The Effect Of Early Enteral Nutrition On The Inflammatory Cytokines TNF-α, IL-6and IL-10in Rats With Severe Acute Pancreatitis

Background and aimsSevere acute pancreatitis (SAP), is one of the most common gastrointestinal diseases of acute hospital admission with a mortality reported to be up to36%-50%.The pathogenesis of SAP, of which process is mainly the development of inflammation is much complex. On the whole, premature activation of pancreatic proteases leads to disruption of the pancreatic and peripancreatic tissues with a resulting local and systemic activation of inflammatory cascade modulated by proinflammatory cytokines and other inflammatory mediators such as TNF-α, IL-1β, IL-6and PAR In some severe cases, this response which was defined as systemic inflammatory response syndrome (SIRS) appears to be unstoppable, Ultimately progressing to the multiple organ dysfunction syndrome (MODS) which is the main casuse of early death in patients with SAP. SIRS in SAP is concomitantly the production of antiinflammatory cytokines such as IL-10,IL-2to inhibit the overexpression of proinflammatory cytokines and protect the body.However,the overproduction of antiinflammatory cytokines can also lead to a compensatory antiinflammatory response syndrome (CARS) which can inhibit the immune response,causing the infectious complication and worsening the process of MODS. Therefore, neither proinflammatory cytokines nor antiinflammatory cytokines predominating in the course of inflammation is favourable.In addition, the imbalance of cytokines may occur as mixed antagenists response syndrome(MARS). So keeping balance between SIRS and CARS is the keypoint to the progress of SAP.As mentioned above, the pathogenesis of SAP is too complex to fully elucidate currently. As a consequence, therapies optioned for SAP are mainly symptomatic treatments. But the important role of nutritional support in patient with SAP is known by doctors gradually. At present,the enteral nutrition (EN) and parenteral nutrition(PN) are two common method of nutrition support in SAP. EN has historically been contra-indicated in an attempt to minimise further secretion of enzymes, In recent years, however,with further research into SAP, the overwhelming evidences prove that PN is inferior to EN regarding bacterial translocation,mortality, length of hospital stay and the occurrence of MODS and systemic infections.Because EN can improve immune system,regulate the relationship between the pro inflammatory cytokines and the antiinflammatory cytokines,maintain the gut barrier against bacterial translocation to alleviate the extent of SIRS and MODS.2013IAP/APA evidence-based guidelines for the management of acute pancreatitis suggests that EN should be the primary therapy in patients with SAP who require nutritional support.While PN can only be administered as second-line therapy when EN is unable to meet the demand. Hence,the conventional management including fasting and PN on patient with SAP is being replaced by EN gradually.Recently, many clinical studies find that early enteral nutrition (EEN) can improve the prognosis of patient with SAP:Sun et al found that EN started within48h after admission can improve the clinical outcome by mitigating the excessive immune response during the early stage of SAP and lightening the development of intra-abdominal hypertension (IAH) without causing subsequent immunosuppression. Marik PE manifests that EN should begin within24h after admission. which can improve the outcome of patient with SAP. Petrov MS et al compared the effect of EN versu PN on SAP patient with eleven randomized controlled trials and found that EN was better than PN when administrated within48h admission, whereas the effectiveness of the these two types of artificial nutrition did not differ significantly when the applicatin of nutrition was delayed. Spanier Bw et al show that EN has a beneficial influence on acute pancreatitis (AP) and should be used within48h after admission if condition is allowed. Li et al report that EN within48h of admission may improve the prognosis of AP by decreasing the risk of complication. In addition, EEN can also improve the prognosis of other types of diseases. Heyland DK put forward that the rate of infectious complications and mortality in critically ill patient given EN within48h admission were significantly decreased by24%and32%respectively, when compared with those patients who administrated over48h admission. Meanwhile, Mclave SA considers that EEN which was provided to critically ill patient who was put in the intensive care unit early after admission could downregulat SIRS and improve patient’s outcome. Consequently,2012International Consensus Guidelines for Nutrition Therapy in Pancreatitis suggest that EN should be used in patient who requires nutrition support as soon as possible.There are many researchs for exploring the optimal timing of EN in SAP, currently, however, the majority of these papers have some common limitations. First, the onset of SAP or AP, which is defined as the time of onset of abdominal pain, is not equated with the time of admission to the hospital. Consequently the time window observing for effect of early EN on patients with SAP or AP is not accurate; secondly, the high mortality and complexity of disorder,together with the difficult problem of tolerance and compliance of patients receiving EN are all the restrictions factors to clinical studies.In view of these limiting factors mentioned above, we use animal model which is well-matched to this study to explore the effect of early EN initiated at12h after the onset of SAP on dynamic change of inflammatory cytokines of TNF-α、IL-6、IL-10of rats with SAP, thus conducting a preliminary evaluation of the development of SIRS,clinical course of SAP as well as the safety and validity of EEN,providing some available evidence for further clinical study in the end. This paper is divided into two parts:Part1The establishment of SAP animal modelMethods1.Twelve male Sprague-Dawley rats were randomly divided into sham operation group (n=6) and SAP model group(n=6). The rats in SAP group were induced by retrogradely injecting5%sodium taurocholate solution(0.1mL/100g) into pancreas via the distal common bile-pancreatic duct. In the sham operation group, incisions were closed immediately following the turning over of the pancreas.2.The level of serum amylase of rat combined with pathological change and histological score of injured pancreas were used to determine whether construction of SAP model was successful.3.Statistics:SPSS version16.0was used for the statistical analysis. Data are expressed as mean±SD. Statistical analysis of data was done by using t test(Students t test).P<0.05was considered to be statistically significant.Results1.The degree of serum amylase of rat in SAP model group was increased significantly after induction when compared with sham operation group (P<0.05).2.Pathological changes in pancreatic tissue:The morphology of pancreas tissue was normal in sham operation group. However, there are hemorrhage, edema, focal necrosis and inflammatory cells infiltration in pancreatic tissue of rats in SAP model group.3.The pathological scores of injured pancreas in the SAP model group was evidently higher than that in sham operation group(P<0.05).ConclusionsThe SAP rat model was successfully induced and it is suit to explore the effect of EN or PN on SAP. Part2Effect of early enteral nutrition on the inflammatory mediators TNF-a, IL-6and IL-10in rats with severe acute pancreatitis Methods1.Fourty-eight male Sprague-Dawley rats were randomly allocated into8groups:sham-operation group (n=6),SAP model group (n=6), EN group was divided into12h,24h,48h subgroup (n=6in each subgroup), PN group was separated into12h,24h,48h subgroup (n=6in each subgroup).2.The rats in SAP model group were induced by retrogradely injecting5%sodium taurocholate solution (0.1mL/100g) into pancreas via the distal common bile-pancreatic duct. In the sham operation group, incision was closed immediately following the turning over of the pancreas five times. After induction of SAP, the rats in EN group were given jejunal feeding catheters which were linked to micro-infusion pumps and the rats in PN group, of which tail vein were inserted with indwelling needles which were also connected to the pumps.3.The rats in sham-operation and SAP model groups were sacrificed when they were confirmed to survive after surgery. While the rats both in EN and PN groups were administrated with nutritional fluid after12h operation, and executed at12h,24h,48h separately after commencement of nutrition support.Serum amylase was examined by Automatic Biochemical Analyzer. The concentration of serum TNF-α、 IL-6、IL-10was measured with ELISA kits.The pancreata were immediately dissected free from their attachments. A portion of these tissue was fixed in10%formaldehyde solution and dehydrated,sliced, paraffin-embedded and H.E stained to determine the histological scores of injured pancreas.The rest was analyzed for the expression of TNF-a,IL-6, IL-10in pancreatic tissue by RT-PCR.The intestinal tissues were made into sections stained with HE for evaluating the degree of intestinal injury and measuring the thickness of mucosa and height of villi.4.Statistics:SPSS version16.0was used for the statistical analysis. Data are expressed as mean±SD. Statistical analysis of data of the overall comparison of effect of EN and PN or different time intervals on indicators tested was done by using factorial design ANOVA. Statistical analysis of the rest of data was done by using t test (Students t test) or LSD-t (least significant difference-t test) according to the group number for uniform variance.P<0.05was considered to be statistically significant.Results1.Concentration of serum amylase:On the whole, there was no statistically difference in the level of serum amylase between EN and PN group,which was the same result in comparison of different time intervals(P>0.05); The degreel of serum amylase of rat in EN subgroups at each time point was not significantly different when compared to PN subgroup (P>0.05); The concentration level of serum amylase was also showed no evidently difference within subgroups of EN group or PN group respectively (P>0.05).2.Concentration of serum TNF-a:The effect of EN in the mass on reducing the level of serum TNF-a was superior to PN (P<0.05); The influence of12h time interval on serum TNF-a was greater than24h and48h time intervals (P<0.05), however, there was no sharply discrepancy between24and48h time intervals (P>0.05). The degree of serum TNF-a of rat in EN12h subgroup was obviously higher than that in PN12h subgroup (P<0.05), however,the significant difference of serum TNF-a was not observed at24h and48h time point after nutrition support (P>0.05). The result of comparison of subgroups within EN or PN group revealed that the concentration level of serum TNF-a of rat in EN12h subgroup or PN12h subgroup was significantly higher than that in24h and48h subgroups respectively (P<0.05),and there was no sharply difference between24h and48h subgroup (P>0.05).3.Concentration of serum IL-6:The effect of EN on decreasing the level of serum IL-6was better than PN as a whole (P<0.05); The impact of different time intervals on serum IL-6was clearly different, the degree of IL-6was the greatest at48h time interval and the least at12h.(P<0.05); The level of serum IL-6of rat in EN subgroup was significantly decreased when compared with PN subgroup at each time point (P<0.05); There was significant difference within subgroups of EN or PN group (P<0.05). In addition,the trend that the expression of serum IL-6which was the highest in EN48h subgroup and the lowest in EN12h subgroup was the same in the comparison within subgroups of PN.4.Concentration of serum IL-10:The effect of EN on increasing the level of serum IL-10was superior to PN on the whole(P<0.05); The influence of different time intervals on serum IL-10was visibly different (P<0.05), the level of serum IL-10was the highest at48h time interval and the lowest at12h. The level of serum IL-10of rat in EN subgroup was significantly increased when compared with PN group at each time point (P<0.05);There was significant difference within subgroups of EN or PN (P<0.05). In addition, the result that the expression of serum IL-10which was the highest in EN48h subgroup and the lowest in EN12h subgroup was the same in the comparison within subgroups of PN.5.Expression of TNF-a, IL-6and IL-10mRNA in pancreatic tissue:The impact of EN on reducing the expression of TNF-a, IL-6mRNA and increasing the expression of IL-10mRNA in pancreas was better than PN (P<0.05); In the comparison of different time intervals, the level of TNF-a mRNA was the greatest at12h time interval(P<0.05), however there was no significantly difference between24h and48h(P>0.05); The degree of IL-6mRNA in the three time intervals was obviously different, which was the highest at48h and the lowest at12h (P<0.05);The expression of IL-10mRNA was the highest at24h time interval and the lowest at12h(P<0.05); The mRNA expression levels of TNF-a was significantly decreased in EN12h subgroup in comparison with that in PN12h subgroup (P<0.05), nonetheless, there was no evidently difference between EN and PN subgroups at24h,48h time point(P>0.05).The IL-6mRNA expression in EN subgroups at each time point was significantly decreased when compared with PN subgroups (P<0.05).On the contrary, expression of IL-10mRNA was obviously enhanced in EN subgroups at each time point in comparison with PN subgroups (P<0.05).The result of comparison of subgroups within EN or PN group revealed that the expression levels of TNF-a mRNA both in EN12h and PN12h subgroups were significantly higher than those in24h and48h subgroups respectively (P<0.05),and there was no sharply difference between24h and48h subgroups(P>0.05); The expression degree of IL-6mRNA in48h subgroup of EN or PN was the highest,which was the lowest in12h subgroup of EN or PN(P<0.05);The expression of IL-10mRNA in24h subgroup of EN was the most and there was no sharply difference between12h and48h subgroups(P>0.05);The expression of IL-10mRNA in24h subgroup of PN was the highest,which was the lowest in PN12h group(P<0.05).6.The histological scores of injured pancreas:On the whole, the effect of EN on postponing the level of pathological score of pancreas was better than PN (P<0.05); The impact of different time intervals was clearly different, the pathological score was the highest at48h and the lowest at12h (P<0.05); The pathological score of pancreas in EN subgroup was significantly lower when compared with PN subgroup at24h and48h time point respectively(P<0.05), however, the socre was showed no statistically difference between EN and PN group at12h time point(P>0.05).The result of comparison of subgroups within EN or PN group indicated that the histological score of pancreas in48h subgroup of EN or PN was the highest and the lowest in12h subgroup respectively(P<0.05).7.Measurement of thickness of mucosa and height of villi:The impact of EN on delaying reduction of thickness of mucosa and height of villi was better than PN as a whole (P<0.05);The influence of different time intervals on thickness of mucosa and height of villi was clearly different, the data of thickness of mucosa and height of villi were the highest at12h and the lowest at48h (P<0.05); The thickness of mucosa and height of villi in EN48h subgroup was significantly higher when compared with PN48h subgroup (P<0.05), however, the result was showed no statistically difference between EN and PN group at12h and24h time point(P>0.05).There was evidently discrepancy within subgroups of EN or PN (P<0.05). In addition,the trend that the thickness of mucosa and height of villi which were the highest in EN12h subgroup and the lowest in EN48h subgroup was the same in the comparison within subgroups of PN. Conclusions1.When compared with parenteral nutrition, the early enteral nutrition support may regulate the balance between proinflammatory and antiinflammatory cytokines and improve immune function by down-regulating the level of proinflammatory cytokines and up-regulating the degree of anti-inflammatory cytokines both in serum and pancreatic tissue in an even better fashion.2.In comparison with parenteral nutrition, the early enteral nutrition support may alleviate the deleterious effect of SIRS on pancreas and jejunal mucous structure and then mitigate the progress of SIRS and MODS to a certain extent.