Role Of Phosphatidylinositol 3-kinase/protein Kinase B(PI3K/PKB) Signal Transduction Pathway In The Activation Of Neutrophils During Acute Necrotizing Pancreatitis

Role of phosphatidylinositol 3-kinase/protein kinase B(PI3K/PKB) signal transduction pathway in the activation of neutrophils during acute necrotizingpancreatitisIntroductionAcute necrotizing pancreatitis (ANP) has become focus of clinical research because of its high mortality and severe features. ANP's pathogenesis is extremly complicated, and now it is believed to be a systemic inflammation involved with many kinds of inflammatory factors and cells, such as neutrophils, mononuclear macrophage, and endothelial cell. As more the more information about ANP has been obtained by clinicans and researchers, people gradually realized that systemic inflammation response syndrome(SIRS) and multiple organ failure(MOF) following SIRS are two main causes of death after ANP. Decreasing the severity of SIRS can reduce the possibility of MOF, therefore decreasing mortality. More and more evidence indicate that pro-inflammatory factors excreted by inflammatory cells are the key factor to lead to SIRS. Researchers suspect that monoclonal antibodies and antagonists to these cytokines may be benefit to the inflammation during ANP. Althougt later research prove these conclusions, therapeutic results are not ideal. Plenty of cytokines which can act and induce each other constitute a complex network, so the strategy that only acts on a single or several cytokines has resulted in limited success. How to regulate the production of cytokines and inhibit the development of SIRS and MOF is very important to management of ANP.Although there are numerous cytokines, signal transduction pathways that can regulate the production of these cytokines are rare. Effective inhibition or regulation to some key pathways may control the production of pro-inflammatory cytokines, furthermore, interrupt the development of SIRS and MOF. So, it is very important to investigate the role of signal transduction during the progression of acute necrotizing pancreatitis. Phosphoinositide 3-kinases(PI3K) and the downstream serine/threonine kinase AKT/protein kinase B have a central role in modulating neutrophil activation, chemotaxis, and apoptosis. PI3K is a heterodemeric complex, comprising a p110 catalytic subunit, of which there are four characterized isoforms(α,β,γ,andδ). The type IA PI3Ks, p110α, p110β, and p110δ, associate with the p85 family of regulatory subunits, but type IB p110γbinds to a p101 adaptor molecule, p110γis activated by engagement of G-protein coupled recaptors.The serine/threonine kinase Akt/protein kinase B is the best characterized target of the PI3K-generated phosphoinositides PtdIns-3,4-P₂ and PtdIns-3,4,5-P₃. PKB has the ability to activate the regulatory IκB kinase, IκBα, through phosphorylation at a PKB phosphorylation-consensus sequence at Thr²³. PKB-dependent activation of IκBαleads to accelerated degradation of IκBαand enhanced translocation of NF-κB to the nucleus. NF-κB binding to special promoter can improve transcription actitvity of inflammatory factor gene, and lead to the production of cytokines. So, PI3K/PKB signal transduction pathway is the main channel to regulate the production of cytokines. Results from studies to trauma,burn,and septic shock indicate that PI3K/PKB signal transduction pathway is the main way to control inflammatory reaction. Inhibiton of PI3K/PKB signal transduction pathway can decrease the production of cytokines and improve survival rate. Now, the role of PI3K/PKB signal transduction pathway in the development of ANP remains unclear, so we investigate its role in order to elucidate the pathogenesis of ANP on signal transduction level and provide new therapy strategy for ANP.Materials and MethodsMaterials and equipmentsSD rats with body weight 250～300g were provided by the experimental animal center of China Medical University.sodium pentobarbital; Dextran T-500; Ficoll-Paque Plus; sodium taurocholate; trypsin: wortmannin; LY294002; RNAout; RT-PCR Test Kit; PKB and p-PKB antibody; PVDF; ELISA Test Kit for rats TNF-αand IL-1β; immunohistochemistry Test Kit by SABC; MPO Test Kit etc.operating boar for cell culture, SANYO MDF-U ultra deep-freeze equipment,OLYMPUS AX70 invert microscope,GFL THERMOLAB oscillative water bath box,hypothermia high speed centrifuger,DYY-6C electrophresis apparatus,Bio-Rad Seimidry transfer system, Chemilmager5500 auto-electrophoresis gel imaging analytical system,PTC-100 PCR amplification device, horizontal plate electrophoresis system,GLS-700D gel imaging analytical system,A-200 Ds electronic balance,B-Brown trace pump,PH meter etc.Methods1,Isolation and identification of neutrophils:Neutrophils were separated by density gradient centrifugation and identified by Wright's staining.2,Establishment of acute necrotizing pancreatitis model:ANP model was eatablished by retrograde injection of sodium taurocholate into biliopancreatic duct.3,Experimental protocols:Firstly, separated neutrophils were divided into four groups: control group(C),trypsin group(T),trypsin＋wortmannin(W) and trypsin＋LY294002 (L). After pretreatment by Wortmannin(final concentration 200nmol/L) (group W) and LY294002(final concentration 100μmol/L) (group L) respectively for 1h, group W and L were stimulated by trypsin(final concentration 40nmol/L) for 1h. At the same time, group T was also stimulated by trypsin (final concentration 40nmol/L) for 1h. After stimulation, we collected neutrophils and culture medium, protein and mRNA levels of TNF-αand IL-1βin cell culture and cells were examined by ELISA and RT-PCR respectively. Levels of phosphorylated PKB(p-PKB) and total PKB in neutrophils were examined by western blotting before and after stimulation with trypsin at 40nM/L. In the second section, forty SD rats were divided into five groups(n=8) randomly: a control group and four ANP groups (representing ANP model has been established for 3,6,12,24h respectively). Neutrophils were isolated at corresponding time spot, and levels of p-PKB and TNF-α, IL-1βmRNA were detected.In the third section, 24 rats were divided into 3 groups(n=8) randomly: control (C), ANP group (S) and ANP＋Wortmannin (W). Wortmannin (1.4mg/kg) or vehicle was administered by intraperitoneal injection 4 hours before duct infusion of sodium taurocholate. Six hours after duct infusion, animals were re-anesthetized. Neutrophils and serum were separated for examination of ELISA,RT-PCRå’ŒWestern later. After resected, pancreas and lung were stored at under 70℃for histopathological evaluation,immunohistochemistry staining, and examination of MPO,W/D.Disposal of lung specimen: After inferior lobe of right pneumectomy, the bronchus was cannulated using a polyethylene catheter, and 10% phosphate-buffered formalin was infused at a pressure of 30 cmH₂O until the lung distended homogeneously, lung was fixed in 10% phosphate-buffered formalin for 36h.4,Test methods(1) Levels of p-PKB and total PKB in neutrophils were examined by western blotting.(2) mRNA levels of TNF-αand IL-1βin cells were examined by RT-PCR.(3) Protein levels of TNF-αand IL-1βin cell culture and serum were examined by ELISA.(4) NF-κB expression in lung was detected by immunohistochemistry staining (streptavidin-biotin complex, SABC).(5) Histopathological evaluation of lung and pancreas were performed through HE staining.(6) Evaluation of MPO and W/D in lung5,StatisticsValues were expressed as mean±SD, group comparison was analysed by t test, statistical significance was set at P＜0.05.ResultsActivation of phosphatidylinositol 3-kinase/protein kinase B(PI3K/PKB) induced by trypsin in neutrophilIn the neutrophils of control group, the basal activity of total PKB was high, whereas, the level of p-PKB was very low. Expression of TNF-αand IL-1βmRNA in the control group was low too. Levels of phosphorylated PKB(p-PKB) in neutrophils after stimulation with trypsin at 40nM/L increased significantly compared with control(P＜0.05). Protein and mRNA levels of TNF-αand IL-1βpresented similar tendency. After neutrophils were pretreated by PI3K inhibitors, Wortmannin (200 nM)and LY294002(100μM), 1 hour ago before the stimulation of trypsin, the activation of p-PKB induced by trypsin could be completely inhibited(compared with trypsin control, P＜0.05,＜0.05). The expression of protein and mRNA of TNF-αand IL-1βdeclined as the activity of p-PKB was inhibited.Dynamic changes of PI3K/PKB signal transduction in neutrophils of rats with acute necrotizing pancreatitisPancreas presented dark red, edema and congestion after model had been established for 3 hours, but necrosis was not definite. Pancreas was in faint color after model had been established for 6 hours, at the same time, edema,congestion and local necrosis was apparent. Moderate bloody ascites and sapo macula could be seen ,too. Pancreas was in pallor and lots of necrosis could be seen after model had been established for 12 hours, meanwhile, severe bloody ascites and sapo macula appeared in the cavity accompanied with gastroentero dilatation. Manifestation of 24 hours was identicial with that of 12 hours.The basal activity of total PKB in neutrophils from control group was high, whereas the level of p-PKB was low. Levels of phosphorylated PKB(p-PKB) in neutrophils after model had been established increased significantly compared with control(P＜0.05) at any time, and reached summit at 24h. Although level of p-PKB at 12h decreased slightly compared with that at 6h, it still maintained rather high level. Levels of p-PKB increased significantly at respective time compared with control(P＜0.05).Expression of TNF-αand IL-1βmRNA in the control group was small. Expression of TNF-αand IL- 1βmRNA in each experiment group increased rapidly after model had been established. Expression of the two cytokines mRNA increased significantly at 3h, and reached summit at 6 and 12h respectively. Untill 24h, they still maintained high levels. Levels of TNF-αand IL-1βmRNA increased significantly at respective time compared with control(P＜0.05).Effect of PI3K / PKB inhibitor on the function of peripheral neutrophil production of pro-inflammatory cytokines and rats with acute necrotizing pancreatitisLevels of phosphorylated PKB(p-PKB) and TNF-α,IL-113mRNA in peripheral neutrophils after ANP model had been established increased significantly compared with control(P＜0.05). Phosphorylation of PKB and production of TNF-α,IL- 1βmRNA in neutrophils could be completely inhibited by wortmannin, meanwhile, the levels of TNF-αand IL-1βin serum decreased significantly. Lung MPO and wet/dry weight ratio from pancreatitis group increased significantly compared with control(P<0.05), and Wortmannin could decrease the level of MPO and W/D. Pathological examination suggested that wortmannin significantly relieved the lung injury and reduced the severity of the lesion in the lung, but it only decreased the infiltration of inflammatory cells in pancreas and lung, and failed to reduce the pancreas bleeding and necrosis. Increased expression of NF-κB in lung tissue from SAP model decreased too after the introduction of Wortmannin. Conclusion1,Neutrophils transmitted message through PI3K/PKB signal transduction pathway after PARs-2 had been activated by trypsin.2,Protein and mRNA levels of TNF-αand IL-1βincreased after PARs-2 on neutrophils had been activated.3,Being PI3K inhibitors, Wortmannin and LY294002 could inhibit the activation of trypsin on neutrophils, therefore decrease the production of inflammatory factors.4,PI3K/PKB signal transduction pathway was activated in neutrophils during early period of acute necrotizing pancreatitis, which took part in the regulation of cell activation.5,Inhibiton of PI3K/PKB signal transduction pathway may effectively control neutrophils' secretion of pro-inflammatory factors TNF-αand IL-1βduring early times of acute necrotizing pancreatitis.6,Inhibitors of PI3K / PKB signal transduction pathway could decrease systemic inflammation response during early period of acute necrotizing pancreatitis.