Mechanical Role Of Poly(ADP-Ribose)Polymerase In Severe Acute Pancreatitis Associated Adrenal Injury In Rats

Objective:To explore the pathology features, cell apoptosis, and function of the adrenal gland in the rat model of severe acute pancreatitis (SAP). To investigate the tendency of change and roles of poly(ADP-ribose) polymerase (PARP) activity in the course of adrenal injury after severe acute pancreatitis. To explore the dose-response relationship of the PARP inhibitor3-aminobenzamide (3-AB) in rats with SAP. The aim of this study is to explore the effects of PARP on the activation of nuclear factor-κB and NF-κB-dependent gene expression, and to investigate the mechanism of PARP inhibitor3-aminobenzamide on pancreatitis-associated adrenal injury in SAP.Method:Part One Pancreatitis model was established by retrograde infusion of5%sodium taurocholate into the bilio-pancreatic duct. In the sham operation group (control group), pancreas and duodenum was reversed after a midline incision. To detect the time courses of PARP activity, rats were randomly divided into control group and group3,6,12,24h after SAP model induced. Serum amylase (AMY) and lipase (LPS) were detected, and the corticosterone level was measured by an ELISA kit. Histology in pancreas and adrenal stained with H&E (hematoxylin and eosin stain) for light microscopy were analyzed. Apoptotic changes of adrenocortical cells were obversed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. The PARP activity analysis was quantitated by western blot.Part Two Sixty SPF male Wistar rats were randomly allocated into six groups (n=10for each group):sham control group (sham+vehicle, CON group), severe acute pancreatitis model group (SAP+vehicle, SAP group),3-AB pretreatment groups (SAP+3-AB, SAP group, marked T1、T2、T3group, respectively) at different dosage (10,20and40mg/kg), and drug-control group (sham+3-AB group, Drug-Con group)(n=10). SAP model was induced by retrograde infusion of5%sodium taurocholate (1ml/kg) into the biliopancreatic duct. The rats of CON group and SAP group were injected with5%DMSO (2ml/kg) by femoral vein30min before the operation.3-AB pretreatment groups were injected different dosage of3-AB dissolved in5%DMSO, drug-control group was chosen optimal dosage according to the effect of3-AB. Rats in each group were sacrificed at12h after operation to measure the quantity of ascites, serum AMY, ALT and Cr and to observe the pathological changes of pancreatic tissues. Rats in each group were sacrificed12h after the operation to measure the amount of ascites, serum AMY, ALT and Cr and to observe the pathological changes of pancreatic tissues.Part Three150SPF male Wistar rats were randomly allocated into two groups: experimental reseach group (N=90) and observation group of survival rate (N=60). Rats in research group were randomly distributed into three groups:Sham-operation control group (CON+vehicle group, CON group, n=10), severe acute pancreatitis group (SAP+vehicle group, SAP group, n=40),3-AB pretreatment group (SAP+3-AB group,3-AB group, n=40). Rats in SAP group and3-AB group were set up to four subgroups:3h,6h,12h and24h time points (n=8), respectively. The rats of CON group and SAP group were injected with5%DMSO (2ml/kg) by femoral vein30min before the operation.3-AB group were injected3-AB (20mg/kg) dissolved in5%DMSO. All rats were sacrificed at each time point, separately. Ultrastructural changes of adrenocortical cells was analyzed by transmission electron microscopy, apoptotic changes of adrenocortical cells were obversed by TUNEL assay, adrenal myeloperoxidase (MPO) activity was detceted by chromatometry (reflects neutrophil infiltration). PARP activation, NF-κB p65transactivation, protein expressions of tumor necrosis factor-a (TNF-a) and intercellular adhesion molecule (ICAM-1) were analyzed by western blot. In another set of experiment,60rats were randomly divided into three groups (n=20): control group, severe acute pancreatitis group and3-AB group (the operation approach, administration and dosage of vehicle or3-AB were same with experimental reseach group), in order to observe the effect of PARP inhibitors3-AB on survival rate (duration of30h) of rats with severe acute pancreatitis.Results:Part one With the progress of pancreatitis, pancreatic enzyme indicators amylase and lipase, and pancreatic histology score were evaluated significantly. In pancreatitis rats, serum corticosterone level was firstly stimulated from basic level to maximal level at3h, declined subsequently to near basic level at12h, and reached to the bottom which was lower than the controls after24h, demonstrated that worsened adrenal function. Adrenal vessels and sinusoids opened obviously, and were hyperemia at3hours. Adrenal gland was damaged progressively with the duration (6-24hours) of pancreatitis, and microscopy showed obvious structural disorder and hemorrhage, cells necrosis and degeneration. Apoptotic nuclei were rarely found in control group. TUNEL-positive cells mainly existed in the fascicular zone, were significantly increased in adreanal cortex after induction of pancreatitis. The total apoptotic index in adrenal cortex increased steeply related with time and peaked at24h after pancreatitis (P＜0.05). Little immunoreactivity of PAR was detected in control group. PAR immunoreactivity began to inerease at3h, reached its peak at6h and kept a decreased but still high level until24h after pancreatitis, compared with control group (P<0.05).Part Two Compared with CON group, the results of ascites, serum AMY, ALT, Cr, and pancreatic pathologic score in SAP group was all significantly increased (P<0.05). T1group can reduce the quantity of ascites, but failed to decrease the index value of serum and pancreatic pathologic score; T3group can reduce serum AMY, ALT, and pancreatic pathologic score, but failed to decrease the levels of ascites and serum Cr (P>0.05), while T2group can significantly reduce the above-mentioned indicators (P<0.05). The difference of the above-mentioned indicators between Drug control group (20mg/kg3-AB) and CON group was not statistically significant (P>0.05).Part Three Compared with SAP model group, PARP inhibitor3-AB reduced serum pancreatic enzyme indicators and histology score, allivated histopathological and ultrastructural changes in adrenal, downregulated adrenocortical apoptotic index and myeloperoxidase activity (P<0.05), improved the12-24hour serum corticosterone level (P<0.05). NF-κB p65transactivation, TNF-a and ICAM-1protein expressions increased in adrenal tissue after induction of pancreatitis, and inhibition of PARP activity reduced the activation of NF-κB, TNF-a and ICAM-1.Conclusion:1. Pancreatitis-associated adrenal injury of function, morphology and apoptotic changes was revealed in severe acute pancreatitis model rats. Adrenal edema, hemorrhagic necrosis, cell apotosis and structure destruction, as well as the adrenal insufficiency took place with inflammation progress in rats with severe acute pancreatitis. The adrenal injury may be responsible for the adrenal insufficiency in severe acute pancreatitis. PARP activation was a time-dependent rise in adrenal tissue in rats with severe acute pancreatitis, has effects on adrenocortical cells apoptosis and necrosis, suggesting that PARP over-activation plays a crucial role in pancreatitis-associated adrenal injury in severe acute pancreatitis. The PARP inhibitor3-AB20mg/kg administered intravenously was safe, effective and optimal dosage for attenuating the severity of severe acute pancreatitis. Blockade of PARP activity exerts the protective effect against adrenal injury associated with severe acute pancreatitis by inhibition of the inflammation signaling pathway mediated by NF-κB and its related inflammatory mediators, neutrophil infiltration, attenuated adrenocortical cells apoptosis and necrosis, allivated histopathological and ultrastructural changes, and apoptotic index in adrenal.