The Expression And Significance Of TLR-4 In Lung Of Wistar Rat With Acute Cholangitis Of Severe Type

PrefaceAcute severe cholangitis (ACST) also known as acute obstructive suppurative cholangitis (AOSC), biliary tract infection is the most serious of a disease, acute onset ,severe condition, fast changes, more complications and high mortality. Cholelithiasis from biliary obstruction secondary bacterial infection, and the release of toxins resulting sepsis (sepsis) is ACST incidence of the major reasons for the further development of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). In addition ACST can cause liver damage, lung involvement cause acute respiratory distress syndrome (ARDS) Clinical common Studies show that bacteria and endotoxin (LPS) - induced inflammatory mediators and cytokines excessive release of proinflammatory media / anti-inflammatory media imbalance is the key cause of ARDS.First of bacterial lipopolysaccharide LPS binding protein (LBP), LPS or LPS-LBP monomer complexes destined for the CD14 cell surface receptor binding sites. CD14 is a known patter-reorganization receptor (PRP) in monocytes, macrophages surface . LPS on macrophages, monocytes and neutrophils in the role played an important role. However, CD14 is a membrane anchor protein, and the lack of intracellular and transmembrane region, not directly mediated transmembrane signal transduction. Therefore, people speculate there may be a transmembrane biological molecules, by which the cells to LPS signal transduction can into the intercellular and aroused the intracellular signal transduction.In 1997,Janeway is the first finding and coding hToll Toll-like receptor protein. Research shows that Toll-like receptors (TLR) to identification of pathogens carried by specific molecules ligand (pathogen associated molecular patterns, They). When TLR They were activated by pathogen-associated molecular pattern(PAMPs), they can trigger cell signaling cascade through activated transcription factor NF-kappa B pathway activated cells or other means to start a series of immune and inflammatory response, the release of a variety of cytokines and inflammatory mediators, not only killing pathogenic organisms, but also causing the organism pathological changes. Therefore the combination of TLR and pathogen associated molecular pattern might be the beginning of the session causing SIRS and MODS, further causing inflammation factor (pro-inflammation) and auxiliary costimulatory molecules (Co-stimulate molecules) reversely , playing the role of regulating inflammatory response. There have been at least 10 TLRs family members, TLR-4 as a major member has drawn greater attention , because it can mediated gram-negative bacteria infection of the LPS receptor signal transduction. Currently, it is a hot topics that the relationship between TLR and sepsis . The research on them may be can find new breakthrough for the pathological mechanism of sepsis and effective interventions to find. But ACST, TLR-4 in the lung tissue expression of different points in time as well as their ACST caused ARDS related to the role of research was rare.Based on discussion the relationship between the pathological change and the expression change of TLR-4and TNF-αin the lung tissue of severe acute cholangitis of rats after postoperative 6 hours and 24hours , to make sure the expression rule of TLR-4 in severe acute cholangitis in the lung tissue of rats.Materials and Methods1 An animal model:Wistar rats, male or female ,weight about 180-220g for each one . Before operation ,the rats need to fast for 12 h, fasting water for 4 h. Operations are carried out under aseptic conditions. After anesthesia, preparing the skin and sterilization , putting the sterile hole shop towel. Admission abdominal incision into the abdominal median exposed liver margin, find and bring duodenum, liver duodenum revealed ligament find its shallow running of the common bile duct, into the pancreatic duct ligation of the common bile duct 3-0 margin. The experimental group (n = 24): on the 3rd casingneedle puncture into the common bile duct configuration good ml E.coli bacteria suspension, puncture hole in the proximal cholechochal again after abdominal clearance. The control group (n = 18) with the law in the common bile duct ml saline injection.2 specimen collection:The rats were sacrificed separately after surgery in 6 hours and 24 hours, before the rats were killed carry out 3ml blood by carotid artery to part used to immediately blood gas detect, part of autoclaves placed in the test tube, after standing for 10 minutes, 5000rpm centrifugation, in the supernatant, -20°C preservation, used to do blood biochemical detection and TNF were detected by ELISA method. Cut part of liver and lung tissue, part of them put into 4% formaldehyde solution ,fixed them. Then undergo histopathology detection and TLR-4 immunohistochemical detection. Part quickly placed in liquid nitrogen and then transferred to -80°C refrigerator preservation, will be TLR-4 RT-PCR. Another cut all left lung, weighing, drying again after weighing, doing lung wet / dry proportion of detection.3. detection indicators:1) mouse liver, lung pathological Detection2) blood detection: WBC count and PLT3) blood gas analysis: PaO2, PaCO24) liver function analysis: ALT, DBIL, IDBIL5) lung wet / dry Detection6) TNF-αperipheral detection: ELISA7) detection expression of TLR-4 location in lung tissue: immunohistochemical method8) the detection of TLR-4 mRNA expression in lung tissue: RT-PCR4. statistical methods:Study result was applied by t-test and analyzed by SPSS14.0 software, P <0.05 indicates that there are statistical difference, P <0.01 indicates significant difference.Results and discussion1.An animal model18/18(100%) survival rate for the control group, the experimental group survival 6h group 12/15 (80%), 24h group 12/20(60%). From survival situation, the control group survival, after surgerying all awake, eating. It's visible that the 24h group mouse's urine were yellow dyeing. The experimental group 6h group did not wake up, the temperature cool, urine stained. 24h group, 3/12 wake up, the body temperature was cold, clear yellow with urine. Liver biopsy, the control group had no significant change in 6h group .It can be see from the 24h group that intrahepatic biliary dilatation duct within cholestasis. The experimental group 6h clearly visible swelling of liver cells, with osteoporosis disorder, obviously steatosis, scattered in the point of necrotic foci, necrotic foci in a more inflammatory cell infiltration; 24h more serious pathological changes, more necrotic foci, necrosis was large, abscess formation, with a large number of inflammatory cell infiltration . These results suggest that ACST mouse modeling success.2. Biochemical Index and Blood-lipid DetectionThe experimental group 6h WBC is increased (the experimental group vs. control group 12.530±5.238 vs 7.215±1.053,), 24 hours increased more obviously (the experimental group vs. control group 16.482±6.105 vs 9. 073±3.201, P <0.01). Platelet decline in 6h (experimental group vs. the control group vs 15.328 850.254 200.478±±20.158, P <0.05), 24 hours more notable differences. (Experimental group vs. the control group vs 13.216 793.523 146.537±±25.418), showed that ACST rats apparent systemic infection, sepsis in the performance. The expression of ALT in ACST rats significantly increased in six hours and 24 hours and the experimental group were significantly higher than the control group. (6h group vs 80.734 200.157 470.255±±20.369, P <0.01,24 h vs 87.428 270.327 550.835±±39.215, P <0.05) Compared with simple common bile duct ligation group, IDBIL and DBIL increased slightly, but have no statistical difference. Pulmonary function testing, PaO2 in ACST group decreased significantly, while CO2 retention. The decline in oxygen and hypercapnia suggested that ACST rats had pulmonary dysfunction. Left lung wet / dry in ACST rats significantly higher than in the control group, suggested that the ACST rats'pulmonary lung tissue water content of the obvious, severe pulmonary edema.3. Mouse lung tissue pathological changesMicroscopic observation: 6h control group of normal rat pulmonary alveolar cells, arrangement in order, no exudation in interstitial cells, no inflammatory cell infiltration; there were slightly exudation in interstitial cells in 24 h group. The experimental group 6h the alveolar wall thickening, exudation in interstitial cells increase, consolidation can be found , inflammatory cell infiltration; the same pathological changes between 24 h and 6h group, but a more serious level in 24 h, and pulmonary hemorrhage and abscess, necrosis foci formation can be seen.4.Immunohistochemistry: expression of TLR-4In alveolar epithelial cells can be see the expression of TLR-4 in 6h group suffered from acute severe cholangitis, but it is not expression in the control group or weak. In 24h, TLR-4 expression was significantly decreased.5. RT-PCR: the expression of TLR-4mRNA in lungSix hours after operation on acute severe cholangitis rats, TLR-4mRNA was over expressed in lung tissue, while no or weak expression in control group. 24-hour decline significantly.Conclusion1. It's arisen pulmonary dysfunction and pathologicalinjury in acute severe cholangitis rats.2. Expression of TNF-αin the acute severe cholangitis rats'peripheral blood increased with the time, and positive correlation with lung injury.3. Expression of TLR-4 in rat lung tissues increased significantly ,with the release of TNF-αwere positively correlated in acute severe cholangitis rats.4. The important reasons caused ARDS is that the increased expression of TLR-4 in acute severe cholangitis rats'lung tissues.