The Effect of Emodin-Assisted Early Enteral Nutrition on Severe Acute Pancreatitis and Secondary Hepatic Injury

Severe acute pancreatitis (SAP) characterized by atrocious progression and numerous complications often leads to a high mortality rate due to hypermetabolism, systemic inflammatory response syndrome (SIRS), and multiple organs dysfunction syndrome (MODS). Studies have revealed that both early enteral nutrition (EEN) and emodin are potent agents in the management of SAP. However, whether the combined strategy is rational and more effective than either one alone remains unknown. In this regard, Wistar rats were treated with emodin-assisted EEN (EAEEN) through enteral nutrient tubes after induction of SAP by retrograde infusion of 5.0% sodium taurocholate into the common pancreatic duct. Serum levels of amylase, tumor necrosis factor-alpha (TNF-α), angiotensin II (AngII), maleic dialdehyde (MDA), glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and C-reactive protein (CRP), intestinal secretory IgA (SIgA), pancreatic and hepatic myeloperoxidase (MPO) activity as well as plasma levels of D-lactate and endotoxin were measured. In addition, pathologic alterations of pancreas and liver were observed microscopically. We found that EAEEN could significantly ameliorate these parameters and prevent pancreas and liver from serious damage. In conclusion, Our results indicated that EAEEN could exert beneficial effects on experimental SAP and obviously abate the severity of secondary hepatic injury. The combined strategy was safe and more effective than either one alone in the acute stage of SAP. This study also provided an experimental base for the clinical treatment of SAP patients with EAEEN.


INTRODUCTION
Severe acute pancreatitis (SAP), characterized by atrocious progression, multicomplications, and difficult treatment [1][2][3] is an acute abdomen that ranges from a mild illness to a life-threatening condition. Clinically, the overall mortality rate for SAP is approximately 38.4% [4]. In spite of decades of intensive study, we are still in lack of etiological therapy. SAP causes such a hypermeolic state that adequate supply of nutrients plays a decisive role to reverse systemic malnutrition, potentiate resistance against infection, block the pathological deterioration, and facilitate recovery [5][6][7]. In the past, it was believed that early enteral nutrition (EEN) in patients with SAP might exacerbate the clinical as well as pathological conditions and provoke serious complications. However, therapeutic strategies have changed dramatically over the past 20 years. The high mortality rate of SAP is often a result of multiple organ dysfunction syndrome (MODS) and appears to be especially related to systemic inflammatory response syndrome (SIRS) or even to infections of the necrotic pancreas. Since MODS and SIRS are apt to be facilitated with gut mucosal dysfunction and abundant Gram-negative bacteria as well as other gastrointestinal pathogens are commonly detected in pancreatic infections, the gut is considered to be the main source of SAP-related septic complications, so that maintaining gut barrier is equally as important as resting the pancreas when the inflammation within the gland resolves.
Though total parenteral nutrition (TPN) has been the standard practice for providing exogenous nutrients during the early stage of SAP, long-term administration of TPN can produce catheter-related complications, weaken systemic immunity, and induce secondary enterogenous infection. In comparison to TPN, EEN helps enhance the immunological and mechanical functions of intestinal mucosa, maintain the balance of gut microflora, reduce oxidative stress, prevent secondary infections, and improve therapeutic results. Emodin, the main effective component of rhubarb, which is a traditional Chinese herbal medicine and has the function of purgation, heat-clearing, and detoxicating as well as promoting blood circulation to remove blood stasis, has proved to be a beneficial pharmacy for SAP [8][9][10][11][12]. Because of the liver's special position in the gut circulation and the unsubstitutable role played in body metabolism, it becomes a major target for extrapancreatic damage of SAP. And liver injury, in turn, deteriorates SAP and amplifies the systemic damage caused by MODS. Therefore, effective prevention of liver injury is a key to shortening the course of SAP and decreasing the related mortality.
EEN and emodin have been fully affirmed by most scholars as an important part of therapy for SAP. However, it is still controversial what substance and dosage in EEN and what timing and route of administration of EEN associated with emodin are more appropriate and whether the combined treatment is more effective than either one alone. In the present experiment, we tried to explore possible mechanisms of EEN and emodin in the treatment of SAP as well as its secondary hepatic injury, and to demonstrate the feasibility and effectiveness of emodin-assisted early enteral nutrition (EAEEN).

Animal models
Sixty male Wistar rats, housed in cages with a controlled temperature of 26 • C and 12-hour light-dark cycles, were fed standard laboratory chow as well as water ad libitum, and allowed to acclimatize for at least a week. The rats were fasted overnight with free access to water before experiments. Surgical anesthesia was performed by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg body weight). SAP was induced by retrograde infusion of 5.0% sodium taurocholate (0.15 mL/100 g body weight) into the common pancreatic duct [13]. The "out" end of the enteral nutrient tube was placed at 7 cm from the far point of Treitz ligament, while the "in" end was dragged backward through the body wall and fixed on the back. The experimental protocol was undertaken in accordance with the guideline for the care and use of laboratory animals in research and was approved by the Ethical and Research Committee of the First Clinical Hospital, Harbin Medical University.

Experimental groups
Sixty rats were randomly divided into four groups (n = 15) after SAP induction: control group (group A), emodin group (group B), EEN group (group C), and EAEEN group (group D). Control group was allowed free access to water without any intervention; Emodin group received emodin (3.0 mg/100 g body weight) through enteral nutrient tube at 12  The remaining living animals in each group were reanesthetized 72 hours after SAP induction using intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg body weight). Laparotomies were performed and samples of blood and tissues were immediately obtained.

Histopathologic examination
A portion of the pancreatic tail and hepatic and ileal tissues from each rat was incised and the pancreatic as well as hepatic tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (HE) for light microscopy. Two experienced pathologists who were blinded to the experimental protocol scored the pancreatic tissue on a scale from 0 to 4 for the degrees of edema, inflammation, hemorrhage, and necrosis in 20 fields. We applied the scoring system defined by Kusske et al. [14] and the scores of each histologic examination were totaled. The ileal tissue was observed to measure the villus height of ileum microscopically.

Secretory IgA (SIgA), myeloperoxidase (MPO) activity, and ascitic fluid
The jejunum and ileum were excised and the intestinal mucus was collected to measure the level of SIgA by radio immunoassay with the kit (Institute of Atomic Energy Physics, Chinese Academy of Sciences, China). The values of MPO activity in pancreas and liver were measured to assess polymorphonuclear leukocyte (PMN) infiltration. A portion of the pancreatic and hepatic tissues was obtained to determine pancreatic and hepatic MPO activity by chromatometry with the kit (Invitrogen, Calif, USA) and ascitic fluid was quantified.

Edema
After removal, fresh samples of pancreas and ileum were immediately weighed and dried at 100 • C for 24 hours and reweighed. The wet-dry weight ratio of pancreas (pww/dw) and ileum (iww/dw) were calculated to determine the degrees of pancreatic edema and ileal edema.
Gang Wang et al.

Laboratory test
Blood samples were collected from abdominal aorta and centrifuged at 3000 rpm for 5 minutes. The serum was captured and stored at −20 • C until assayed. Tumor necrosis factoralpha (TNF-α) and angiotensin II (AngII) were measured by radio immunoassay with the kits (Institute of Atomic Energy Physics, Chinese Academy of Sciences, China). Maleic dialdehyde (MDA), C-reactive protein (CRP) (Jiancheng Biotech Ltd., Nanjing, China), and plasma D-lactate (Sigma, USA) were assessed by enzymatic-spectrophotometric method. Plasma endotoxin (Shanghai Med & Chem Institute, Shanghai, China) was tested by chromogenic limulus amebocyte lysate technique. Glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), and amylase were determined by automated HITACHI-7150 analyzer.

Statistical analysis
Results were expressed as mean ± standard deviation (SD). The significance of differences in histopathologic scores was assessed by Kruskal-Wallis test. Other continuous data were analyzed by factorial design ANOVA test. Statistical analysis was finished by SPSS10.0 statistical program and difference was considered statistically significant when P < .05.

RESULTS
Before sampling 72 hours after the induction of SAP, the ratio of survival to death was 7 to 8 in control group, 9 to 6 in emodin group, 8 to 7 in EEN group, and 12 to 3 in EAEEN group. Compared with control group, there were significant differences in the following data as described in emodin, EEN, and EAEEN groups (P < .05, Tables 1-4, Figures 1-5).

Macroscopic findings
In group A, severe edema, diffuse hemorrhage, and necrosis in pancreas were observed. The gastrointestinal tract was obviously ectatic and edematous. There was much bloody ascites in the peritoneal cavity. Compared with group A, the severity of macroscopic changes was significantly abated in groups B, C, and D.

Laboratory test
In contrast to emodin and EEN, EAEEN significantly reduced the levels of TNF-α, AngII, MDA, ALT, AST, CRP, and amylase in serum as well as the levels of plasma endotoxin and D-lactate (P < .05, Tables 1, 2, and 4, Figures 1 and 5). Differences in these data were not considered statistically significant between group B and group C.

Edema, SIgA, MPO, and ascitic fluid
The levels of MPO in pancreas and liver, pww/dw, iww/dw, and the amount of ascitic fluid were significantly lower, while the level of SIgA was obviously higher in group D than those in group B and group C (P < .05, Tables 2, 3,  2-4). However, no significant differences were found in these data between group B and group C.

Histopathologic examination
The degrees of edema, inflammatory infiltration, hemorrhage, and necrosis and the scores of histopathologic alterations in pancreas were significantly reduced in group D as compared with those in group B and group C (P < .05, Table 3, Figures 6-9). EAEEN could also attenuate the severity of secondary liver injury obviously (Figures 10-14). The villus height of ileum was significantly increased by EAEEN compared with emodin and EEN (P < .05, Table 3). There were no significant differences in the scores of pancreatic  histopathologic alterations as well as the ileal villus height between group B and group C.

DISCUSSION
Emodin characterized by the main active monomer of rhubarb is a derivative of anthraquinone (3-methyl-1, 6, 8trihydroxyanthraquinone). Studies have proved that emodin is a potent agent in the management of clinical and experimental acute pancreatitis (AP) [8]. Zhang et al. [9], and Wu et al. [12] found emodin had significant therapeutic effects on SAP rats by correcting intestinal flora disturbances, promoting intestinal peristalsis, inhibiting inflammatory cytokine release and pancreatin activity, and scavenging oxygen free radicals (OFR). Wu et al. [10] proved the mechanisms of emodin in the treatment of SAP included modulation of abnormal eicosanoid metabolism, promotion of pancreatic cy- toprotection, and improvement of pancreatic microcirculation. Kumar et al. [11] found emodin was a potent inhibitor of nuclear factor-kappaB (NF-κB) activation as well as expression of adhesion molecules and could be useful in treating various inflammatory diseases. In a word, emodin has multiple beneficial effects on SAP. In our experiment, administration of EAEEN could significantly abate the severity of SAP as compared with other groups, which was reflected by the reductions in levels of serum amylase, the amount of ascitic fluid, pancreatic MPO activity, pww/dw, and the pathologic scores of pancreas. Our results indicated that the advantages of EAEEN for the treatment of SAP existed in (1) EEN alone could enhance gut immunity, promote hyperplasia of intestinal mucosa, improve Gang Wang et al.    A vicious cycle of pancreatic microcirculatory changes such as capillary stasis and vasoconstriction has been shown to occur in the course of SAP [15]. Renin-angiotensin system (RAS) is an important stress system in pancreatic microcirculatory disturbances. RAS in systemic circulation is activated to a great extent and produces massive angiotensinII (AngII), which subsequently combines with its receptors predominantly localized to the epithelia of pancreatic ducts, vascular endothelia, and pancreatic acinar cells [16], results in the strong vasoconstriction of pancreas, and aggravates the pancreatic ischemia and necrosis in SAP. In this study, our results proved that administration of EAEEN enhanced colloid osmotic pressure, prevented excessive peripancreatic and retroperitoneal fluid exudation followed by hypovolemic shock, and inhibited the activation of pancreatic enzymes [9,12] so as to eliminate the main factors that induced the abnormal activation of RAS, further significantly reduced the level of serum AngII and ameliorated pancreatic microcirculatory disturbances.  Gut barrier dysfunction and intestinal immunosuppression are directly related to the severity of SAP, so that how to protect the intestinal mucosa from being damaged might play a key role in preventing bacterial and toxin translocation and deterioration of SAP. D-lactate is the indigenous metabolic product of intestinal resident flora. Normally, the level of blood D-lactate is quite low and the measurement of the increased plasma D-lactate level may be a useful marker to assess the intestinal injury [17]. SIgA, the predominant immunoglobulin present in mucosal secretions, is the major ingredient of the intestinal immunological function on mucosal surfaces. In this study, the levels of intestinal SIgA, plasma D-lactate, the ileal villus height, and iww/dw showed obvious differences in EAEEN group as compared with those in other groups. Our data demonstrated that administration of EEN associated with emodin could significantly attenuate the severity of gut damage and enhance the intestinal immunity by restoring the microecology of the intestinal flora, maintaining the intestinal epithelia integrity, abating edema of the intestinal wall, and promoting the secretion of intestinal immune substances so as to reduce the incidences of enterogenous infection and endotoxemia.

5
In SAP, bacterial and endotoxin translocation through the damaged gut barrier is the culprit for the development  of secondary septic complications and a second peak of mortality. Endotoxin is the lipopolysaccharide (LPS) present in the Gram-negative bacterial wall. Abundant plasma endotoxin intensively remotivates mononuclear-macrophage system and accelerates the production of many cytokines as well as inflammatory mediators, which induce septic shock and amplify SIRS followed by the development of MODS after releasing into the blood. Atrociously amplified SIRS is a hallmark for SAP with elevated circulating proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1. TNF-α is the most prominent "first-line" cytokine in SIRS and plays a central role in the pathogenesis of AP and related systemic complications [18]. TNF-α can induce infiltration and activation of neutrophils, upregulate cellular adhesion molecules, promote the secretion of OFR, initiate and aggravate the cascade of other proinflammatory cytokines and inflammatory mediators. MPO, a member of peroxidases secreted by neutrophils, is an indicator for the count and activity of neutrophils.
In our study, administration of EAEEN significantly reduced the levels of plasma endotoxin, serum TNF-α, and MPO in pancreatic tissue, so as to inhibit both local and systemic inflammatory reaction as well as oxidizing reaction and abate the severity of pancreatic damage. We enumerated the  following explanations as the rationales: (1) emodin could inhibit the overgrowth of pathogenic organisms including Gram-negative bacteria in the intestinal tract [9] and accelerate their excretion by promoting intestinal peristalsis, so that the direct source of endotoxin was eradicated and the level of plasma endotoxin depressed; (2) EEN could potentiate plasma protein synthesis, increase the blood perfusion in portal system, promote hepatoenteral circulation, and enhance hepatic detoxification, which in turn greatly reduced the level of plasma endotoxin; (3) EEN improved the gut barrier function and maintained the balance of gut microflora so as to decrease the production of endotoxin and to prevent it from entering the blood, which consequently abated the remotivation of mononuclear-macrophage system, so that the releases of TNF-α and other cytokines were reduced; (4) emodin inhibited the activation of pancreatic enzyme [9] so as to prevent mononuclear-macrophage system, activated by pancreatic enzyme after its entering the plasma, from producing TNF-α; (5) AngII is capable of promoting the releases of proinflammatory cytokines and stimulating the migration and infiltration of monocytes and neutrophils [15,19,20]. Administration of EAEEN significantly reduced the level of serum AngII, which further decreased the concentration of serum TNF-α and pancreatic MPO activity so as to attenu-  ate the severity of inflammation in pancreas; (6) TNF-α induces aggregation and activation of neutrophils in the pancreas. Administration of EAEEN could significantly reduce blood TNF-α level, which consequently reversed this process and abated the pancreatic inflammatory reaction.
Secondary liver injury (SLI) has a remarkable incidence in SAP, and the severity of hepatocyte injury is proportional to the severity of SAP. Several mechanisms are involved in the initiation of SLI: (1) in the acute stage, massive proinflammatory factors excessively activate the liver Kupffer cells, which then intensely release even more profactors, so that a cascade amplifying cycle inside and outside the liver is formed, bringing heavy inflammatory injury to the liver; (2) TNF-α and endotoxin induce massive neutrophils infiltration and activation, producing great amount of OFRs, which initiate intensive lipid peroxidations aggravating liver injury; (3) intestinal mucosal damage leads to a remarkable increase of endotoxin level in the portal circulation bringing enormous damage to the liver. Sameshima et al. [21] found that LPS could significantly elevate serum TNF-α activity and induce more severe liver damage in AP rats, which suggested that both LPS and TNF-α played critical roles in the development of secondary hepatic injury during AP. Tadao et al. [22] found that activated neutrophils and macrophages produced large 8 Mediators of Inflammation amount of OFRs in SAP and they were closely involved in the secondary liver disease. MDA is the product of lipid peroxidation of OFR, which indicates the amount of OFR and indirectly reflects the severity of tissue damage. CRP is an acute phase reactive protein produced by the liver especially in the context of SAP. The increase of serum CRP is proportional to the severity of hepatocyte injury. Our experiment demonstrated that EAEEN could remarkably decrease TNFα, MDA, and endotoxin levels in the blood and MPO activity in the liver, so that protect the liver from the tremendous inflammatory and oxidizing injuries brought upon by SAP, as manifested by the decreases of ALT, AST, and CRP levels in the blood.
In a word, administration of emodin prior to EEN formed a firm base for subsequent EEN therapy of SAP by clearing bacteria and toxin, promoting exsufflation and defecation, abating abdominal distension and systemic inflammatory reaction, so that the enteral nutrient solution could be fully absorbed. Furthermore, EEN could "compensate for" the massive loss of nutrients caused by administration of emodin, so they two could benefit each other in combination and obviously improve the therapeutic efficacy. In this experiment, administration of emodin and EEN in the early stage of SAP was safe and rational, and the combined strategy was more effective than either one alone. Our results showed that administration of EAEEN could control multiple deteriorating factors of SAP and provided an effective treatment for SAP and its secondary hepatic injury by protecting intestinal mucosal barrier, enhancing body immune competence, inhibiting systemic inflammatory reaction and reversing microcirculatory dysfunction, and so forth. This study suggested a broad potential value for EAEEN in the clinical treatment of SAP. However, further studies are needed to determine whether administration of EAEEN can also protect other organs from being damaged in SAP.