Research On The Influence Of Pre-B-cell Colony Enhancing Factor In Rats With ARDS

Background： Acute respiratory distress syndrome (ARDS) is acommon disease in intensive care unit.It can be caused by pulmonaryinfections, sepsis, severe acute pancreatitis, trauma, transfusions and soon,while it is a manifestation for the excessive inflammatory reaction inlung of body.Although the morbidity and mortality of ARDS havedecreased with the development of the medical science, it remainsunsatisfactory.Therefore, researches on the pathogenesis and treatment ofARDS are still popular and the existing researches have shown acomplicated pathogenesis of ARDS referred to masses of signal pathwayand cytokines.The Pre-B-Cell Colony Enhancing Factor(PBEF), also known asvisfatin or niacinamide phosphoribosyl transferase (Nampt), is aninflammatory cytokine found in recent years.It is primarily secreted bymature adipocytes as well as activated inflammatory cells, and it can beexpresssd in several organs. Vitro experiments found the alveolar epithelialcells and the pulmonary artery endothelial cells induced by inflammatorystimuli could largely express PBEF, and it is also found that the cell damagecaused by inflammatory stimuli attenuated by reducing the expression of PBEF.Thus it could suggest that PBEF may be involved in multiplepathophysiological processes of ARDS, including the injury of lungmicrovascular and alveolar epithelial cells, the increased permeability ofalveolar epithelial cells and so forth. In addition, some researchers foundPBEF as a potential biomarker of ARDS by the study of several kinds ofARDS models of dogs and rats.Therefore,PBEF may be a new target toresearch the pathogenesis and the treatment of ARDS.Objective: To investigate the effects of pre-B cell colony enhancingfactor(PBEF) on the lung pathological scores, wet/dry weight ratio, arterialoxygen pressure, the levels of TNF-α and IL-1β in the BALF, the mRNAexpression of TNF-α,IL-1β,ICAM-1,VCAM-1as well as the proteinexpression of ICAM-1,VCAM-1and NF-κB p65in lung tissue in rats withARDS induced by oleic acid tail vein injection and drugintervention.Referring to the phenomena observed by other scholars invitro,this tries to potentially verify the role of PBEF in ARDS, and providenew ideas and evidence for further study of the pathogenesis,pathophysiological processes and treatment of ARDS.Methods:40healthy adult male SD rats weighing (214±26) g wererandomly divided into control group (group C), model group (group OA),drug intervention group (group D), and solvent control group (group S).Ratsin group OA, group D and group S were induced ARDS models by oleic acidtail vein injection at the dose of0.15ml/kg.Rats in group D received PBEF inhibitor FK86610mg/kg and group S also received the same volume of itssolvent dimethyl sulfoxide by intraperitoneal injection,respectively, half anhour,12and24hours respectively before modeling.Whether the models hadbeen successfully made was preliminary determined by rats’ respiratoryrates, colour of skin and lips as well as their activity.Rats were anesthetizedby intraperitoneal injection of3.5%chloral hydrate(10ml/kg)6hours aftersuccessfully modeling.Then trachea were isolated, aorta blood obtainedfrom the abdominal aorta arterial is used to detect arterial oxygenpressure.The lavage fluid was collected after taking left lung lavage, and thefluid was centrifuged to get supernatant for the determination of the contentof inflammatory cytokines TNF-α and IL-1β (ng/L).The right upper lobe oflung was immediately doused in formalin for histopathologic andimmunohistochemical detections while the right middle lobe was used fordetection of wet to dry weight ratio and the right lower lobe was immediatelyfrozen at-80℃refrigerator or liquid nitrogen for detecting the expressionof PBEF,TNF-α,IL-1β,ICAM-1and VCAM-1mRNA by RT-PCR andRT-qPCR,as well as the protein expression of PBEF、ICAM-1, VCAM-1andNF-κB p65by Western Blot.Results:1.Observation of behavior and appearance.Rats in Group C were generally in good condition.They were morelively and didn’t bristle.Their lips and skin were pink with no dyspnea, which was significantly different compared to that of other groups(p<0.001).In a short period of time after modeling, rats in group OA, groupD and group S showed cyanosis of the lips and skin, the whole bodypiloerection, activity reduced, and a significant increase in respiratory rate.The respiratory rate of rats in group D showed a difference with that of groupOA and group S (p <0.05).The difference of respiratory rate of rats in groupOA and group S were not statistically significant (p>0.05), and there was nodeath in each group.2.arterial oxygen pressure.The arterial oxygen pressure of rats in group C was103.2±11.2mmHg,which showed significant difference with other groups(all p<0.001).Thearterial oxygen pressure of rats in group D were73.8±6.8mmHg, whichshowed a difference with group OA and group S (both p<0.05).The arterialoxygen pressure of rats in group OA were59.6±6.7vs59.8±11.0mmHg ingroup S,but the difference was not statistically significant (p>0.05).3.The pathology and wet/dry weight ratio of lung tissue.Lung tissue of rats in group C was pink grossly, with no significantcongestion, bleeding and swelling. The structure of the lung tissue was intactand clear under light microscope, with less inflammatory cells infiltration inthe alveolar interstitium.There is no significant congestion,hemorrhage,alveolar septa widened nor cavity edema fluid, and the alveolar wall was alsointact.In group OA, the lung tissue was dark red and swollen grossly, with visible bleeding and congestion. A large area of the alveolar wall wasdestructed with the alveolar cavity filled with pink edema fluid, alveolarseptum widened and edema.Inflammatory cells infiltration, leakage of redblood cells as well as pulmonary vascular congestion were also seen in thisgroup. Lung tissue of rats in group D was also swollen grossly, with visiblebleeding and congestion,but less significant than that of group OA.The samepathological changes observed in group OA could be seen in group D,but thedamaged area was significantly reduced, with less alveolar space edemafluid, alveolar interstitial edema, inflammatory cells infiltration and redblood cells leakage. Lung tissue of rats in group S was similar to that ofgroup OA both grossly and by light microscopy. The wet/dry weight ratio ofgroup C was4.174±0.311, which showed statistically significant differencewith other groups (all p<0.001). The ratio of group D was5.855±0.200,andthe difference was statistically significant compared to that of group OA andgroup S (both p<0.05), which were6.527±0.239and6.664±0.324respectively. However, the difference between the two groups was notstatistically significant (p>0.05).4.The expression of PBEF, ICAM-1and VCAM-1detected byImmunohistochemistryThe protein expression of PBEF could be found in the bronchialepithelia, vascular endothelia, alveolar wall and alveolar edema fluid of ratswith ARDS by immunohistochemical detection.The expression levels of all index the were relatively the lowest in group C,which were statisticallysignificant (all p<0.001).The index in group D were relatively lower thangroup OA and group S, and the difference was significant (bothp<0.05).These index in group OA and group S were relatively the highest,but the difference between the two groups was not significant (p>0.05).5.Levels of TNF-α and IL-1β in the bronchoalveolar lavage fluid.The level of TNF-α and IL-1β in the bronchoalveolar lavage fluid ofrats in group C were lowest respectively, which showed statisticallysignificant difference with other groups (p<0.05or p<0.001).In group Drespectively showed a significant difference with group OA and group S(p<0.05).The difference of the level of TNF-α and IL-1β in the thebronchoalveolar lavage fluid of rats in group OA and group S was notsignificant (p>0.05).6.Detecting the mRNA expression of PBEF,TNF-α,IL-1β,ICAM-1andVCAM-1in lung tissue by RT-PCR and RT-qPCR.The detection of PBEF,TNF-α,IL-1β,ICAM-1and VCAM-1mRNA inlung tissue by RT-PCR and RT-qPCR showed similar results. The expressionlevels of all index the were relatively the lowest in group C,which showedstatistically significant difference with other groups (p<0.001orp<0.05).The index in group D were relatively lower than group OA andgroup S, and the difference was significant (p<0.05or p<0.001). These ingroup OA and group S were relatively the highest, but the difference between the two groups was not significant (p>0.05).7.Detecting the protein expression of NF-κB p65in lung tissue byWestern Blot.The detection of NF-κB p65in lung tissue by Western Blot showed thatthe expression of NF-κB p65in group C was relatively the lowest, whichshowed a significant difference with other groups(all p <0.001). NF-κB p65in group D was relatively lower than group OA and group S, and thedifference was also significant (p<0.05or p<0.001). NF-κB p65in group OAand group S was relatively the highest, but the difference between the twogroups was not significant (p>0.05).Conclusion:1.PBEF could promote the the expression and production of TNF-α andIL-1β in lung tissue of rats with ARDS;2.PBEF could promote the expression of adhesion molecules ICAM-1andVCAM-1in lung tissue of rats with ARDS;3.PBEF could promote the activation and expression of NF-κB in lung tissueof rats with ARDS.Therefor,PBEF may induce the inflammatory injury of the lung tissueby activating the inflammatory response and promoting the expression ofinflammatory cytokines.It also induces the infiltration and migration ofinflammatory cells by promoting the expression of adhesion molecules.Thusit may play an important role in the development of ARDS.