Study On The Mechanism Of Damage In Intestinal Mucosa Induced By Tumor Necrosis Factor Alpha In Fulminant Hepatitic Failure

Introduction and ObjectiveSpontaneous bacterial peritonitis (SBP) is one of common and serious complication in patients with fulminant hepatitic failure ( FHF). Its incidence is 8 — 27% , and the rate of complicated SBP in FHF is 17. 7%. The mortality of SBP is 48 -57%. Because of the high morbility, variable symptom, difficulty to diagnosis and poor prognosis, SBP is paied more and more attention. The damage of intestinal mucosal barrier was a key link induced SBP and multiple organ failure in FHF. Recently, pathogensis of SBP has been recognized. During hepatitis gravis, SBP was closly correlated with increased permeability of intestinal mucosa and ischemia, bacterial translocation, hypoxia of intestinal mucosa, va-sopermeability increased, epithelium mucosae edema, disruption of intercellular junction complex. However, the pathogenesis of SBP is still no clear. The mechanism of intestinal bacteria reach the ascites is not well known. The phenomenon called bacterial translocation has been implicated in the pathogenesis of SBP in recent years and is now considered a key step. It is not known how bacteria can pass through intestinal mucosal barrier to other organs. The high levels of cytokines such as tumor necrosis factor ( TNF) , γ - interferon, Interleukin (IL) are related to the development of SBP.In this study, we observated the alterations of intestinal mucosal morphology , the changes of the tight junction and the patterns of bacteria entering intestinal mucosa in mice, the relationship between expression of key epithelial intercellular junction proteins and bacteria invasion intestinal mucosa in mice model with FHF, and the expressive level of ZO - 1 (Zonula Occluden 1) decreased after incubated with TNFct in cell culture. We approach the action of intestinal mucosal barrier in mice model with FHF to explore the mechanism of SBP inhepatitis gravis complicated.Materials and MethodsReagent:Tumor necrosis factor alpha (TNFα), anti - TNFR1 - IgG, LPS GalN TNFα serum detection kit ZO - 1 antibody FITC marker goat - anti - rabbit IgG fetal bovine serum glutamine nonessential amino acid DMEM culture liquid TRI -zol lysate of cell and tissues anti -TNFα - IgG Caco -2 cell strain.Animals;Six - week - old male BALB/c mice,body weight was from 18 to 22 gram.Methods:1. Animal model: Mice with FHF were injected intraperitioncally Galac-tosamine ( GalN ) (800 mg/kg body weight) , followed by lipopolysaccharide (LPS) (100 μg/kg body weight). For preparation of mice with GalN/ TNFa- treated induced mice model with FHF were given an intraperitoncal injection of GalN (800 mg/kg body weight) , followed by TNFa ( 10μg/kg body weight). In control groups, mice were given an intraperitoincal injection of GalN (800 mg/kg body weight) , LPS (100μg/kg body weight) , NS (equal volum GalN and LPS, body weight) , respectively. Anti -TNFa monoclonal antibody (100μg/mouse) was injected intravenously into GalN/LPS - treated mice 30 minutes before GalN/LPS administration and anti - TNFR1 - IgG was injected intravenously into GalN/LPS - treated mice 15 minute before GalN/ LPS administration.2. The levels of alanine transaminase (ALT) in serum were determined by biochemistry method.3. The level of TNFa in serum was determined using ELISA kit.4. Liver and intestinal specimens were taken and stained with hematoxylin- eosin. Morphological change was observed under light microscope.5. Ultrastructures of intestinal mucosa were observed under transmission e-lectron microscope (H.-600; HITACHI, Japan).6. Immunohistochemistry method was used to detecte the distribution of ZO- I protein in frozen tissue sections.7. ZO - 1 protein in intestinal tissues was determined by Western blot method. The protein bands were detected by using the alkaline phosphatase (AKP) system according to the manufacturer' s instructions (Sigma, USA). (3 - actin was used as a protein loading control.8. Caco -2 cell was cultured according to the methods of described by Elizabeth Willott. Cells were seeded in plates in DMEM until confluence. Experiments were conducted at 7 days after plating. We examined the effects of TNFa on Caco -2 cell at different dosages and different time points.9. Immunofluorescence method was used to detecte the distribution of ZO -1 protein.10. ZO -1 protein in Caco -2 cell was determined by Western blot method. The protein bands were detected by chemiluminescence system according to the manufacturer' s instructions ( Sigma, USA). (3 - actin was used as a protein loading control.11. RT - PCR method was used to detecte the level of ZO -1 mRNA.Results1. Animal model:Administration of LPS/ GalN and TNFa/GalN respectively induced FHF models. Nine hours after GalN/ LPS or GalN/TNFa administration, the mortality of mice reached 59. 3% (54/91) and 62.9% ( 66/105 ). However, the mortality of mice with acute liver necrosis induced by GalN/ LPS did not incease after 12h. The total mortality was 66.7% (80/120) and 69. 6% (94/135). There were no dead animals in other control groups.2. liver functionThe levels of ALT in serum of NS, LPS, GalN and TNFa - treated mice were normal at different time points. But the levels of ALT in serum of GalN/ LPS and GalN/TNFa - treated mice began to increase at 6h(2513. 15 ±874. 18U/L)and(204.1 ±82.05 U/L)after GalN/LPS and GalN/TNFa administration respectively,9h ( 6235.45 ±912.43 U/L) and(4774. 82 ±1117.95 U/L) ,and reached a maximal level at 12h( 10215.75 ±967.71 U/L)and (6177. 84 ± 1280.96 U/L). It began to decrease at 24h ( 10251. 94 ± 1045. 81U/L) and(4204. 58 ±1118. 63U/L). There was significantly difference between in two FHF groups and the other control groups. The levels of ALT in serum of anti - TNFa - IgG and anti -TNFR1 - IgG treated mice at 9h were significantly reduced in mice with FHF at 9 h after GalN/LPS administration (257. 1 ±83. 17U/L)and (904 ±583.00U/L)respectively, P <0.01).3. Liver biopsy:Nine hours after injection GalN/LPS and GalN/TNFa, liver tissues from most mice with FHF presented severe haemorrhage and hepatic necrosis, but a-cidophil degeneration was found in some residual hepatocytes. The hepatic cord disappeared and the structure of liver leaflet deranged. In normal control group, the pathology of liver tissues were normal. And in other control groups, the pathology of liver tissues were slightly alterated.4. The levels of TNFa in serum of mice after treatmentThe levels of TNFa in serum of GalN and NS - treated mice were normal at different time points except of LPS - treated mice at 2h (61. 0982 ±34. 5281 pg/ml) ,and 6h (22.2294 ±13.7507 pg/ml). But the levels of TNFa in serum of GalN/LPS - treated mice began to increase at 2h (446. 1800 ±55. 4914 pg/ ml) and decreased at 6h (14. 8171 ±9. 0108 pg/ml) , and again soared at 9h (319.4310±33.7261pg/ml) and at 12 h (303.7810 ±31.7939 pg/ml). It began to decrease at 24 h (137. 8759 ±46.2482 pg/ml) . There were significant difference at 2h, 9h, 12h comparing with other different time points, P <0.01.5. Assessment of intestinal mucosal injury under light microscope and electron microscope.Pathomorphology of colon mucosa was not remarkably alterated in mice with FHF compared with other control groups under light microscope. Epithelial cells were arranged normally. There were not exuviations and detritus, but just slightly edema and inflammatory cell infiltration.When these TJs in FHF mice were examined by TEM, there were significant differences in the appearances of TJ strands at 9h after GalN/LPS and GalN/ TNFa administration and control groups at 9h after NS, LPS, D - GalN/TNFa administration, and the appearance of TJ strands in anti - TNFct - IgG and anti - TNFR1 - IgG treated groups respectively were not change comparing with normal control group. The morphological abnormalities at 9h in FHF group included that the integrity of tight junctions was disrupted, and microvilli shorted , broken, sheded. The disrupted tight junctions were hardly found at the other time points and the other control groups.6. Bacteria invade intestinal mucosaObservating under TEM, bacteria invasion through the intestinal mucosa started at about 6h and 9h after GalN/LPS administration, but not be seen in other control groups. At the same time, we found the disruption of TJs just at the position where the bacteria invaded. It was obvious that the pattern of bacteria invading intestinal mucosa was pinocytosis, but it was not sure that it entered enterocytes through paracellular pathway.7. ImmunohistochemistryLocal high intensity of ZO - 1 protein was seen in the apical region of the lateral membrane representing TJs of intestinal epithelial cells. There were significant differences in the appearance of ZO -1 protein among experiment group and control groups. The results under light microscopy revealed thatZO -1 protein was exclusively concentrated on the intestinal epithelial cells. In experiment group, the distribution of ZO - 1 protein at 2h and 24h after GalN/LPS and GalN/ TNFa administration were almost unchanged comparing with NS, LPS, GalN, TNFa, anti - TNFa - IgG and anti - TNFR1 - IgG treated groups. Decreased intensity of ZO - 1 protein staining in epithelial cells was observed at 6h and 9h after GalN/LPS administration.8. Western blot analysisWe performed Western blot in colonic mucosa tissue lysates of mice. Results from these experiments demonstrated consistent with the results of immunohistochemistry. There were more significant reduction of ZO - 1 protein expression at 6h (35. 30 ±7.33)% and 9h (25. 62 ±7. 03)% in mice with FHF than at 2h (80. 93 ± 16. 93)% in mice with FHF and at 9h in mice after NS 100% , LPS (117. 70 ± 12. 35)% , GalN (115. 27 ± 17. 11)% , anti - TNFa - IgG ( 83. 17 ± 15. 16) % and anti - TNFR1 - IgG (90. 76 ± 9. 45) % administra-tion, respectively (P<0. 01). These findings are consistent with the results of immunohistochemistry showing down - regulation of ZO — 1 protein in the intestinal mucosa of mice model with FHF. The levels of ZO - 1 protein showed a tendency of diminished protein expression in FHF group at 6h and 9h after GalN/LPS administration.9. The experiment result about TNFa - treated on Caco -2 cells in vitro9. 1 After Caco -2 cells were treated for 24h with the indicated concentrations (50ng/ml) of TNFa, the high expression of the immunofluorescence signal of ZO -1 was in cell nucleus, but the low expression was on cell membrane. In the opposition, when high concentration of TNFa (200ng/ml) treated intestinal mucosal cell, the low signal of the immunofluorescence of ZO -1 was decreased on cell membrane ,? and the same result occured in cell nucleus. When the incubated with TNFa lOOng/ml for Oh, 4h, 8h and 24h, the immunofluorescence signal of ZO - 1 for 24h was decreased in Caco -2 cells.9. 2 Western blot analysis showed that the level of ZO — 1 protein in Caco -2 cells for 24h (43. 84 ±26. 87% ) was significantly decreased than for the other time points (Oh 100% , 4h (91.02 ±4.74)% , 8h(81.90 ±11.10)% ), when incubated with TNFa lOOjig/L for Oh, 4h, 8h and 24h. P <0.01.9. 3 The RT - PCR results showed that after Caco - 2 cells were treated for 24h with the indicated concentrations (0,50,100 and 200 ng/ml) of TNFa, the level of ZO -1 mRNA decreased from 1. 1926 (TNFa:0ng/ml) to 0.7834 (TNFa: lOOng/ml) and 0.7081 (TNFa:200 ng/ml). When TNFa treated with lOOjxg/L for Oh, 4h, 8h and 24h, the levels of ZO - 1 mRNA in Caco -2 cells were significantly decreased at 24h (67. 66 ±5. 65) % , vs control (Oh: 100 %, 4h (.95.45 ±5. 52)%, 8h(81.99 ±5.39 )%) (P<0.01).Conclusions(J)The adopted animal model was beliverable. TNFa plays an important role in animal model with FHF.(2)There was morphological abnormality in the intestinal mucosa in mice model with FHF. The ultrastructural changes included the less of intestinal vil-lus, disruption of tight junction, etc.(3) The bacteria in gut can invade into intestinal epithelial cell by the pattern of endocytosis, and the intercellular tight junction was disrupted just near the region where the bacteria invaded.0 Animal experiment confirmed that high levels of TNFa in serum was one of main causes of the the tight junction disrupted and the expression of ZO -1 decreased in intestional epithelial cells of mice model with FHF. Anti - TNFa -IgG and anti - TNFR1 - IgG can prevent the disruption of intestinal epithelial intercellular tight junction in mice model with FHF and the low expression of ZO — 1 protein in intestional epithelial cells of mice model with FHF.?Cell culture confirmed that the expression of ZO - 1 protein in cell nucleus was increased, but not on cell membrane where intestinal epithelial cell treated with low concentration of TNFa. In the opposition, the expression of ZO - 1 protein was decreased on cell membrane, and the similar result occurred in cell nucleus where intestinal epithelial cell incubated with high concentration of TNFa.?Cell culture confirmed that TNFa decreased the expression of ZO - 1 protein by down - regulation of ZO - 1 mRNA. A dose - dependent relation existed between the concentration of TNFa and the expression of ZO - 1 protein.