An Study On Model Establishment And Interaction Between Aspergillus Fumigates And HPMVECs And Its Mechanism

Background and AimsInvasive pulmonary aspergillosis(IPA)is a severe invasive and infectious disease as a result of aspergillus fumigatus(AF)’s entering immunocompromised host cells.It is characterized by AF invasion of blood vessels.Human pulmonary microvascular endothelial cells(HPMVECs)may play a key role in the in the pathological process of IPA disease.This study intends to build AF-induced HPMVEC infection model to probe into the interaction of AF with pulmonary microvascular endothelial cells thoroughly and relevant mechanisms.Methods:We conducted this study by co-culture human lung microvascular endothelial cells with aspergillus fumigatus strains so as to establish AF-induced HPMVEC infection model.The aim was to observe change of human lung microvascular endothelial cell morphology and cell vitality.During the methodology,by using flow cytometry and laser confocal microscopy we observed the AF induced toxicity on HPMVECs and the impact of the change of actin cytoskeleton.What’s more,we measured trans-endothelial electrical resistance(TER)to study the AF’s influence on human lung microvascular endothelial cell permeability.To explore the effect of AF on HPMVECs secretion function and related mediated signal transduction mechanisms,we also respectively observed AF-induced HPMVECs release of inflammatory factor from the level of protein and mRNA level.Furthermore,by using different types of MAPK inhibitors and Western blot,we explored the role of MAPK signaling pathways in the influence of AF on HPMVECs secretion function,illustrating the intervention of MAPK pathway in mechanism of HPMVECs inflammation induced by AF.Finally,we also discussed the role of a promising anti-inflammatory property silibinin in this model from the level of protein and mRNA level.Results:We successfully constructed AF-induced HPMVEC infection model and mice macrophage J744 model,noticing change of HPMVEC actin filament,increase in cell permeability and decrease of cell activity.It was also observed macrophage phagocytosis of AF.There was an unexpected outcome that HPMVECs doesn’t engulf AF obviously.Besides,we observed AF bring about HPMVECs changes of morphology,change of intracellular actin and increase the permeability of HPMVECs.SB203580(p38 inhibitors),Y27632(ROCK inhibitors)and LY317615(PKC inhibitors)could inhibit the changes above to varying degrees.AF was shown to induce HPMVECs to secrete a variety of cytokines(IL-6,IL-1beta,ICAM-1,E-selectin)in time and dose dependent manner.SB203580 could inhibit the secretion of cytokines induced by AF.What’s more,we observed increase in the degree of activation of p38.Simultaneously,silibinin was shown to inhibit the secretion of cytokines induced by AF,resulting in a decrease in degree of p38 activation.Conclusions:This study has demonstrated that aspergillus fumigatus have toxic effects on HPMVECs;HMVECs does not have a role in phagocytosis of AF.Aspergillus fumigatus has been shown to increase the cell permeability and change cytoskeleton in HPMVECs,which is mediated by p38 MAPK,ROCK and PKC signaling pathway.Aspergillus fumigatus can promote PMVECs secretion of cytokines mediated by p38 MAPK signaling pathway.Silibinin has been proved to have inhibitory action to this process.This study conducted by discussing the interaction between AF and pulmonary microvascular endothelial cells is expected to provide a new valuable clues for the IPA development of pathogenesis research and renewed target for potential treatment.