Protective Effect And Mechanisms Of Melatonin On Severe Acute Pancreatitis Associated A Cute Renal Injury

Abstract:Objective:Observation of superoxide dismutase (SOD), malondialdehyde (MDA),kidney tissue Livin protein and Caspase-9 expression about severe acute pancreatitis rat serum in order to explore melation on severe acute pancreatitis related protective effect of renal injury and related mechanisms. Method:nighty female Sprague-Dawley(SD) rats were randomly divided into normal 12 hours group(n=10),normal 24 hours group(n=10),normal 48 hours group (n=10),SAP(severe acute pancreatitis) 12 hours group(n=10),SAP 24 hours group (n=10),SAP 48 hours group (n=10),MT(melatonin treatment) 12 hours group(n=10),MT24 hours group(n=10),MT48 hours group(n=10). Prior to the experiments, rats were deprived of food with free access to water. All procedures including 3% pentobarbital sodium intra-peritoneal injection (40mg/kg) for anesthesia were performed under sterile conditions. Common disinfection, 1mL/kg body weight of 5% sodium taurocholate was retrograde injected into the biliopancreatic duct of the rats to induce SAP. Rats in the normal group underwent operation with nothing infused. The pancreas was flipped and striked gently. After operation, rats were free access to food and water. After operation 10 minites,3ml melatonin solution put to use for melation groups with subcutaneous injection.Melatonin selected in accordance with literature data of high dose(50mg/kg).Melatonin solution was first dissolued in ethanol,and then diluted with normal saline,so that the final ethanol concentration was 1%.The serum amylase,creatinine (Cr),blood urea nitrogen (BUN) were measured by automatical analyzer.The MDA was measured by thiobarbituric content,and the determination of SOD activity was xantline oxidase. Immunohistochemistry was used to detect kidney Livin and Caspase-9. The determination of renal cell apoptosis index was in situ end labeling (TUNEL) detection. Result:1. Pathological change of pancrea:Normal control group have no significant pancreatic lesions in general.The panacrea tissue surrounding pancreatic duct was redden immediately and expanded gradually and deepen. After 12 hours, there was obvious hyperemia and edema, the pancreatic tissue was darken and there were a little necrosis and hemorrhage and moderately ascites. In the melatonin treatment 12h group,the degree of color,hyperemia and edema of pancreatic tissue were less than that in the SAP 12h group,there was no obvious necrosis and hemorrhage. After 24 hours, rats were abdominal distension obviously, pink bloody ascites could be finded in abdominal cavity, conglutination of pancreas and surrounding tissue was serious. The color of surface was beige. But in melation group,the bloody ascites were moderately,intestinal was engorgement,conglutination of pancrea and surrounding tissue was less serious, the color of pancreatic was violet and dark. After 48 hours, rats were abdominal distension obviously, bloody ascites could be finded in abdominal cavity,conglutination of pancreas and surrounding tissue was serious. The color of surface was beige. But in melation group,the bloody ascites were moderately,intestinal was engorgement,conglutination of pancrea and surrounding tissue was less serious, the color of pancreatic was violet and dark. Observation under light microscope:Texture of normal pancreaic tissue was intact, without inflammatory cell in interstitial tissue. After 12 hours,widen of pancreatic lobule gap,leafage gap,gland alveolus and infiltration of less inflammatory cell could be found. The state is more similar in melation group,but the degree was lower than that in the SAP 12 hours group. After 24 hours, pancreatic tissue became edema,hemorrhage,foliated necrosis, infiltration of a great quantity of inflammatory cell, disappearance of parts of normal texture. In the melatonin 24 hours, the pancreatic interstitial substance became rarefaction,wild edema, the red blood cell exude, the pancreatic lobule was found a little necrosis, infiltration of a small quantity of inflammatory cell. After 48 hours, pancreatic tissue became edema,hemorrhage,foliated necrosis, infiltration of a great quantity of inflammatory cell,disappearance of most parts of normal texture. In the melatonin 48 hours, the pancreatic interstitial substance became rarefaction,wild edema, the red blood cell exude, the pancreatic lobule was found a little necrosis, infiltration of a small quantity of inflammatory cell.2.Pathological changes in kidney:Normal control group rat kidneys in general have seen no obvious lesions. SAP model group 12H appearance of edema in kidney, was dark purple; SAP model group 24H kidney congestion and edema, and bleeding points can be see. SAP model group 48H kidney have more congestion and edema,and most bleeding points can be see. At each time point of renal pathological changes in SAP groups of melatonin groups were less than SAP groups. Light microscope observation:The normal control group was no exception. Each time point of renal cortex and medulla structure were clear. SAP Group 12H the glomerular mildly swell, The tubular swell more obvious, renal interstitial infiltrated of inflammatory cells, At SAP 24H the glomerular,tubular swelling increased,glomerular congestion can be seen, visible necrosis of renal tubular epithelial cells, and the tube can be seen. At SAP 48H the glomerular,tubular swelling increased,glomerular congestion can be seen, visible necrosis of renal tubular epithelial cells, an increase in renal interstitial inflammatory cells. At each time point in melatonin group those of pathological changes in light microscope were less that of the SAP groups.3. Biochemical Indicators:SAP-made module at different time points of serum AMY, Cr, BUN and MDA levels at all time points compared with the normal group was significantly higher (P<0.01), gradually increased with time. SAP-made module at different time points SOD activity was significantly lower than normal(P<0.01), gradually decreased with time. Melatonin in the intervention group at different time points of serum AMY, Cr, BUN and MDA content compared with SAP-made module at different time points significantly lower(P<0.05), decreased gradually with time; Melatonin in the intervention group at different time points the SOD activity was significantly higher than SAP-made modules (P<0.01), increased gradually with time. 4.Apoptosis-related indicators:SAP-made module at each time point the positive rate of Livin protein was lower than the normal group(P<0.01),Caspase-9 positive rate was higher than the normal group(P<0.01); Melatonin in the intervention group at each time the Livin protein positive rate was increased than the SAP model group(P<0.01), Caspase-9 positive rate was lower than the SAP model group(P <0.01).5.Correlation Analysis:5.1 SOD, MDA and renal damage in analysis of results:SOD was negatively correlated with serum Cr(r=-0.916, P<0.01), MDA was positively correlated with serum Cr(r=0.807, P<0.05).5.2 SOD, MDA and its correlation analysis:renal cell apoptotic rate was negatively correlated with SOD(r=-0.827, P<0.01),Was positively correlated with MDA(r=0.607, P<0.05).5.3 SOD, MDA and apoptosis-related protein Livin, Caspase-9:SOD and Caspase-9 was negatively correlated(r=-0.382, P<0.05), MDA and the expression of Caspase-9 was positively correlated (r=0.356, P<0.05); SOD had no correlation with Livin expression (r=-0.572, P> 0.05), MDA and Livin expression was not correlated (r=-0.489, P> 0.05).Conclusion:The study demonstrates that melatonin plays an protective role on SAP with Acute kidney injury, The mechanism exerts this effect may depend on the inhibition of lipid peroxidation and reduce apoptosis in the kidney.