severe acute pancreatitis ( sap ) , characterized by atrocious 
 progression , multicomplications , and difficult treatment 
 [ 13 ] 
 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% . 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 
 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 . 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 [ 812 ] . 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 ) . sixty healthy male wistar rats , weighing 190250 g , 
 were purchased from animal center , the first clinical hospital 
 of harbin medical university ( harbin , china ) . emodin was 
 obtained from pharmacy , the first clinical hospital of harbin 
 medical university . sodium taurocholate was obtained 
 from sigma ( st . louis , mo , usa ) . sixty male wistar rats , housed in 
 cages with a controlled temperature of 26c 
 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 . 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 . 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 . 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 h , 22 h , 
 32 h , 42 h , 52 h , and 62 h , 
 respectively ; een group received pepti-2000 
 variant through enteral nutrient tube at 15 h , 25 h , 
 35 h , 45 h , 55 h , and 65 h , 
 respectively . the enteral nutrient solution was continuously 
 infused at each 
 time point and the dosage at 15 h , 25 h , and 35 h 
 was 120 ml / kg ( body weight ) 
 followed by that increasing to 240 ml / kg ( body weight ) 
 at 45 h , 55 h , and 65 h ; 
 eaeen group received emodin ( 3.0 mg/100 g body weight ) 
 at 12 h , 22 h , 32 h , 42 h , 
 52 h , and 62 h and pepti-2000 
 variant at 15 h , 25 h , 35 h , 
 45 h , 55 h , and 65 h. the dosage and 
 infusion method of 
 pepti-2000 variant at each time point in eaeen group were 
 the same as those in een group . 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 ) . 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 . 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 . after removal , fresh samples of 
 pancreas and ileum were immediately weighed and dried at 
 100c 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 . blood samples were collected from abdominal aorta and 
 centrifuged at 3000 rpm for 5 minutes . tumor necrosis factor - alpha ( tnf- ) 
 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 . the significance of differences in histopathologic scores was 
 assessed by kruskal - wallis test . statistical analysis 
 was finished by spss10.0 statistical program and difference 
 was considered statistically significant 
 when 
 p<.05 . 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 
 14 , 
 figures 15 ) . in group a , severe edema , diffuse 
 hemorrhage , and necrosis in pancreas were observed . 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. in contrast to emodin and een , eaeen significantly reduced 
 the levels of tnf- , 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. 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 , and 4 , 
 figures 24 ) . however , no significant differences were found in 
 these data between group b and group c. 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 69 ) . eaeen could 
 also attenuate the severity of secondary liver injury obviously 
 ( figures 1014 ) . 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. emodin characterized by the main active monomer of 
 rhubarb is a derivative of anthraquinone 
 ( 3-methyl-1 , 6 , 8-trihydroxyanthraquinone ) . studies have proved that emodin is a potent agent in 
 the management of clinical 
 and experimental acute pancreatitis 
 ( ap ) . zhang et al . , and wu et al . 
  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 .   proved the mechanisms 
 of emodin in the treatment of sap included 
 modulation of abnormal eicosanoid metabolism , promotion of pancreatic 
 cytoprotection , and improvement of pancreatic microcirculation . found emodin was a potent inhibitor of nuclear 
 factor - kappab ( nf-b ) 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 
 microcirculation of intestinal mucosa , decrease the 
 permeability of intestinal mucosa , and reduce the incidence 
 of translocation of intestinal bacteria ; ( 2 ) pepti-2000 variant , 
 which is an elemental diet with low - fat content and can 
 be absorbed easily , contains adequate nutritional ingredients , 
 scanty residue and has low viscosity . administration of 
 pepti-2000 variant by enteral nutrient tube at different time 
 points conformed to the daily dietary habit , placed the pancreas at a 
  full rest ,  and inhibited  rebound  
 of the inflammatory reaction ; ( 3 ) emodin could clear the intestinal 
 bacteria and toxin , promote intestinal peristalsis and 
 recovery of the intestinal function , so as to reduce the 
 incidence of secondary enterogenous infection . taken together , this 
 combined strategy was methodologically more rational and 
 purposive , with undoubtedly higher efficacy . a vicious cycle of pancreatic microcirculatory changes such 
 as capillary stasis and vasoconstriction has been shown to occur 
 in the course of sap . 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 
 , 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 . 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 . 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 . 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- ( tnf- ) and interleukin-1 . cytokine in sirs and plays a central role in 
 the pathogenesis of ap and related systemic 
 complications . tnf- 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- , 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  
 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- and 
 other cytokines were reduced ; ( 4 ) 
 emodin inhibited the activation of pancreatic enzyme 

 so as to prevent mononuclear - macrophage system , 
 activated by pancreatic enzyme after its entering the plasma , 
 from producing tnf- ; ( 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- and pancreatic mpo activity 
 so as to attenuate the 
 severity of inflammation in pancreas ; ( 6 ) tnf- 
 induces aggregation and activation of neutrophils in the pancreas . administration of eaeen could significantly reduce 
 blood tnf- 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- 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 . found that lps could 
 significantly elevate serum tnf- 
 activity and induce more severe liver damage in ap rats , 
 which suggested that both lps and tnf- played 
 critical roles in the development of secondary hepatic 
 injury during ap . found that activated 
 neutrophils and macrophages produced large 
 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 
 tnf- , 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 . 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 .