Leeder PC, King RF, Barclay GR, Martin IG, Larvin M, et al. Early increase in intestinal permeability in patients with severe acute pancreatitis: correlation with endotoxemia, organ failure, and mortality. J Gastrointest Surg 1999; 3: 252-262, doi: 10.1016/S1091-255X(99)80067-5.
https://doi.org/10.1016/S1091-255X(99)80... . Normally, the gastrointestinal tract absorbs nutrients and acts as a barrier to exclude luminal bacteria from the internal environment ²⁴24. Braga M, Gianotti L, Gentilini O, Liotta S, Di Carlo V. Feeding the gut early after digestive surgery: results of a nine-year experience. Clin Nutr 2002; 21: 59-65, doi: 10.1054/clnu.2001.0504.
https://doi.org/10.1054/clnu.2001.0504... . However, SAP-induced increase of intestinal permeability, which promotes bacterial and endotoxin translocation, leads to sepsis and MODS ²⁵25. Juvonen PO, Alhava EM, Takala JA. Gut permeability in patients with acute pancreatitis. Scand J Gastroenterol 2000; 35: 1314-1318.. It has been suggested that stabilization of intestinal integrity can reduce pancreatic infections in experimental and clinical pancreatitis ²⁶26. Wang X, Wang B, Wu J, Wang G. Beneficial effects of growth hormone on bacterial translocation during the course of acute necrotizing pancreatitis in rats. Pancreas 2001; 23: 148-156, doi: 10.1097/00006676-200108000-00005.
https://doi.org/10.1097/00006676-2001080... .

In this study, the rats with SAP showed an increased inflammatory response of the ileum compared with the sham-operated group, and the subsequent endotoxin level was significantly elevated in our experimental SAP model. In fact, extensive inflammatory cell infiltration and focal or patchy necrosis of pancreas existed in the SAP model ²⁷27. Van Laethem JL, Marchant A, Delvaux A, Goldman M, Robberecht P, Velu T, et al. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology 1995; 108: 1917-1922, doi: 10.1016/0016-5085(95)90158-2.
https://doi.org/10.1016/0016-5085(95)901... , and inflammatory cell infiltration is known to facilitate the release of endotoxin ²³23. Ammori BJ, Leeder PC, King RF, Barclay GR, Martin IG, Larvin M, et al. Early increase in intestinal permeability in patients with severe acute pancreatitis: correlation with endotoxemia, organ failure, and mortality. J Gastrointest Surg 1999; 3: 252-262, doi: 10.1016/S1091-255X(99)80067-5.
https://doi.org/10.1016/S1091-255X(99)80... . This is consistent with the observed increase in inflammatory cell infiltration and up-regulated endotoxin level.

In addition, the humoral mediators released by excessive activation of macrophages/monocytes and neutrophils have been reported to lead to remote organ injury ²⁸28. Sawa H, Ueda T, Takeyama Y, Yasuda T, Shinzeki M, Nakajima T, et al. Blockade of high mobility group box-1 protein attenuates experimental severe acute pancreatitis. World J Gastroenterol 2006; 12: 7666-7670.. Here, the expression of HMGB1 and the levels of inflammatory cytokines in serum, including TNF-α, IL-1β, and IL-6, were increased significantly in the pancreas of rats with SAP compared with those in sham-operated rats. HMGB1 was recently identified as a late mediator of lethal systemic inflammation ²⁹29. Wang H, Yang H, Czura CJ, Sama AE, Tracey KJ. HMGB1 as a late mediator of lethal systemic inflammation. Am J Respir Crit Care Med 2001; 164: 1768-1773., which contributes to its potential as a target for the development of anti-inflammatory treatments. In addition, HMGB1 has been shown to be a downstream cytokine of early-phase inflammatory cytokines such as TNF and IL-1, which are involved in SAP-associated SIRS ¹1. Yuan H, Jin X, Sun J, Li F, Feng Q, Zhang C, et al. Protective effect of HMGB1 a box on organ injury of acute pancreatitis in mice. Pancreas 2009; 38: 143-148, doi: 10.1097/MPA.0b013e31818166b4.
https://doi.org/10.1097/MPA.0b013e318181... . Therefore, it is likely that HMGB1 is produced and released by damaged organs in SAP. Another study also found that the modulation of HMGB1 and other inflammatory cytokine responses was associated with a reduction in the severity of SAP by ethyl pyruvate, which may play a therapeutic role in liver inflammation in this SAP model ³⁰30. Luan ZG, Zhang H, Ma XC, Zhang C, Guo RX. Therapeutic treatment with ethyl pyruvate attenuates the severity of liver injury in rats with severe acute pancreatitis. Pancreas 2012; 41: 729-737.. Furthermore, in acute pancreatitis, proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, are produced within the pancreas and subsequently within distant organs, and the severity of acute pancreatitis is well correlated with the levels of these cytokines ³¹31. Norman JG, Fink GW, Denham W, Yang J, Carter G, Sexton C, et al. Tissue-specific cytokine production during experimental acute pancreatitis. A probable mechanism for distant organ dysfunction. Dig Dis Sci 1997; 42: 1783-1788, doi: 10.1023/A:1018886120711.
https://doi.org/10.1023/A:1018886120711... ,³²32. Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg 1998; 175: 76-83.. That evidence is consistent with our results, suggesting that overexpression of HMGB1, TNF-α, IL-1β, and IL-6 may be related to the intestinal damage occurring in experimental SAP, and these gene products could be potential markers to diagnose the progress of SAP.

Moreover, effects of growth hormone and somatostatin on SAP were detected in this study. Administration of growth hormone and somatostatin to the rats with SAP resulted in significant reductions of HMGB1, IL-1β, IL-6, TNF-α and endotoxin levels as well as in the alleviation of histological changes. In previous investigations, growth hormone has been reported ¹⁵15. Andiran N, Yordam N. TNF-alpha levels in children with growth hormone deficiency and the effect of long-term growth hormone replacement therapy. Growth Horm IGF Res 2007; 17: 149-153, doi: 10.1016/j.ghir.2007.01.002.
https://doi.org/10.1016/j.ghir.2007.01.0... ,²⁶26. Wang X, Wang B, Wu J, Wang G. Beneficial effects of growth hormone on bacterial translocation during the course of acute necrotizing pancreatitis in rats. Pancreas 2001; 23: 148-156, doi: 10.1097/00006676-200108000-00005.
https://doi.org/10.1097/00006676-2001080... to alleviate acute necrotizing pancreatitis (taurocholate model) in rats, and the mechanism of the protective function of growth hormone in acute pancreatitis might be related to the improvement of intestinal barrier permeability through down-regulation of apoptosis in intestinal cells ³³33. Wang X, Wang B, Wu K, Xu M, Gong Z. Growth hormone downregulated the excessive apoptosis of ileal intestinal epithelial cells in rats during the early course of acute necrotizing pancreatitis. Pancreas 2002; 25: 205-209, doi: 10.1097/00006676-200208000-00016.
https://doi.org/10.1097/00006676-2002080... . Our results are in agreement with this in demonstrating that treatment with growth hormone can inhibit the release of TNF-α in SAP rats and humans to some extent ¹⁵15. Andiran N, Yordam N. TNF-alpha levels in children with growth hormone deficiency and the effect of long-term growth hormone replacement therapy. Growth Horm IGF Res 2007; 17: 149-153, doi: 10.1016/j.ghir.2007.01.002.
https://doi.org/10.1016/j.ghir.2007.01.0... ,³⁴34. An Y, Xiao YB. Growth hormone prevents acute liver injury induced by cardiopulmonary bypass in a rat model. J Thorac Cardiovasc Surg 2007; 134: 342-350, doi: 10.1016/j.jtcvs.2007.02.036.
https://doi.org/10.1016/j.jtcvs.2007.02.... . Additionally, in this study, both growth hormone and somatostatin reduced the mortality of SAP, which is in line with previous studies showing that the mortality and local complications of acute pancreatitis ³⁵35. Choi TK, Mok F, Zhan WH, Fan ST, Lai EC, Wong J. Somatostatin in the treatment of acute pancreatitis: a prospective randomised controlled trial. Gut 1989; 30: 223-227, doi: 10.1136/gut.30.2.223.
https://doi.org/10.1136/gut.30.2.223... declined after treatment with somatostatin ³⁶36. Buchler MW, Binder M, Friess H. Role of somatostatin and its analogues in the treatment of acute and chronic pancreatitis. Gut 1994; 35: S15-S19, doi: 10.1136/gut.35.3_Suppl.S15.
https://doi.org/10.1136/gut.35.3_Suppl.S... . Somatostatin treatment has also been associated with a slight reduction in the need for surgery due to local complications ³⁷37. Planas M, Perez A, Iglesia R, Porta I, Masclans JR, Bermejo B. Severe acute pancreatitis: treatment with somatostatin. Intensive Care Med 1998; 24: 37-39, doi: 10.1007/s001340050512.
https://doi.org/10.1007/s001340050512... . Meanwhile, in this study, the effects of somatostatin in SAP rats were similar to growth hormone. Both attenuated levels of HMGB1, IL-1β, IL-6, TNF-α, and endotoxin, as well as the inflammatory response in pancreas and ileum tissue. Mortality of SAP rats was also lower in those treated with growth hormone and somatostatin. However, an inhibitory effect of somatostatin on growth hormone release has been reported by Arimura et al. ³⁸38. Arimura A, Sato H, Dupont A, Nishi N, Schally AV. Somatostatin: abundance of immunoreactive hormone in rat stomach and pancreas. Science 1975; 189: 1007-1009, doi: 10.1126/science.56779.
https://doi.org/10.1126/science.56779... . Hence, the clinical effects of growth hormone and somatostatin on SAP need to be further confirmed. Their specific functions in SAP, and whether they are related to a synergistic effect require further examination.

In conclusion, this study showed that both growth hormone and somatostatin could attenuate the inflammatory response in pancreas and ileum tissues in SAP model rats, and significantly decrease HMGB1, TNF-α, IL-1β, IL-6 and endotoxin levels. Moreover, the improvement of G+S on the severity of acute pancreatitis and the survival rate of SAP rats was greater than only treating with growth hormone or somatostatin. However, further studies should be performed to evaluate protein and plasma levels of HGMB1 to verify the protective effects of growth hormone and somatostatin on SAP, elucidate the role of HMGB1 in SAP, and explore whether the combination of growth hormone and somatostatin can improve the survival rate of SAP patients clinically. Moreover, the possible mechanisms of growth hormone and somatostatin on SAP need to be further investigated.

Acknowledgements

Research supported by a grant from the Science and Technology Commission of Shanghai Municipality (#11nm0503900).

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