The Role And Mechanism Of JAK/STAT3 For Injury Of AT-Ⅱ Of Rat Of Severe Acute Pancreatitis In Vitro

Background :Severe acute pancreatitis (SAP) is the disease that have threatened to life,which occurs in 30% of all patients with SAP at least. Acute pancreatitis associated lung injury (APALI) is the main cause of death in earlier period of disease (1,2). Its pathogenesy is multiple. Firstly,pancreatin that has been activated induced cytokines activation,activated leucocytes released massive cytokines and mediators of inflammation and damaged local tissue. Secondly,plasmic cytokines exaggerated inflammatory reaction and induced distant organic leucocytes to aggregate and activate. Thirdly,leucocytic respiratory burst released oxyradical and damaged distant organ. Cytokines and mediators of inflammation are core mechanisms and straight factor in the course of lung injury. The research discovered that inflammatory reaction was activated by several signal transduction channels and Janus kinase/signal transducers and activators of transcription 3(JAK/STAT3) is mutual and the most significant pathway. Phosphorylated JAK/STAT3 activated nuclear factor and protein,then regulated genetic transcription of cytokine and mediators of inflammation. Majority of cytokines and mediators of inflammation transmited signal from cellular membrane to nucleus to switch on a trail of inflammation reaction (3,4). Respecting this research finding indicated that JAK/STAT3 is main switch of starting inflammation reaction. We could inhibit waterfall of amplify in earlier period of inflammation reaction and prevent acute lung injury if we accommodate availably this signal pathway. In this study,we made rat of SAP model by hypodermic injecting asodium taurocholate(STC) in capsule of pancreas and made primary culture alveolar typeⅡepithelial cells (AT-Ⅱ) by improved method in vitro. AT-Ⅱwere treated with serum from rat model of severe acute pancreatitis(SAP group) and pre-treated JAK enzyme inhibitor AG490 (SAP+AG490 group) , while the normal cell control was set with Dulbecco's Modified Eagel's medium(DMEM). AT-ⅡSTAT3 DNA binding activity,mRNA and protein's expression were detected by lectrophoretic mobility shift assay(EMSA),Reverse transcriptase polymerase chain reaction(RT-PCR) and Western blotting method. The level of Surfactant protein C in AT-Ⅱand the apoptosis of AT-Ⅱwere detected by flow cytometry(FCM),Na~+-K~+-adenosine triphosphatase(Na~+-K~+-ATPase) activity was detected by chromatometry. To study the effect of JAK/Stat3 on injury of AT-Ⅱin severe acute pancreatitis,new route for APALI of intervention will be provided in early period.Method1.Rat of SAP model were made by hypodermic injecting 5% asodium taurocholate in capsule of pancreas.2.AT-Ⅱwere made primary culture by improved method in vitro.3.AT-Ⅱwere treated with serum from rat model of severe acute pancreatitis(SAP group) and pre-disposal treat JAK enzyme inhibitor AG490 (SAP+AG490 group),while the normal cell control were set with Dulbecco's Modified Eagel's medium(DMEM).4.AT-ⅡSTAT3 DNA binding activity,mRNA and protein's expression were detected by EMSA,RT-PCR and Westernblotting method.5.The level of Surfactant protein C in AT-Ⅱand the apoptosis of AT-Ⅱby flow cytometry and Na-K-adenosine triphosphatase(Na~+-K~+-ATPase) activity were detected by chromatometry.Results1.All rats were killed at 24h.Then,in SAP groups,the rats can be observed such symptom after dissection as flatulenet gastrointestinal tube,part of intestinal necrosis,a large number of bloody ascites and pleural fluid,swelling of the congestive pancreas obviously,visible and disseminated necrotic focus,there was peripancreatic saponif maculation. In microscope there was pancreatic tissue's hydrosarca. Lobular septum,interlobular septum and gland alveolus septum's distance was widen,hemangiectasia and congestion,hemorrhagic focus in region,diverging focal necrosis,and a bulk of inflammatory cell infiltration.2.There were a lot of aniso-distributing aniso-size blue particle when AT-Ⅱwere cultured and stained by alkaline phosphatase after 2 days,There were a lot of perinuclear aniso-size fuscescent osmiophilic globuli when AT-Ⅱwere cultured and stained by digallic acid phosphatase after 5 days.3.After 2 hours,compared with control group,activation of stat3 was enhanced in SAP group,expression of stat3 mRNA and protein was also enhanced. After 2 hours,compared with SAP group,activation of stat3 attenuated in SAP+AG490 group, expression of stat3 mRNA and protein was decent.4.Compared with control group,level of SP-C protein declined[4h SP-C fluor index (0.69±0.02) vs (1.02±0.03),P<0.01] in SAP group,the apoptosis of AT-Ⅱincreased[2h (11.55±1.90)% vs (5.30±0.36)%,P<0.05],activation of Na~+-K~+-ATPase attenuated [2h(0.0477±0.001) vs (0.0390±0.001),4h(0.0512±0.001) vs (0.0402±0.002),P<0.05)]. Compared with SAP group,level of SP-C protein declined[4h SP-C fluor index (0.69±0.02) vs(0.48±0.01),P<0.01] in SAP+AG490 group,the apoptosis of AT-Ⅱincreased[2h(11.55±1.1) % vs (13.92±0.82%),P<0.05],activation of Na~+-K~+-ATPase attenuated[4h(0.0390±0.001) vs (0.0312±0.001),(0.0402±0.002) vs (0.0322±0.002),P<0.05].Conclusions1. Rat of SAP model by hypodermic injecting 5% asodium taurocholate in capsule of pancreas were made successfully. This way is reliability,simple and reproducibility,which can make a lot of animal model.2. The improved method of primary culture of alveolar typeⅡepithelial cells (AT-Ⅱ) in vitro was made successfully. It is simple and can boost AT-Ⅱconcentration. The way can provide fine AT-Ⅱresource for in vitro experiment.3. JAK/STAT3 was activated and participated in pathophysiology of process in SAP injury of lung. JAK enzyme inhibitor AG490 can inhibited availably JAK/STAT3 in vitro.4. JAK/STAT3 maintained AT-Ⅱto secrete SP-C,inhibited apoptosis and activated Na~+-K~+-ATPase,and it could sustain alveoli of lung stability.