Study On Pulmonary Pathological And Immunological Changes Of SP-D Deficient Mice And Roles Of Exosome Treatment In Lung Restoration

Background Critically ill patients have a high incidence of infection due to their severe illnesses,multiple diseases and immunity deficiency.The most common infection that occurs in critically ill patients is pulmonary infection as a result of lying in bed,invasive mechanical ventilation and other risk factors.Generally,anti-infection strategies for critically ill patients are more comprehensive,more intensive and more complicated than those for non-critically ill patients.The selection and withdrawal of antibiotics is often a tough issue for physicians,and the recurrence of pulmonary infection after anti-infection treatment is also a big problem.The previous stage of this research carried out clinical studies on risk factors for recurrence of pulmonary infection.The results not only provide guidance for physicians to make more reasonable clinical decisions on anti-infection,but also inspire the following ideas for experimental research on critical lung disease from the perspective of patient host defense.Pulmonary surfactant is a phospholipid-protein mixture secreted by alveolar typeⅡ epithelial cells,of which pulmonary surfactant protein(SP)is an important component.SP-D plays an important role in reducing alveolar surface tension,and SP-D is also a type of host defense protein,playing an important role in innate immunity of the lungs.Targeting the animal model of SP-D deficient mice,this thesis studied the pathological status and immunological changes of mouse lungs.Meanwhile,this thesis conducted mesenchymal stem cell(MSC)-derived exosome intervention on SP-D deficient mice,so as to explore the possibility of lung restoration for severe pulmonary diseases.Objectives1.To study lung histopathology,lung inflammasome,serum inflammatory cytokines and alveolar macrophages on SP-D KO mouse models.2.To explore the possibility of lung restoration by treatment of MSC-derived exosomes in pathological and immunological changes caused by SP-D deficiency.Methods1.WT mice and SP-D KO mice with the same genetic background(C57BL/6)were established as the animal models.Aged SP-D KO mice(>6 months old)were mainly studied.2.MSCs were cultured and sub-cultured.Exosomes from MSCs were isolated by kit method,and the exosomes were determined by protein assay and transmission electron microscopy.3.MSC-derived exosomes were used to treat the aged SP-D KO mice.Different doses and courses of exosome treatment were given to mice through nasal administration.Mice were divided into different groups and sub-groups.4.Blood,BALF or lungs were taken from mice after the course end of exosome treatment.5.HE staining was performed to study lung histopathology.6.Activation of lung NLRP3 inflammasome and p-NF-κB p65 was detected using immunofluorescence analysis.7.Serum expression of IL-6 and IL-1β was detected by ELISA.8.BALF cytology and the in vitro phagocytosis of alveolar macrophages were preliminarily studied.9.All data were presented as means±SD.Data were compared using Student t test or ANOVA by SigmaStat software.For all comparisons,P<0.05 was considered statistically significant.Results1.HE stain showed normal lung in WT mice,and severe emphysema in the aged SP-D KO mice.Aged SP-D KO mice with MSC exosome treatment showed only mild emphysema in HE stain.The average alveolar size of the treated group was smaller than that of the untreated group(P<0.05).2.Immunofluorescence analysis showed that NLRP3 activation in the lung of SP-D KO mice was increased compared with WT mice.Then,the treatment groups with different exosome doses were compared with the untreated SP-D KO group,finding that NLRP3 activation decreased with the increase of doses(P<0.05).3.Immunofluorescence analysis showed that p-NF-KB p65 expression in the lung of SP-D KO mice was increased compared with WT mice.Then,the treatment groups with different exosome doses were compared with the untreated SP-D KO group,finding that p-NF-κB p65 expression decreased significantly with the increase of doses(P<0.01).4.ELISA results showed that the expression levels of serum IL-6 and IL-1β were very low in all the groups.5.Analysis of inflammatory cells showed BALF of SP-D KO mice consisted of mainly alveolar macrophages and lymphocytes.Quantitative analysis showed the percentage of each inflammatory cell was not different in BALF of SP-D KO mice with or without exosome treatment.Experimental phagocytosis showed occasional phagocytosis of alveolar macrophages.Conclusion1.Aged SP-D KO mice(>6 months old)showed severe emphysema in the lung histopathology,indicating that SP-D could maintain alveolar homeostasis.2.Both NLRP3 inflammasome and p-NF-κB p65 were up-regulated in SP-D KO mice,indicating that SP-D inhibited the activation of NLRP3 inflammasome and NF-κB signaling pathway.3.MSC exosomes played a certain role in restoring the pathological status of emphysema caused by SP-D deficiency.4.Increase of MSC exosome doses could inhibit the activation of NLRP3 inflammasome and NF-κB signaling pathway in SP-D KO mice,indicating that MSC exosome intervention could restore some immunological changes in the SP-D deficient lung.