Severe Acute Pancreatitis Vascular Endothelial Dyfunction In Rats

THE FIRST PART OF THESIS THE INDUCTION OF MODEL OF SEVERE ACUTE PANCREATITIS IN RATS AND THE CHANGES OF VASCULAR EDOTHELIAL FUCTIONObective To set up a animal model of severe acute panctreatitis SAP that was of easy replicated、operability, and good stability; and to observed the morphology and functional changes of vascular endothelial cells (VEC) under SAP pathological state.Methods Sixty-four male SD rats were divided into two groups randomly, including sham operation group(group C) and severe acute pancreatitis group(group A),each group32. Rats in group C were only turned the pancreas and duodenum after laparotomy;in group A were induced by retrograde injection of1.5%deoxycholate.The rats were sacrificed at6,12,24and48hrs after operation (8rats once for each time).Observed the pancreas pathological changes and grading;observed ultrastructural pathological changes in pancreatic acinar cells and vascular endothelial cells;detected in serum amylase;detected vascular endothelium-dependent relaxation and contraction of superior mesenteric artery.Results1.The typical of necrotizing pancreatitis pathological changes,such as Inflammatory cell infiltration,edema of the acinar cells,granular coagulation necrosis,and the lobular structure damage were observed in group A;the pancreas pathological score were significantly increased than group C (P＜0.05),the lesions gradually increased with the time goes by(P＜0.05), thrombus formation in pancreas were observed at6hrs in group A;pancrea morphology were normal in group C.2.Swelling of the acinar cells, a significant expansion in the rough endoplasmic reticulum, many vacuoles in the acinar cells were observed under transmission electron microscopy(TEM) in group A;vascular endothelial cells in the pancreas were observed folding, swelling, basement membrane loosened,membrane damage and even cell death, loss in group A; the sub-cell structure of acinar cells and vascular endothelial cells in group C under were normal under TEM.3.Group A at each time of serum amylase was significantly higher than group C (P＜0.05), and reached a peak at12hrs, no significant difference with the same group of24,48hrs.4. In group A, vascular endothelial relaxation degree at6hrs were significantly decreased compared with group C (P＜0.05), followed by slow decline over time;6,12hrs of vascular endothelial contraction reactivity were no significant difference but lower compared with group C at24h after (P＜0.05).Conclusions Retrograde injection of1.5%sodium deoxycholate to pancreatic duct can be prepared a SAP animal model.The serum amylase increased and the pathological changes were in line with SAP under microscopy and TEM.In early stage of SAP,the vascular endothelial were damaged with the morphological and functional changes. THE SECOND PART OF THESIS SEVERE ACUTE PANCREATITIS ENDOTHELIAL DYFUCTION IN PATHOGENESIS INFLUENCING FACTORSObjective To investigate the mechanism and influencing factors of severe acute pancreatitis endothelial dysfunction.Methods Sixty-four male SD rats were divided into two groups randomly, including sham operation group(C) and SAP group(A),each group32,according to the first part of the method of manufacturing SAP model.The rats were sacrificed at6,12,24and48hrs after operation (8rats once for each time),with detection of inferior vena venous blood serum levels of TNF-α,ET-1content by ELISA;detection of pancreatic tissue MDA content and MPO activity by colorimetric method;detection of the pancreas of ICAM-l,VCAM-1,E-selectin gene expression with RTQ-PCR;detection pancreatic tissue ICAM-1,VCAM-1,E-selectin in the protein expression by Western-blot.Results1.The serum TNF-alpha levels rapidly rised at the early stage in the A group, and over time, gradually increasing tendency;serum ET-1levels were significantly increased at6hrs, and continued to increase until12hrs to the peak, remained relatively high level until48hrs, TNF-alpha, ET-1compared with group C was significantly increased (P＜0.05).2.Compared with group C, the same point in time, A group of pancreatic tissue MDA content of MPO was significantly increased (P＜0.05) and increased with time trend.3.CAM-1、 VCAM-1and E-selectin gene were in low level expression in group C;rat pancreas of group A at each time ICAM-1gene expression was significantly higher (P＜0.05) compared with group C, sustained high expression reached the peak at12hrs and no significant difference between12,24,48hrs.VCAM-1gene expression was significantly higher (P＜0.05) compared with group C,the expression increased rapidly after6hrs,reached the peak at12hrs and sustained high expression up to48hrs;E-selectin gene expression each time compared with group C was significantly higher (P＜0.05) at all time points showed no significant difference between the two groups.4.ICAM-1protein in group C expressed little,6hrs in group A expression compared to group C was significantly increased,showed a higher expression till48hrs; trace amounts of the expression of VCAM-1protein in group C, group A model was significantly increased at6hrs,reached its peak at24hrs,the expression level is sustained high expression till48hrs;E-selectin in group C were only a small amount expression,in group A,the expression level was markedly increased at6hrs and showed a high level of expression maintained up to48hrs.Conclusions The serum levels of TNF-α, ET-1level, pancreatic MPO activity, MDA content and ICAM-1、VCAM-1、E-selectin expression was significantly increased suffering SAP;inflammatory factors, oxidative stress and adhesion molecules involved in the SAP vascular endothelial dysfunction in the pathological process. THE THIRD PART OF THESIS THE PROTECTIVE EFFECT OF LOW MOLECULAR WEIGHT HEPARIN IN SEVERE ACUTE PANCREATITISObjective To investigate the protective effect of low molecular weight heparin in acute necrotizing pancreatitis in rats.Methods Nighty-six male SD rats were divided into tree groups randomly,including sham operation group(group C) and SAP group(group A) and Low molecular weight heparin intervention group (group)G, each group32, according to the first part of the method of manufacturing SAP model. Rats In group G, took a subcutaneous injection of low molecular weight heparin (10U/100g) after modeling of A. The rats were sacrificed at6,12,24and48hrs after operation (8rats once for each time).The pathologic changes of pancreas were observed and graded under the microscope; the level of serum amylase was detected; the level of serum TNF-a and IL-6were detected;pancreatic tissue MDA content and MPO activity were detected;The expression of E-selectin mRNA and protein expression in pancreatic tissue were detected; vascular endothelium-dependent relaxation and contraction of superior mesenteric artery was detected.Results1. Rats in group G at each time pancreas pathological score was significantly decreased than group A (P＜0.05), but still higher than in group C;2. Rats in group G at each time of serum amylase compared with group A was significantly decreased (P＜0.05), but still higher than in group C;3. Rats in group G serum TNF-α and ET-1levels than group A was significantly decreased (P＜0.05), but still higher than in group C;4. MDA levels of group G at each time compared with group A at the same time without significant differences, compared with group C at the same time of pancreatic MDA content was higher (P＜0.05);rats in group G at each time MPO levels compared with rats in group A at the same time was significantly lower,6、12hrs compared with group C the same time were difference,24、48hrs compared with group C increased (P＜0.05);5. Rats in group G at each time E-selectin mRNA and protein expression levels compared to group A was significantly decreased, but still higher than in group C;6. Rat in group G vascular endothelial relaxation degree increased at6,12hrs compared with group A (P＜0.05), no significant differences with group C, compared with group C no significant differences and compared with group A increased at24、48hrs; endothelial contraction reactivity was no significant difference in group C and group at6、12hrs, compared with group C no significant difference and compared with group A increased (P＜0.05)at24hrs, compared with group C lower but higher than group A (P＜0.05).Conclusions The low molecular weight heparin can significantly reduce the rat necrotizing pancreatitis serum amylase, TNF-a and ET-1levels, lowered the expression of adhesion molecules, reduce the MPO activity, improve endothelial function and pancreatic pathological damage.