| BackgroundSevere acute pancreatitis (SAP) has multiple complications and a hazardous prognosis. It results in mortality rates ranging from30to40%. Acute lung injury (ALI)and acute respiratory distress syndrome (ARDS) is the most common and severe complication in SAP. It is also one of the important death reasons in clinical patients with SAP. The main pathological features and cause of ALI is excessive release of cytokines and inflammatory mediators that affect the permeability of the lung microvascular endothelial cells and lead to pulmonary edema. Pre-B cell clones enhancement factor (PBEF) is a novel peptide hormone, which is closely related lung injury. As current research data shown, PBEF is involved in a variety pathological process of lung injury. However, the role of PBEF in SAP induced ALI remains larger unknown and this study is the first to investigated it in the world.Chapter I study of the relationship between the serum PBEF and SAP with ALI/ARDSObjective:The expression level of PBEF, TNF-α and IL-8in serum samples were detected by ELISA method.Method:19serum samples were collected from SAP patients with ALI,and13samples from patients with ARDS while32serum samples were collected from mild acute pancreatitis as control. The expression level of PBEF, TNF-a and IL-8were detected by double sandwich ELISA method. The relationship of PBEF, TNF-α and IL-8expression level with the development of SAP patients with ALI were comparative analysis.Results:The serum PBEF of SAP patients complicated by ARDS was significantly higher than that of SAP complicated by ALI and mild acute pancreatitis, the difference was significant (P<0.05). Further analysis showed that serum TNF-α and IL-8level were higher as PBEF and three of them were positive correlated (r=3.215, P=0.012; r=4.247,P=0.019,P<0.01).Conclusion:We found the correlation of the level of serum PBEF and the level of serum TNF-a and IL-8in patient with SAP complicated by ALI or ARDS and mild acute pancreatitis.Chapter Ⅱ The expression of PBEF in the lung of animal model of SAP with ALI/ARDSObjective:The mRNA and protein of PBEF, TNF-α and IL-8were examined in lung tissue of SAP complicated ALI rat model.Method:Total of48rats were randomly divided into sham operation group (SHAM group, n=24) and pancreatitis with acute lung injury group (ALI group, n=24). SAP complicated ALI in animal models was constructed by sodium deoxycholate retrograde injected into the rat pancreatic bile duct method. To determine the success of SAP complicated ALI animal model, Pancreatic amylase, blood gas analysis,lung wet/dry ratio and the lung histology injury scoring system were detect at4h,8h,12h after the animal models were constructed. And then,the expression of PBEF, TNF-α and IL-8mRNA and protein were detected by real-time quantitative PCR and Western blot at the same time.Results:The pathological changes of the pancreas, pancreatic amylase, and blood gas analysis results suggested that animal models met the pathological changes of severe acute pancreatitis. In the light microscope, we made a preliminary comparison of the lung pathological change of experimental animals of the SHAM group and the ALI group,and found pulmonary edema, inflammatory cell infiltration, and hemorrhage in the SAP with ALI group.Total lung tissue injury score increased four hours after the modeling, deteriorated gradually within12hours,the difference was statistically significant (P<0.05);In the SAP with ALI group,lung wet to dry ratio was significantly increased, and gradually increased with time, compared with the SHAM group, the differences were significant(P<0.05)We used real-time PCR to detect the expression of PBEF, TNF-α and IL-8mRNA in the lung tissues of SAP model complicated by ALI at4h,8h,12h after the animal models were constructed. The expression level of PBEF mRNA as well as TNF-a mRNA and IL-8mRNA in ALI group was significant higher than control group; We tested the expression of PBEF, TNF-α and IL-8protein by using Western blotting at the same time as above in the same batch of samples, the results showed:the trend of PBEF, TNF-α and IL-8protein were alike as mRNA,and the difference was significant(P<0.05).Conclusion:The expression of PBEF, TNF-alpha and IL-8in lung tissue of SAP complicated ALI rat model are higher than that of sham operation group.CHAPTER ⅢThe molecular mechanism of PBEF mediated acute injury of human pulmonary microvascular endothelial cellObjective:To investigate the molecular mechanism of PBEF in mediating acute injury of human pulmonary microvascular endothelial cell.Method:Human pulmonary microvascular endothelial cells (PMVECs) were transfected with pEGFP-PBEF recombinant plasmid and lentiviral pLKO.1-PBEF-SiRNA interference vector in vitro. The expression of PBEF were detected by real-time quantitative PCR and Western blot. The effect of PBEF over-expression on apoptosis of PMVEC cells were determined by flow cytometry. We treated the PMVECs with pEGFP-PBEF recombinant plasmid and lentiviral pLKO.1-PBEF-SiRNA interference vector with TNF-α, and tested the expression of IL-1β,IL-6and IL-8by real-time quantitative PCR and Western blot. After the PMVECs with PBEF over expression were treated by TNF-α, the P38, ERK, JNK and PI3K signaling pathways were inhibited with corresponding signaling pathway-specific inhibitor blockers (U0126, SB203580, of SP600125, LY294002respectively). The expression of IL-1β,IL-6and IL-8were detected by real-time quantitative PCR and Western blot.Results:(1)The results of digestion and sequencing of Construc-tion of recombinant plasmid pEGFP-N1-PBEF showed the plasmid comply with following experiments., we verified PBEF mRNA and protein expression levels were much higher after transfected with ove r expression vector than the control group, and the the PBEF mRN A was10times of that in the control group, the difference was sig nificant (P<0.05). The test results of flow cytometry showed that PB EF overexpression promotes apoptosis of human HPMEC, the differenc e was significant (P<0.05).(2)Transfected with PBEFover-expression v ector or treated with TNF-α, the expression levels of IL-1β, IL-6an d IL-8were significantly increased, especially after induced by TNF-α, the expression of IL-1β, IL-6and IL-8will be further enhanced, the difference was significant (P<0.05).(3)The inhibiting ratio of PB EF was85%after cells transfected by pLKO.1-PBEF-SiRNA, siRN A of PBEF significantly down regulated the expression of PBEF, Transfection of SiRNA PBEF after induced by TNF-α could still sig nificantly inhibited the expression of PBEF,against the induced effect of SiRNA PBEF,the difference was significant (P<0.05).(4)The expr ession of IL-1β, IL-6and IL-8decreased significantly when using SB203580inhibition of P38pathway,the difference was significant (P<0.05);when using PD98059inhibited ERK pathway, the inflammatory fa ctor expression was also apparent,the difference was significant (P<0.05); when using JNK inhibitor SP600125, only IL-8expression decrease d obviously, the difference was significant (P<0.05); and when using t he LY294002to inhibit the PI3K pathway, the expression of inflamma tory cytokines did not change significantly (P>0.05).Conclusion:1,In PMVEC,TNF-a and PBEF have synergistic reaction;2,It is the first time to find that PBEF promoted the expression of IL-1β, IL-6and IL-8mainly through P38and ERK pathway in PMVEC in vitro... |
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