Paeoniflorin Ameliorates Acute Necrotizing Pancreatitis And Pancreatitis-induced Acute Renal Injury

Objective:To observe the dose-effect relationship of paeoniflorin(PF) on acute necrotizing pancreatitis(ANP). The optimal dose of PF was used to observe the protective effect on acute renal injury following ANP and the probable mechanism of PF on acute renal injury following ANP.Method:Part One:There are 40 SPF male Sprague Dawley(SD) in the first part of this research. All the rats were randomly divided into 5 groups, such as Sham-operation group, ANP group, and the PF+ANP groups and the PF+ANP was subdivide into ANP+P1(50mg/kg),ANP+P1(100mg/kg)and ANP+P1(150mg/kg).Each groups had 8 rats. The PF+ANP groups were that after successfully made the ANP models for 30mintues the differently dose of PF was injected into the femoral vein. The ANP group was only used the 5% sodium taurocholate solution(lmg/kg) into the biliary-pancreatic duct. The sham-operation group was only open the abdomen and flip the duodenum and pancreas. All the groups were sacrificed at the 12 hours time point after the induction of ANP, as the pancreatic damage reached a climax at that time point. The effect of PF was evaluated by determining the levels of serum amylase (AMY), serum lipase (LIPA), aspartate-transaminase (AST), alanine-aminotransferase (ALT), BUN?Cr and the pathological changes of pancreas shown by hematoxylin-eosin (H&E) staining.Part Two:There are 72 SPF male Sprague Dawley(SD) in the second part of this research. All the rats were randomly divided into 3 groups:the Sham-operation group, ANP group, and the PF+ANP groups(the optimal dose of PF is 100mg/kg) and each group had 24 rats. The sham-operation group was only open the abdomen and flip the duodenum and pancreas. The ANP group was lead by the 5% sodium taurocholate solution(lmg/kg). The PF+ANP group was that after successfully made the ANP models for 30mintues the differently dose of PF was injected into the femoral vein. All the groups were sacrificed at the3hour,6hour and 12 hours time point after the induction of ANP, as the pancreatic damage reached a climax at that time point. The serum levels of TNF-?, IL-1? and IL-6 were measured by ELISA kits according to the manufacturer's instructions. The serum levels of BUN and Cr were measured by the automatic biochemistry analyzer with standard techniques. The kidneys were fixed in 4% paraformaldehyde and the hematoxylin-eosin (H&E) staining was used for pathological examination.Part Three:To explore the probable mechanism of PF on acute renal injury following ANP. The NO detection kit was used for measuring the NO production in different groups. The immunohistochemistry staining analysis of NF-?Bp65 in kidney tissues were also in this part of this part. Western blot analysis was used in the part for detected the P38 MAPKs signal way in kidney tissues and discuss if the P38 MAPKs signal way take part in mechanism of the PF on acute renal injury following ANP. The expression of Caspase-3 in kidney tissue was measured by the immunohistochemistry staining and the TUNEL kit was also used to detected the renal cell apoptosis.Results:Part One:To obtain the optimal dose of PF for preventing the acute necrotizing pancreatitis. The results shown that the optimal dose of PF is 100mg/kg. The 50 mg/kg can lower the serum level of AMY and LIPA than those in ANP group and the pathological examination by H&E staining also expressed that the 50mg/kg PF can low the pancreatic pathological scores, the 50mg/kg PF had a small effect on the liver and renal function in rats. The serum level of AMY and LIPA in 100mg/kg PF group is lower than those in ANP and 50 mg/kg group. The serum level of AST?ALT?BUN and Cr had a little change compared to the sham-operation group. Compared to the 50mg/kg group the 100mg/kg group had a better expression in lower the AMY and LIPA level and the serum level of AST?ALT had a little different than 50mg/kg group. The pancreatic pathological scores in 100mg/kg group had a better result than that in 50mg/kg and sham-operation group.150mg/kg group can lower the serum level of AMY, LIPA and pancreatic pathological scores. However the serum of AST,ALT, had a obvious raise than the 100mg/kg,50mg/kg and the sham-operation group. The 100mg/kg group is the optimal dose for preventing the ANP.Part Two:To observe the protective effect of PF on acute renal injury following ANP. In the first part of this research the 12 hour time point is the peak time point of the inflammatory reaction of ANP. In the second part the outcome shown that the PF had the protective effect on acute renal injury following ANP. PF can lower the level of serum of TNF-?, IL-1? and IL-6. The H&E staining of kidney tissue shown that the PF can decrease the pathological scores of kidney. In this way, the PF had a protective effect on acute renal injury following ANP.Part Three:To explore the probable mechanism of PF on acute renal injury following ANP. The levels of NO in renal tissues from the ANP group were markedly increased compared with those in the sham-operated group. However, treatment with PF significantly reduced the NO production. Immunostaining of NF-?B was measured in the kidney samples at the 12 h time point in each group. In the ANP+PF group, the expression of NF-?B was lower than in the ANP group. A marked increase in caspase-3 staining was found in the nucleus in the ANP group. In the ANP+PF group the expression of caspase-3 was significantly decreased. The ANP+PF group exhibited a significant decrease in the ratio of p-p38/p38 compared with the ANP group at the 12 h time point through the western blot.Conclusion:The results of this study showed that PF can ameliorate ANP and protect the kidneys in ANP. 100mg/kg dose of PF is the optimal dose for the treatment. The probable mechanism of PF on acute renal injury following ANP may associated with the release of NO in kidney tissues. PF can inhibit the release of NO. The Immunohistochemistry analysis, western blot and TUNEL assay shown that the effect of PF on acute renal injury following ANP may associated with the p38MAPKs signal pathway and then inhibit the level of NF-?B. In this way to protect the kidney cells.