| Objective Although Orthotopic liver transplantation (OLT )have gained tremendous advancement in the treatment of end-stage liver disease, early postoperative infection (within 30 days after liver transplantation) is still important factor of impact on survival rate of patient. Among infections, sepsis is constantly one of the important causes of postoperative decease. Some studies have confirmed that sepsis is possible related to the bacterial translocation and enterogenous endotoxemia resulted from intestinal mucosa barrier injury by total hepatic vascular exclusion and reperfusion.The intestinal mucosal barrier is composed of mechanical barrier, chemic barrier, biologic barrier and immune barrier, each barrier plays a substaintial defense role on enteron mucous membrane. Intestine is a core organ of surgery stress, bacteria and endotoxin translocation resulted from intestinal mucosal barrier injury plays a center role in systemic inflammatory response (SIRS) and multiple organ dysfunction syndrome (MODS) resulted from various kinds of stress injury. Hence, it has important clinical sense to protect intestinal mucosal barrier.At present, most liver transplantations adopt classics OLT without veon-venous bypass, during which congestion for 45 to 60 minutes or more may occure in the portal vein system and gastrointestinal tract when donor liver is planted in the anhepatic phase. The mucosa of the small intestine is likely to have congestion and ischemia-reperfusion injury, which results in generous oxygen-derived free radicals releasing and Nuclear factor-κB (NF-κB) activation, the latter can cause transcribe and expression of a series of cytokine such as Tumor necrosis factor-α(TNF-α), Intercellular adhesion molecule-1 (ICAM-1) and so on, which make more serious for intestinal mucosal barrier injury and increase intestinal membrane permeability, which causes bacterial and endotoxin translocation, endogenous infection, SIRS and MODS, these raise the mortality of the liver transplantation patients. So, for liver transplantation patients, above all who are in immunosuppression after OLT, it is important to maintain the mucosal barrier function, to improve mucosal nutrition status and to reduce enterogenic infection.Now, there are no effective measures to treat the dysfunction of intestinal mucosal barrier. In recent years, some studies have made clear that glutamine (Gln)-enriched early enteral nutrition (EEN) for serious illness patients of grave wound and grave infection can not only content the nutritional need of human body, but also provide nutrition substrate for intestinal mucosa, improve the blood supply of intestinal mucosa, maintain the intestine immune activity, normal structure of intestinal mucosal cells and intercellular conjunction and the height of villus, so it can prevent beforehand and lessen the intestinal mucosal injury. Meanwhile, Gln-enriched EEN is in line with metabolic physiology which is in favour of protein synthesis and metabolic regulation. Gln-enriched EEN can promote intestinal flora growth and the normal secrete of sIgA of intestinal cells which is in favour of prevention of the endotoxin releasing and reducing enterogenic infection. Hence, Gln-enriched EEN can achieve the purpose of increasing the survival rate of serious illness by the control of SIRS and MODS from spring-head.Now, it is less to study about whether Gln-enriched EEN has safeguard effect on intestinal mucosal injury after OLT. In this study, we successfully established the rat model of orthotopic liver transplantation, utilized Gln-enriched EEN at perioperative period. To investigate the condition of intestinal mucosal injury after OLT and the safeguard effect on intestinal mucosal injury of Gln-enriched EEN from immunology, molecular biology and minute ecology by detecting the changes of correlated index reflected intestinal mucosal barrier, it could provid animal experimental basis for taking effective measures to lessen the intestinal mucosal injury, to reduce the complication of OLT and to raise the survival rate of OLT.Methods Healthy male wistar rats, weighing 220-250g, were randomly divided into three groups: Control group, orthotopic liver transplantation group (OLT group) and OLT plus Gln-enriched EEN group (EEN group). The rats of Control group were not subjected to any treatment. The recipients of EEN group were supplied with Nutrison Fiber (125ml/d/kg) plus Gln (0.3g/d/kg) for 3 days before operation by gastric perfusion, the daily total amount were given six times evenly. From postoperative 3h, Nutrison Fiber (125ml/d/kg) plus Gln (0.3g/d/kg) was again given by gastric perfusion until the animal was put to death. The recipients of OLT were supplied with equivalent volumes Sodium Chloride by gastric perfusion at the same time. The rat models of OLT were established by modified two-cuff method. At 12h,24h and 72h after OLT, the animal was put to death and the blood sample and the tissue sample were collected. The levels of endotoxin in portal vein blood plasma were determined by Limμlus Amebocyte Lysate(LAL) Pyrochrome Kit, the contents of D-lactic acid in portal vein blood plasma were detected by enzymology spectrophotometric method, the contents of tumor necrosis factor-α(TNF-α) in inferior vena cava serum were determined by enzyme assays (ELISA), morphological study of intestine tissues were also analyzed by light and electron microscopy respectively, the content of malondialdehyde (MDA) of intestinal mucosa were observed, the ratio of translocation of intestinal bacteria in liver, spleen, mesenteric lymph nodes (MLN) and portal vein blood were measured, the expression of Nuclear factor-κB (NF-κB) and Intercellular adhesion molecule-1 (ICAM-1) were determined by immunohistochemical method, the expression of TNF-αmRNA and TNF-αprotein were detected by fluorescence quantitive–polymerase chain reaction (FQ-PCR) and Western blotting respectively.Results (1) At 12h, 24h and 72h after liver transplantation, the levels of endotoxin of OIL group were 0.427±0.092, 0.907±0.057 and 0.803±0.052EU/ml respectively and those of EEN group were 0.413±0.056, 0.617±0.049 and 0.406±0.043 EU/ml respectively. The levels of endotoxin of OLT group and of EEN group were obviously higher than that of Control group 0.109±0.017 EU/ml (P<0.01). Which EEN group compared with OLT group at 24h or 72h respectively, there were significant difference (P<0.01), the former decreased 32.0%, 49.4% respectively. (2) At 12h, 24h and 72h after liver transplantation, the contents of D-lactic acid of OLT group were 1.23±0.12, 2.30±0.12 and 1.92±0.11μg/ml respectively and those of EEN group were 1.22±0.11, 1.78±0.10 and 1.23±0.08μg/ml respectively. The contents of D-lactic acid of OLT group and of EEN group were obviously higher than that of Control group 0.48±0.07μg/ml (P<0.01). Which EEN group compared with OLT group at 24h or 72h respectively,there were significant difference (P<0.01), the former decreased 22.6%, 35.9% respectively. (3) At 12h, 24h and 72h after liver transplantation, the contents of TNF-αin serum of OLT group were 107.06±7.47, 759.04±24.14 and 569.17±26.70pg/ml respectively and those of EEN group were 102.28±9.61, 715.50±25.73 and 521.13±20.86pg/ml respectively. The contents of TNF-αin serum of OLT group and of EEN group were obviously higher than that of Control group 15.98±2.07 pg/ml (P<0.01). Which EEN group compared with OLT group at 24h or 72h respectively,there were significant difference (P<0.01), the former decreased 5.74%, 8.44% respectively. (4) At 12h, 24h and 72h after liver transplantation, the pathologic lesions of ileum mucosae under light microscope showed that mucosae were diffusely severe erosion, necrosis and hiatus, and villus interspaces were obviously broadened, and edema in lamina propria, and generous inflammation cells infiltrated in the intestinal wall, and mucosae were attenuation in OLT group, these above-mmentioned phenomena were most severely at 24h after liver transplantation. But the lesions of ileum mucosae under light microscope in EEN group showed that mucosae were slight erosion, and local villi were necrosis and hiatus, and only slight edema in lamina propria, and a small quantity inflammation cells infiltrated in the intestinal wall. The lengths of villi of OLT group were 276.5±16.9, 152.1±11.3 and 199.3±11.6μm respectively and those of EEN group were 283.9±15.7, 228.1±14.9 and 289.9±10.8μm respectively. The lengths of villi of OLT group and of EEN group were obviously lower than that of Control group 383.3±14.1μm (P<0.01). Which EEN group compared with OLT group at 24h or 72h respectively,there were significant difference (P<0.01), the former were 1.50, 1.45 times to the latter respectively. The ultrastructure under transmission electron microscope (TEM) of OLT 24h group showed that epithelial cells were swelling and dissolved, epithelial cell interspaces were enlarged and microvilli were necrosis and hiatus, and those of EEN group showed that epithelial cell interspaces were fundamentally normal, mitochondria were slight swelling, microvilli became slight alveolation and local necrosis and hiatus. (5) At 12h, 24h and 72h after liver transplantation, the contents of MDA in intestinal mucosae of OLT group were 18.95±1.33, 37.13±1.46 and 31.30±1.12nmol/mg.prot respectively, and those of EEN group were 17.91±1.12, 24.88±1.10 and 19.40±0.91nmol/mg.prot respectively. The contents of MDA of OLT group and of EEN group were obviously higher than that of Control group 8.21±0.61 nmol/mg.prot (P<0.01). Which EEN group compared with OLT group at 12h, 24h or 72h respectively, there were significant difference (P < 0.01), the former decreased 5.5%, 33.0% and 38.0% respectively. (6) At 12h, 24h and 72h after liver transplantation, the contents of sIgA in intestinal mucosae of OLT group were 0.733±0.036, 0.546±0.042 and 0.580±0.037μg/ml respectively, and those of EEN group were 0.755±0.039, 0.722±0.029 and 0.905±0.030μg/ml respectively. Besides EEN 72h group, the contents of sIgA of the rest group were lower than that of Control group 0.887±0.045μg/ml (P<0.01). Which EEN group compared with OLT group at 24h or 72h respectively, there were significant difference(P<0.01), the former were 1.32, 1.56 times to the latter respectively. (7) At 12h, 24h and 72h after liver transplantation, the ratio of translocation of intestinal bacteria of OLT group were 52.50%, 82.50% and 72.50% respectively, and those of EEN group were 47.50%, 45.00% and 32.50% respectively. The ratio of translocation of intestinal bacteria of OLT group and of EEN group were higher than that of Control group 5.00% (P<0.00227), those of EEN group were obviously lower than those of OLT group at 24h or 72h respectively (P<0.00227), the former decreased 45.5%, 55.2% respectively. (8) at 12h, 24h and 72h after liver transplantation, the results of immunohistochemistry showed that the optical density of NF-κB expression in OLT group were 0.1494±0.0167,0.2018±0.0139 and 0.1623±0.0142 respectively, and those of in EEN group were 0.1119±0.0091,0.1437±0.0102 and 0.1087±0.0109 respectively,the expression of NF-κB in OLT group and in EEN group were higher than that in Control group 0.0606±0.0147 (P<0.01); the optical density of ICAM-1 expression in OLT group were 0.1380±0.0136,0.1750±0.0086 and 0.1604±0.0077 respectively, and those of in EEN group were 0.1097±0.0064,0.1289±0.0075 and 0.1086±0.0050 respectively, the expression of ICAM-1 in OLT group and in EEN group were higher than that in Control group 0.0464±0.0053 (P<0.01). NF-κB expressed chiefly on the superficial epithelium or crypt epithelium. ICAM-1 expressed chiefly on the epithelium or endothelial cell of blood vessel. The expressions of NF-κB and ICAM-1 of EEN group were lower than those of OLT group at 12h, 24h or 72h respectively (P<0.01). (9) The results of FQ-PCR showed that the expressions of TNF-αmRNA in intestinal tissues of OLT group were 4.20±0.23, 9.40±0.19 and 5.43±0.27×104copies/μgRNA,and those of EEN group were 3.01±0.10, 5.54±0.19 and 2.61±0.14×104 copies/μgRNA, the expressions of TNF-αmRNA of OLT group and of EEN group were obviously higher than that of Control group (P<0.01). But the expressions of EEN group were obviously lower than those of OLT group at12h, 24h or 72h respectively(P<0.01), the former decreased 28.3%, 41.1% and 51.9% respectively. (10) From the analysis of Western blotting, the expressions of TNF-αprotein were shown at 12h, 24h and 72h after liver transplantation, but the expression intensity of EEN group were obviously weakend. Semiquantitative analysis showed that the expressions of TNF-αprotein of OLT group were 1.75, 3.40 and 2.79 respectively,and those of EEN group were 1.32, 2.08 and 1.36 respectively,the latter decreased 24.6%, 38.8% and 51.3% respectively.Conclusions⑴Intestine congestion and ischemia-reperfusion injury resulted from orthotopic liver transplantation during anhepatic phase causes lipid peroxidation of intestinal mucosa epithelium, increases the contents of MDA in intestinal mucosa, reduces the levels of sIgA in intestinal mucosa, meanwhile raises the activity of NF-κB of macrophage in intestinal mucosa and produces a series of cytokine such as TNF-α, ICAM-1 and so on. Which resulted in serious damage of intestinal mucosal function, the damage shows off a few respects:①mechanical barrier was damaged: epithelium swelling, intercellular spaces were widened, villi were necrosis and hiatus, atrophy and shortened.②biologic barrier and immune barrier were damaged and bacterial tanslocation and endotoxemia can be observed.③intestinal mucosal permeability was increased and the contents of D-lactic acid in blood were rised.⑵Gln-enriched EEN can improve the blood supply of intestinal mucosa and reduce the creations of MDA in intestinal mucosa after liver transplantation in rats, which makes for normal secretion of sIgA of intestinal mucosal cells and inhibition of NF-κB activity of intestinal mucosal macrophage and inhibition of creation of TNF-α, ICAM-1 etc. cytokine, thereby improve intestinal mucosal injury and obviously inhibit bacterial translocation and endotoxemia. But the action mechanism of Gln-enriched EEN must further study and investigate. According to the experimental consequence, we inferred that Gln-enriched EEN at perioperative period for liver transplantation patients can reduce enterogenic infection and have substaintial clinical significance to raise the survival rate of liver transplantation.
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