Experimental Research On The Effect Of Glycyl-glutamine Dipeptide By Enteral Feeding On The Recipient Metabolism, Intestinal And Hepatic Structure And Function And Long-term Survival Following Allogenetic Liver Transplantation In Rat

Part One:Experimental Research on the Effect of Glycyl-glutamine Dipeptide by enteral feeding on the recipient metabolism following allogenetic liver transplantation in rat.IntroductionAlmost of the recipients of liver transplantation were malnutrition in a different degree. Orthotopic liver transplantation(OLT) led to a serious wound. The recipient underwent OLT was in a state of serious catabolism, with a metabolic turbulence in a rather high degree. It would underwent a postoperative negative nitrogen state of balance in a long period of time. How to improve the recipient and the graft metabolism , especially anabolism of small intestine, how to reduce the recipient's response, and how to protect the function of important viscera and make the recipient recover all right, were very important to clinical liver transplantation.Gin is the special nutrient of small intestine's mucosa and of various kinds of cells which proliferate rapidly. Under those circumstances like wound, infection, and heavy operations, the recipient has a larger necessary of Gln. But Gln monomer has a low solubility and will form a kind of poisonous pyroglutamic acid. It is the limitation of Gln on clinical practice, there is a lack of Gln in the nutrition support following OLT. Gly-Gln and Ala-Gin are two kinds of most in use synthetic dipeptide which conclude Gln. They have a high solubility and can bear the sterilization under high temperature, thus could be conveniently used in clinical practice. Experiments and documents verify that there is a special carrier for dipeptide in small intestine. This carrier still transfers dipeptide even if the function of the intestine is impaired. This research for the first time observes the effect of glycyl-glutamine dipeptide on the recipient metabolism following allogenetic liver transplantation in rat.Materials and MethodsTwelve male inbred LEW (RT1') rats were random as the donor, 24 male inbred BN (RTln) rats were random as the recipients of allogenetic liver transplantation.The recipients were divided into two groups randomly: the control group (group ALA, n=12) and the experimental group (group GLN, n=\2). In each group, 6 normal BN rats were drawn to collect the samples and done xylose experiment as the normal parameter on the 3rd preoperation day , the residual 6 recipients in the control group received alanine 0.6 g/kg.d for three days before operation and 7 days postoperatively by means of perfusing stomach, the residual 6 rats in the experimental group were given Gly-Gln 0.6g/kg.d in the same way. These 12 BN recipients underwent 3 days fasting (free to water) and orthotopic liver transplantation on an aseptic occasion and use CsA2 mg/kg.d postoperation by means of hypodermic injection.The postoperative general state, survival rate and weight of all the rats were recorded.The 12 BN rats were drawed after transplantation on the 8th postoperative day. the content of Gin and albumin in plasma, the content of Gin, glutathione, and protein in small intestine mucosa , activity of GSH-PX, activity of Gin, protein, GS in musculi skeleti were measured postoperatively.ResultsThe recipient rats had a fasting for three days before the operation and lost weight significantly, while they could still underwent the operation and were alive 8 days after the operation with the weight losed evidently. As for plasma Gin and albumin, and the content of GSH, Gin and the content of Gin and GSH in small intestine mucosa, the content of protein, activity of GSH-PX, Gin and protein in musculi skeleti, group GLN was much higher than group ALA while the latter had a much better activity of GS in musculi skeleti than the former on the 8th postoperative day.DiscussionAfter fasting, the rats were acutely hungry and their response to the operation stress was reduced. After the operation, the rats' feeding returned to normal slowly, weight lost evidently and had an obvious malnutrition. Mucosa had priority to absorb Gly-Gln dipeptide in the intestine and then Gly-Gln dipeptide was decomposed invivo. Gly-Gln increased the content of Gin in plasma, in small intestine mucosa and in musculi skeleti, the content of GSH, protein in small intestine mucosa , the activity of GSH-PX and the content of protein in musculi skeleti then through anabolism enhancement. Gly-Gln also decreased the activity of GS in musculi skeleti and remarkably improved the recipient metabolism following allogenetic liver transplantation in rat.ConclusionGly-Gln dipeptide by enteral feeding improves the Gin in plasma , mucosa and imusculi skeleti and the metabolism of recipient protein and mucosa GSH in rat following allogenetic liver transplantation.Part Two :Experimental Research on the Effect of Glycyl-glutamine Dipeptide by enteral feeding on the Intestinal Structure, the Absorption and Barrier Function Following Allogenetic Liver Transplantation in Rat IntroductionMost of the recipients of liver transplantations were patients of liver cirrhosis and had malnutrition of different degrees before operations. For the time being most transplantation operations adopt the surra orthotopic liver transplantation without a veno-venous bypass. During this operation, congestion about 45 to 60 minutes or more took place in the gastrointestinal tract while there was anhepatic phase. The small intestine mucosa would underwent congestion, anoxia and a reperfusion injury. After the operation, the following problems appeared in gastrointestinal tract: mucosal structure injury and decline of the absorption of the intestine, micro-ecological disorder of the intestinal canal, damage of the mucosa's barrier function, bacteria and endotoxin translocation in intestina cavity. These problems all increased the possibility of infection, thus would initiate systemic inflammation response syndrome (SIRS), even primary nonfunction (PNF) and multiple organ dysfunction syndrome(MODS).How to maintain the mucosal structure , to improve its absorption, to protect itsbarrier function and to reduce postoperative enterogenous infection and thus speed up the repair of the graft's function and patient's recovery, these are of much significance to patients who are in immunosuppression after liver transplantations.This experiment for the first time did a research on the effect of Gly-Gln by enteral feeding on the structure, the absorption and barrier function of the intestine following allogenetic liver transplantation in rat.Materials and methodsExperimented animals, randomly divided groups, perioperative period treatment and drawing experimental materials, were follow as the research one.To use electron microscope observe structure and ultramicro structure of mucosa . the activity of enzyme Na+ - K+ - ATP in small intestine mucosa, the activity of diaccharidase, experimentation of xylose absorption, all these things of each rat should be detected. Its liver, spleen, mesenteric lymph node underwent the cultivation, species identification and counting of germs, the micro-ecological change in ileocecus intestinae(species and amount specifically) should also be detected.ResultsOn the 8th postoperative day, the structure of mucosa, the activity of enzyme Na+- K+-ATP in small intestine mucosa and the activity of diaccharidase, experimentation of xylose absorption of recipient group GLN were significantly better or higher than that of group ALA. On the contrary, as for the cultivation number of germs in liver, spleen, mesenteric lymph node and kidney in group GLN was much lower than that in group ALA. Referring to the micro-ecological change in ileocecus intestinal canal, group GLN had a remarkably higher content of anaerobe than group ALA and a remarkably lower content of aerobe in intestinal canal on the 8th postoperative day.DiscussionAfter the operation, there were swellings of different degrees and mucosa injury in gastrointestinal tract, a decline of the absorption of the intestinal canal, a damage of the mucosa's barrier function, a micro-ecological disorder of the intestinal canal, shifts of bacteria and endotoxin in intestina cavity. These problems all increased thepossibility of infection after OLT, thus would initiate a MODS and would probably had a certain effect on chronic graft dysfunction. This research approved that using Gly-Gln in intestine maintained the organizational structure, especially the ultramicro structure of small intestine following a liver transplantation in rat. It also improved the activity of enzyme Na+- K+-ATP in small intestine mucosa of the recipient and the activity of diaccharidase, strengthened the absorption of small intestine, decreased shifts of bacteria in intestinal cavity, regulated the micro-ecological disorder after a liver transplantation, protected the barrier function of the mucosa .Probably because Gly-Gln dipeptide in intestine could be absorbed effectively by the mucosa and be decomposed in vivo, this remarkably improved the metabolism of Gin and protein in small intestine mucosa and in musculi skeleti. It was carried out through direct nutritious function of mucosa and the anti-oxidational injury function of GSH, through indirectly regulating the intestinal canal's micro-ecology, maintaining the structure of the mucosa, promoting the small intestine's absorption and barrier function.ConclusionGly-Gln dipeptide by enteral feeding improves the structure, the absorption and barrier function of the intestinal canal and regulated the intestinal micro-ecology following allogenetic liver transplantation in rat.Part Three:Experimental Research on the Effect of Glycyl-glutamine Dipeptide by enteral feeding on the Grafted Liver Metabolism > Structure and Function Following Allogenetic Liver Transplantation in Rat.IntroductionIn the course of liver transplantation, the graft liver underwent an inevitable ischemia reperfusion injury(I/RI) which had something to do with many postoperative complicating diseases, and some worse ones would lead to primary graft dysfunction. Over recent years, although some medicines such as calcium ion antagon, protease inhibitor and free radical scavenger had been in use to safeguard the graftfrom I/RI, the effect was not very satisfactory.In the documents it were reported that Gin and Gly had effect on protecting intestinal canal, myocardium and musculi skeleti from I/RI. This research observed the effect of Gly-Gln dipeptide by enteral feeding on the metabolism, structure and function of the grafted liver following allogenetic liver transplantation in rat.Materials and MethodsExperimented animals, randomly divided groups, perioperative period treatment and drawing experimental materials, were follow as the research one. Endotoxin in the blood of portal vein, the content of GSH in plasma, endotoxin in the blood of portal vein, the content of MDA in the graft, the activity of GSH-PX, the change of liver enzyme Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST), the pathology and ultramicro structure of liver, all these samples of each rat should be measured on the 8th postoperative day.ResultsOn the 8th postoperative day, GSH in blood plasma, the content of and the activity of GSH-PX in the grafted liver, the recipients in group GLN were significantly higher than that of group ALA. While the content of MDA in the grafted liver, the activity of ALT and AST, endotoxin in the blood of portal vein all became higher after operation. Group GLN was remarkably lower than group ALA. The morbid pathology and ultramicro structure showed that the liver had damage of cell organelle in two groups ,while the liver structure in group GLN was better than that in group ALA.DiscussionThe grafted liver would underwent I/RI inevitably and the exact mechanism of the liver's I/RI was still unclear. It was mainly considered to be of relationship with the overloading of oxygen-derived free radicals and calcium ion. GSH is very important protective agent of the organism to protect enzyme and other protein hydrosulfide group against the damage of oxygen-derived free radicals.Gly-Gln by enteral feeding can protect the grafted liver both in an direct way and in an indirect way. This research verified that on the 8th postoperative day, theprotein in liver organism and albumin in plasma of group GLN were remarkably higher than that of group ALA in corresponding time. This showed Gly-Gln could improve liver cell's anabolism. This may be of much significance to the repair of the liver underwent I/RI.GSH is very important protective agent of the organism to protect enzyme and other protein hydrosulfide group against the damage of oxygen-derived free radicals. On the 8th postoperative day, GSH in plasma and activity of GSH-PX in graft of group GLN were much higher than that of group ALA. This showed that Gly-Gln facilitated the synthesis of liver GSH, this may be one of its important ways to safeguard liver from I/RI.During liver transplantation, the period in anhepatic phase would result in following injuries: one was congestion in portal vein and intestinal ischemia, the other was reperfusion of artery blood and blood stasis in portal vein, and this course of reperfusion would simultaneously lead to injury of the grafted liver. The documents showed that if the time of congestion in portal vein was prolonged, then bacteria translocation, inflammatory factors and active oxygen release to blood of portal vein, thus make the injury of liver. Gly-Gln produces Gin by means of synthesis and through protecting the intestinal canal directly,Gln alleviates the pathological and physiological changes of mucosa blood of stasis in portal vein, thereby alleviates the trauma of liver cells resulted by the reperfusion of portal vein blood, that is to say, it safeguard the liver from I/RI indirectly. In the experiment, there was a significant decline of endotoxin in blood of portal vein, with agreement as the significant decrease of cultivation number of bacteria in liver, spleen, mesenteric lymph node and kidney. This showed Gly-Gln dipeptide improvement of the structure, absorption and barrier function of intestinal mucosa and regulation of the intestinal micro-ecology may be the important system of improvement the metabolism, structure and function of the grafted liver following allogenetic liver transplantation in rat.ConclusionGly-Gln dipeptide by enteral feeding improved the metabolism, structure and function of the grafted liver following allogenetic liver transplantation in rat.Experimental Research on the Effect of Glycyl-glutamine Dipeptide by enteralfeeding on the recipient and graft long-term survival under immunosuppression following allogenetic liver transplantation in rat.IntroductionOgan transplantation had made marvelous achievements in recent years. Above 90 percents of the recipients following OLT could survive beyond one year.Now as for the recipients the main aim is to enhance life quality and long term survival.Chronic graft dysfunction was defined as the graft function declined chronically and irreversiblly ,it was called chronic rejection and late graft loss or late graft dysfunction before.The UNOS(united network of organ share) in America declared about from 30 percents to 70 percents recipients would suffered from chronic graft dysfunction and confront death or retransplantation under regular immunosuppressant therapy without certain etiological factor based on their long-term survival outcome data.This research was based on Kamada model of allogenetic liver transplantation in rats. One focus was to represented the effect of nonspecific immunity such ischemia reperfusion injury, barrier function of the mucosa, micro-ecology in intestinal canal, bacterial translocation, postoperative metabolism, the toxic side effects of immunosuppressant etc. and of nonimmunity factor on the graft which lives on function for a long period of time .Another focus was to explore whether Gly-Gln dipeptide by enteral feeding for a long period of time may enhance the survival in rat following OLT and the relationship between Gly-Gln and chronic grafted liver dysfunction .Materials and Methods24 Male LEW rat (RTl') of inbred line as the donor, 24 male BN (RTl") rat of inbred line as the recipient of the allogenetic transplantation. Experimental animals were all SPF grade. They were divided randomly into two experimental groups. In control group(group ALA, n=12), the recipient received alanine 0.6g/kg.d for three days and a fasting for one day before operation and 130 days postoperatively.In experiment group( group GLN, n=24), the recipient received Gly-GlnO.6g/kg.d forthree days and a fasting for one day before operation and 130 days postoperatively by means of perfusing stomach. There were 24 BN rats left after orthotopic liver transplantation. One month after the operation, used CsAlmg/kg.d as the immunosuppressant, by means of hypodermic injection. One month after the operation, used CsA2mg/kg.d as the immunosuppressant, by means of hypodermic injection. Observed the survival rate of all the rats postoperatively, monitored the micro-ecological change in intestinal canal and death dynamically, made pathologic anatomy of the dead rats and explored the reason of chronic graft dysfunction.ResultsThe statistics of the reasons of the death in the comparison group (group ALA) included: 2 died of putrescence of lobes of liver, 2 of infection of abdominal cavity and 4 of chronic obstruction of biliary tract and hyperplasia pathologic change. The statistics in the experiment group (group GLN) included: 2 died of putrescence of lobes of liver, 1 of infection of abdominal cavity and 2 of chronic obstruction of biliary tract and chronic hyperplasia pathologic change. Made an analysis of the survival curve in the way of Kaplan-Meier: group GLN was obviously better than group ALA.DiscussionThe focus of this research was to represent the effect of nonspecific immunity factors such as ischemia reperfusion injury, barrier function of the mucosa, micro-ecology in intestinal canal, bacterial translocation and of nonimmunity factors such as postoperative metabolism, the toxic side effect of immunosuppressant etc.on the graft which lives on function for a long period of time. Made an analysis of the survival curve in the way of Kaplan-Meier, the reasons of the deaths of the rats concluded: putrescence of lobes of liver, infection of abdominal cavity and chronic obstruction of biliary tract and chronic hyperplasia pathologic change, which respectively represented the important pathologic changes after liver transplantation: putrescence of lobes of liver resulted from liver I/Rl, infection of abdominal cavity resulted from the intestinal bacterial translocation, chronic obstruction of biliary tract and chronic hyperplasia pathologic change and the prostration of liver functionresulted from the complicating diseases of biliary tract and chronic inflaming reactive change. These may be the important theoretical knowledge of the chronic graft dysfunction. Biliary tract lost blood supply and lacked innervation, so on the basis of recessive infection ,it was very easy for the bacteria and endotoxin translocation and for the products of micro-ecological unbalance to enter into the liver to erode the graft, thus caused chronic obstruction of biliary tract and chronic hyperplasia pathologic change, finally resulted in hepatocirrhosis and prostration of liver function. It was possible that chronic obstruction of biliary tract and hyperplasia pathologic change was one important type of chronic graft dysfunction. Now there are no relevant research documents in this field, so it needs further research.Conclusion1: Gly-Gln dipeptide by enteral feeding for a long period of time improved its survival rate under immunosuppression following allogenetic liver transplantation in rat.The Next Research1: Chronic obstruction and hyperplasia pathologic change of biliary tract was probable one important type of graft dysfunction?2: Gly-Gln dipeptide by enteral feeding for a long period of time may reduce the chronic graft dysfunction caused by infection and chronic obstruction of biliary tract and hyperplasia pathologic change?.