| Objectives:Severe acute pancreatitis(SAP) is a common clinical acute abdomen operation, the mortality rate as high as 30%, the main clinical manifestations included pancreatic tissue edema, hemorrhage, necrosis, infiltration of inflammatory cells, pancreatic tissue exudation. Ischemia reperfusion injury(IRI) can significantly increase the SAP condition, which leads to the systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction syndrome(MODS).Pancreatic microcirculation disorder is one of the important pathogenesis of SAP and the pathophysiological changes in the performance.The microcirculation disorder include mailly of pancreatic microvascular blood flow changes; micro artery and arteriole diameter change; capillary diameter,density, the perfusion changes; pancreatic microvascular permeability;hemorheology; cell adhesion factor change etc.Improve the microcirculation is the important measures and key steps in treatment of SAP, although many drugs try to improve pancreatic microcirculation, but the effect is not ideal, so to look for new ways to improve pancreatic microcirculation is necessary.Vascular Bradykinin is also named Pancreatic Kininogenase(PK), as an activator, which is composed of 18 amino acids and 4 kinds of sugar, the in vivo degradation of kininogen kinin release, which directly effecton the small blood vessels and capillaries to expand, increasing the vascular network node in the dilation of blood vessels at the same time, improve the permeability of blood vessels and blood flow; inhibition of angiotensin I converting to angiotensin II, reduce microvascular contraction; by relieving the kinin and histidine peptide of XII a negative feedback, the inhibition of calcium and blood coagulation time, promote vascular endothelial cells to produce PGI2, inhibit platelet aggregation, to prevent blood coagulation, and plasminogen-activator plasminogen into fiber, improve fibrinolytic activity, the content of fibrinogen decreased, prevent thrombosis; decomposition LMWK generatepeptide, is involved in many physiological processes, and is able to regulate the inflammatory reaction process.Although PK has broad pharmacological effects, but whether it can improve SAP pancreatic microcirculation has not been reported. This experiment is to investigate the effect of PK on pancreatic microcirculation in SAP with IRI, and provide experimental basis and theoretical basis for its clinical application in SAP.Methods:1 Experimental animal:Male Wistar rats of clean grade, 3-4 months of age, body weight 300-350 g, Beijing Longan experimentalanimal center, free feeding and drinking water.2 Animal grouping:The first part: 60 rats were divided into two groups,and each group wdivided into three little groups, named the control group, SAP with Igroup, PK treatment group,all rats were sacrificed 12 hours after themodeThe serum AMY/LIP, serum inflammatory mediators of TNF-a/ILexpression, plasma endotoxin,and pancreatic pathological score wede-tected.The second to fifth parts: there were 30 rats in each part, named the control group, SAP with IRI group, PK treatment group,all rats were sacrificed 12 hours after the model. The pancreatic microcirculation indexes of rats was detected in the second group, the pancreatic microvascular permeability was detected in the third group, the blood rheology of rats was detected in the fouth group, the white blood cell adhesion factor CD18, vascular endothelial cell adhesion factor CD54 expression was detected inthe fifth group.3 Preparation methods of animal model:3.1 Preparation methods of SAP with IRI model:SAP model preparation: by the method of Aho and improved,30mg/kg, 3% sodium pentobarbital intraperitoneal injection of anesthesia, after the success of fixed on the operating board, minus the abdominal hair, abdominal skin disinfection, with sterile towels. The median incision 3cm into the abdomen, duodenum, pancreatic tissuefound in the inner sheet, find the pancreatic duct. In the hepatoduodenal ligament of liver door to small artery clip to prevent sodium taurocholate into bile, liver. 24 G in pancreatic duct to the duodenum at the opening of the needle puncture, into the pancreatic duct of 0.5cm and a temporary suture needle, connecting micro infusion pump, through the pancreatic duct, 0.2ml/min rate, 0.1ml/kg weight of 5% sodium taurocholate into thepancreatic, 5 minutes after the needle, hyperemia and edema, removed artery clamp, sterile cotton swabpressure puncture point in 5 minutes, and the reduction of abdominal organs.IRI model preparation: according to Fujimoto ’method, the SAP immediately after the preparation was clamped for 30 minutes due to release of splenic artery, pancreatic ischemia, temporary abdominal closure. Before model 12 h fasting water deprivation; rats immediately after subcutaneous injection of normal saline 5ml,free drinking water, pay attention to keep warm. The blank control group in the same way retrograde pancreatic duct injection of physiological saline, more than the same conditions. All the experimental animals by medical ethics management standard.3.2 Preparation method of the PK treatment after SAP with IRI model:After the SAP with IRI model,the rats exposed the tail, and then use the 40-45 of warm water for half a minute, the vascular congestion, dry spare. Choose 5 needles, 30 degree angle to the tip of the tail 2cm into the needle, parallel move, see back to the blood injection. According to drug instructions immediately after the model, PK by 2U/ only, soluble in 5ml NS, continuous infusion of caudal vein injection of drugs for 12 hours.3.3 Preparation methods of the control model:The control group in the same way retrograde pancreatic duct injection of physiological saline, more than the same conditions. All the experimental animals by medical ethics management standard.4. Detection of PK on the indexs of SAP with IRI model of rats:4.1 Detection of PK on serum AMY/LIP of SAP with IRI rats: The detection were measured by automatic biochemistry analyzer. The rats after the model of 12 h drew blood in the abdominal aorta 2.5ml about, put into the sealed coagulation promoting tubes in static 1h,centrifugal 10min(3000r/min), analyzed AMY/LIP by using CHEMIX-800 automatic biochemical.4.2 Detection of PK on serum inflammatory mediator expression ch-anges of TNF-a/IL-1 of SAP with IRI rats:Detection of serum TNF-a/IL-1 express-ion level by using the method of ELISA. At the same time of detection of serum enzyme, drew blood in the abdominal aorta 2.5ml into the sealed coagulation promoting tubes in static1 h, centrifugal 10min(3000r/min), serum preserved in refrigerator to be-20℃, serum TNF-a/IL-1 was dete-cted in strict accordance with the kit instructions requirements.4.3 Detection of PK on endotoxin of SAP with IRI rats: Plasma endotoxin detection by using tachypleus amebocyte lysate matrix azo chromogenic method. The rats were sacrificed 12 hours after model, blood was collected from the abdominal aorta 5ml, in strict accordance with the limulus kit requirements for test equipment used to do heat treatment, blood was obtained from the serum levels of 0.1ml, the configuration agent, mixing with limulus reagent 0.05 ml, 37 ℃water bath after the removal of 3min, adding sodium nitrite solution 0.5ml, after mixing evenly add sulfamic acidamonia 0.5ml mixing, adding nano 0.5ml mixed with 723 spectrophotometer at the wavelength of 545 nm colorimetric readings agent. Formula: A=As-(Ab-Abb), according to the above formulas, the absorbance A value, in the investigation of the endotoxin content standard curve of endotoxin.4.4 Changes of PK on pancreatic pathological score of SAP with IRI rats:Evaluation from two aspects of general and the pancreas pathological section. Before the detection of blood serum endotoxin, observe the changes of pancreatic tissue and pancreas first, then drew pancreatic tissue 100 mg, fixed by 10% formalin, routinelyembedded in paraffin, HE staining, to observe the pathological changes under the light microscope, the method according to the modified Schmidt and Pozsar method score from edema, hemorrhage, necrosis andinflammatory infiltration and so on four aspects, each was respectively 0, 1, 2, 3, 4 and five scores, andcalculate the total score summary.5 Detection of PK on the pancreatic microcirculation of SAP with IRI rats: Detection of the blood flow of pancreatic microcirculation of ischemia reperfusion injury in rats with severe acute pancreatitis:The blood flow pancreatic microcirculation was detected by laser Doppler flowmetry. By diameter 0.25 mm, wavelength 780 nm, the probe touch the surface of pancreas at the pancreatic head, the tail of the pancreas, the midpoint of pancreatic body and tail, select the measurement line, avoid the large blood vessels or hematoma.Including pancreatic blood flow and blood flow velocity changes; micro artery and arteriole diameter change;capillary diametr, density, the perfusion changes, finally take the arithmetic mean value as a result.6 Detection of PK on the pancreatic microvascular permeability of SAP with IRI rats :Pancreatic microvascular permeability was measured by Evans blue method. By the method of Keiji and improved, 12 h after modeling for detection of pancreatic microcirculation in the rats after femoral vein at the doseof 2ml/kg injected 1% Evans blue, take all the pancreatic tissue after 30 minutes, and weighed after clipping about 150 mg after weighing into 5ml tubes, in accordance with the 0.03/mg addition of formamide. 24 hours after taking the leaching solution by 752 C 1ml, UV visible spectrophotomety determination of Evans blue, and calculate the Evans blue leakage; the remaining tissue was cut in 160 degree heat drying box, 24 hour after weighing, the wet / dry weight ratio of pancreas tissue dry weight calculationof 150mg; and finally to Evans blueleakage/pancreatic tissue dry weight(ug/g) said the microvascular permeability.7 Detection of PK on hemorheology of SAP with IRI rats:The hemorheology were detected by BV-100 blood rheology test instrument. The rats after the model of 12 h drew blood in the abdominal aorta 5ml, shaked and detected by blood rheology test instrument. Including the whole blood high, low cutting rate, plasma viscosity, whole blood reduced viscosity;erythrocyte aggregation index, erythrocyte sedimentation rate, equation K value of ESR; erythrocyte deformation index, erythrocyte rigidity index; hematocrit, fibrinogen.8 Detection of PK on white blood cell adhesion factor CD18 and vascular endothelial cell adhesion factor CD54 expression of SAP with IRI rats:Detection of white blood cell adhesion molecules by flow cytometry. The rats after the model of 12 h drew blood in abdominal aortic 5ml, heparin, 100 ul anticoagulant, add FITC mark Dan Kang(anti CD18, CD54) 20 ul at room temperature, the dark incubation with 40 min, the hemolytic agent for lysis of red blood cells, 1500r/mincentrifugal 10 min, go to the supernatant, and PBS buffer(4ml PH7.4) mixing, 1500r/min centrifugal 15 min,adding 1g/L paraformaldehyde fixed, 24 h analysis, the analysis of each cell line CD18 and the expression level of CD54 receptor, expressed by the fluorescence density of FITC mark Dan Kang.9 Statistical method:With SAS 8.0 statistical software package, the results of serum AMY/LIP, TNF-a/IL-1, endotoxin,and blood flow of pancreatic microcirculation, pancreatic microvascular permeability, hemorheology, white blood cell adhesion factor CD18, vascular endothelial cell adhesion factor CD54, were expressed by " x ±s ",using the method of variance, P < 0.05 was statistically significant. The pathological scores are grade data, using Chi square test,P < 0.05 was statistically significant.Results1 Effect of PK on the monitoring index of SAP with IRI rats:1.1 Effects of PK on serum AMY/LIP of ischemic reperfusion injury in rats with severe acute pancreatitis:Compared with the blank control group, the serum AMY/LIP in SAP and IRI group increased significantly. Compared with the SAP and IRI group, serum AMY/LIP of PK group decreased significantly.1.2 Effects of PK on serum inflammation media of TNF-a/IL-1 expression of ischemic reperfusion injury in rats with severe acute pancreatitis:Compared with the blank control group, the serum inflammation media of TNF-a/IL-1 in SAP and IRI group increased significantly.Compared with the SAP and IRI group, serum inflammation media of TNF-a/IL-1 of PK group had no obvious change.1.3 Effects of PK on serum endotoxin of ischemic reperfusion injury in rats with severe acute pancreatitis:Compared with the blank control group, the serum endotoxin in SAP and IRI group increased significantly.Compared with the SAP and IRI group, serum endotoxin of PK group had no obvious change.1.4 Effects of PK on pancreatic pathological changes of ischemic reperfusion injury in rats with severe acute pancreatitis:The naked eye observation of anatomy of rat pancreatic tissue: blank control group rats had no edema, expansion of gastrointestinal and abdominal ascites. Pancreatic tissues in SAP with IRI group were associated with congestion and edema, hemorrhage, focal necrosis of bloody ascites, saponified spots significantly. Pancreatic tissues in PK group the above-noted index relieved significantly, especially the hemorrhage and focal necrosis of bloody ascites. Observation of pathological section under light microscope: the blank control group had normal pancreatic acinarstructure integrity, closely arranged, no hemorrhage and necrosis. SAP with IRI group,had pancreatic interstitial edema, hemorrhage, microthrombosis in small vein,interstitial vascular hemorrhage, coagulation necrosis and obvious with patchy infiltration of inflammatory cells. PK group also had pancreatic interstitial edema, hemorrhage, microthrombosis in small vein,interstitial vascular hemorrhage, coagulation necrosis and obvious with patchy infiltration of inflammatory cells,butrelieved significantly.2 Effect of PK on the pancreatic microcirculation of SAP with IRI rats:2.1 Effects of PK on pancreatic microvascular perfusion of blood flow volume and blood flow velocity of ischemic reperfusion injury in rats with severe acute pancreatitis:Compared with the blank control group, the blood flow volume of pancreatic microcirculation in SAP and IRI group decreased and the blood flow velocity slows down gradually. Compared with the SAP and IRI groups, the blood flow volume and blood flow velocity of pancreatic microcirculation of PK group increased.2.2 Effects of PK on pancreatic microvascular diameter of ischemic reperfusion injury in rats with severe acute pancreatitis:Compared with the blank control group, the pancreatic micro artery diameter in SAP and IRI group was thinner significantly; pancreatic micro vein diameter did not change significantly. Compared with the SAP and IRI groups, pancreatic micro artery diameter of PK group increased significantly; but pancreatic micro vein diameter did not change.2.3 Effects of PK on capillary of ischemic reperfusion injury in rats with severe acute pancreatitis:Compared with the blank control group,the capillary diameter and density in SAP and IRI group reduced,capillary perfusion form changed from stable to intermittent and irregular. Compared with the in SAP and IRI group, the capillary diameter and density in PK group increased.3 Effect of PK on the pancreatic microvascular permeability of SAP with IRI rats: Compared with the blank control group, pancreatic microvascular permeability in SAP and IRI group increased rapidly. Compared with the in SAP and IRI group, pancreatic microvascular permeability of PK group decreased significantly.4 Effect of PK on hemorheology of SAP with IRI rats :4.1 Effect of PK on the blood viscosity of ischemic reperfusion injury in rats with severe acute pancreatitis : Compared with the blank control group, the whole blood low, middle,high rate viscosity, plasma viscosity, whole blood reduced viscosity in SAP and IRI group increased significantly. Compared with the SAP and IRI group, the whole blood low, middle,high rate viscosity, plasma viscosity, whole blood reduced viscosity of PK group decreased significantly.4.2 Effect of PK on red blood cell aggregation of ischemia reperfusion injury in rats with severe acutepancreatitis:Compared with the blank control group,the erythrocyte aggregation index,erythrocyte sedimentation rate, ESR equation K value in SAP and IRI group increased significantly. Compared with the SAP and IRI group, PK group erythrocyte aggregation index, erythrocyte sedimentation rate, ESR equation K value of PK group decreased significantly.4.3 Effects of PK on red blood cell deformability of ischemia reperfusion injury in rats with severe acutepancreatitis:Compared with the blank control group,the erythrocyte deformation index,erythrocyte rigidity index in SAP and IRI group increased significantly.Compared with the SAP and IRI group,the erythrocyte deformation index,erythrocyte rigidity index of PK group decreased significantly.4.4 Effects of PK on hematocrit and fibrinogen of ischemia reperfusion injury in rats with severe acutepancreatitis:Compared with the blank control group, the hematocrit and fibrinogen in SAP and IRI group increased significantly.Compared with the SAP and IRI group,hematocrit and fibrinogen of PK group decreased significantly.5 Effects of PK on the white blood cell adhesion factor CD18 and vascular endothelial cell adhesion factor CD54 expression of SAP with IRI rats:Compared with the blank control group, the CD18 and CD54 in SAPand IRI group increased significantly. Compared with the SAP and IRI group, CD18 and CD54 of PK group decreased significantly.Conclusions:1 Retrograde cholangiopancreatography pump into the 5% sodium taurocholate of preparing SAP rat model. The method is stable and reliable to operation and pathological changes of pancreas.2 After SAP model set up, occlusion of splenic artery for 30 min and then open to set up IRI, The method is high stable, and serum enzyme changes and pathological changes can meet experimental requirements.3 PK can reduce the expression of serum AMY/LIP levels in rats of SAP with IRI.4 PK can not reduce the expression of serum inflammatory mediators TNF-a and IL-l levels in rats of SAP with IRI.5 PK can not reduce the expression of serum endotoxin level in rats of SAP with IRI.6 PK can relieve pancreatic tissue edema, especially the hemorrhage and focal necrosis of bloody ascites.7 PK can increase pancreatic microcirculatory blood flow volume and blood flow velocity of micro artery; expand capillaries, increase capillary density, stable capillary perfusion in rats of SAP with IRI.8 PK can reduce pancreatic microvascular permeability in rats of SAP with IRI.9 PK can reduce whole blood high,middle, low whole blood viscosity, plasma viscosity, whole blood reduced viscosity; reduce red blood cell aggregation index, erythrocyte sedimentation rate, equation K value of ESR; reduce the erythrocyte deformation index, erythrocyte rigidity index reduced pressure; red blood cell volume, fibrinogen in rats of SAP with IRI.10 PK can reduce serum levels of white blood celladhesion factor CD18 and vascular endothelial cell adhesion factor CD54 expression in rats of SAP with IRI. |
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