Effects Of Recombinant Polypeptide Of Heparin-binding Domain Of Fibronection On CAMP/PKA Signal Transduction Pathway In Severe Acute Pancreatitis-associated Lung Injury

This study includes two parts, we through the establishment of SAP model of ratto study in each part. We investigated the mechanism of action of cyclic adenosinemonophosphate/protein kinase A signal transduction pathway in severe acutepancreatitis-associated lung injury in the first part; observed the therapeutic benefitsby recombinant polypeptide of heparin-binding domain of fibronection on the lunginjury in severe acute pancreatitis and to analyze the possible mechanisms in thesecond part.We try to find a treatment by experiment to prevent the lung injury inSAP.Part1The role of cyclic adenosine monophosphate/protein kinase A signal transductionpathway in severe acute pancreatitis-associated lung injuryObjective To investigate the mechanism of action of cyclic adenosinemonophosphate/protein kinase A signal transduction pathway in severe acutepancreatitis-associated lung injury.Methods Fifty-six male healthy SD rats were completely randomized intothree groups: sham operation(SO) group, SAP group and SAP plus H89(cAMPinhibitor) group, then the latter two groups were divided into three sub-groups witheight rats in each sub-group according to the sampling time of6,12and24h,and thetotal numbers of groups were seven.The content change of TNF-α and IL-1β in serum,protein levels of cAMP-dependent protein kinase catalytic subunit(PKA C) andphosphorylated vasodilator-stimulated phosphoprotein(p-VASP)and the expression of VSAP mRNA in lung tissue were detected by enzyme-linked immunosorbentassay(ELISA),immunohistochemistry and quantitative real timePCR,respectively.Pathological changes of the pancreas and lung tissues were alsoobserved.Results Compared with the SO group,the serum levels of TNF-α and IL-1βwere obviously increased at different time points（P<0.05）. pathological changes ofthe pancreas and lung tissues were aggravated significantly.the protein levels of PKAC and p-VASP and the expression of VSAP mRNA in lung tissue were increasedsignificantly（P<0.05）which peaked at12h in the SAP group，TNF-α（266.07±17.14）pg/mL、IL-1β（169.17±25.92）pg/mL、PKA C（210.69±6.32）×103、p-VASP（56.62±0.57）×103、VASPmRNA（2.06±0.21），which had positive correlation withthe serum level of TNF-α、IL-1β.In comparison with the SAP group, pathologicalchanges of the pancreas and lung tissues were alleviated significantly,the proteinlevels of PKA C and p-VASP and the expression of VSAPmRNA in lung tissuewere decreased significantly in the SAP plus H89group at different time points（P<0.05）.Conclusion The cyclic adenosine monophosphate/protein kinase A signaltransduction pathway is found to participate in the pathological process of severeacute pancreatitis-associated lung injury through the up-regulations of TNF-α, IL-1βand phospho-VASP.Part2The effect of recombinant polypeptide of heparin-binding domain of fibronectionon lung injury in severe acuteObjective to observe the therapeutic benefits by recombinant polypeptide ofheparin-binding domain of fibronection on the lung injury in severe acute pancreatitisand to analyze the possible mechanisms.Methods Fifty-six male healthy SD rats were completely randomized intothree groups: sham operation(SO) group, SAP group and recombinant polypeptide ofheparin-binding domain of fibronection (recombinant FN) group, then the latter two groups were divided into three sub-groups with eight rats in each sub-group accordingto the sampling time of6,12and24h,and the total numbers of groups were seven.AMY was measured.Pathological changes of the lung tissues were also observed.TNF-α and FN were examined by enzyme-linked immunosorbent assay(ELISA). Thephosphorylated vasodilator-stimulated phosphoprotein(p-VASP) was positioned usingimmunohistochemical staining. The changes of p-VASP protein and PKA C proteinwas detected by Western Blotting.Results Compared with the SO group，the levels of AMY,TNF-α,p-VASPprotein and PKA C protein increased significantly in the SAP group at different timepoints (P<0.05)，but the serum levels of FN were decreased significantly(P<0.05).Pathological changes of the lung tissues were aggravated significantly．Comparedwith the SAP group，the level of AMY,TNF-α,p-VASP protein and PKA C proteindecreased significantly in the recombinant FN group at different time points(P<0.05)， but the serum levels of FN were increasedsignificantly(P<0.05).Pathological changes of the lung tissues were alleviatedsignificantly．Conclusion Recombinant polypeptide of heparin-binding domain offibronection may ameliorate lung injury of severe acute pancreatitis and alleviate thecapillary leak，which may play a role in the capillary leak caused by reducing theconsumption of plasma FN,changing the level of TNF-α and suppressing thecAMP/PKA signal transduction pathway.