Protective Effect And Molecular Mechanism Study Of FGF21 On Ventilator Induced Lung Injury In Mice

Objective:Mechanical ventilation is an integral part of general anesthesia and an important means of respiratory support therapy for critical patients.However,mechanical ventilation itself may also induce or aggravate lung injury,also known as ventilator-induced lung injury（VILI）,which is a clinical problem that needs to be solved.A growing number of studies suggest that VILI is not only a kind of mechanical trauma,but also a kind of biotrauma,which is closely related to the over-activation of inflammatory response in the lung.Fibroblast growth factor 21（FGF21）is a metabolic regulator secreted by liver into blood and acting on multiple organs in the body.It has been proved to play an important role in various pathophysiological process like glucose and lipid metabolism disorders,microvascular impairments,oxidative stress and inflammatory diseases.However,there are few studies on the role of FGF21 in lung diseases,and the effect of FGF21 on VILI has not been reported at home or abroad.Therefore,the purpose of this study was to investigate the role of FGF21 in VILI and preliminarily explore its mechanism.Materials and methods:After ventilation,patients’serum FGF21 levels were assessed by ELISA.The serum levels of FGF21 and inflammatory cytokines were assessed by ELISA after mechanical ventilation in mice.Fgf21 knockout mice were bred and mechanical ventilation model was established to compare the severity of lung injury and inflammatory response between Fgf21^-/-mice and wild-type mice.The indicators used included Hematoxylin&Eosin staining and pathological scores,TUNEL staining,lung wet/dry weight ratio,Evans blue assay of vascular permeability,bronchial alveolar lavage fluid cell count and protein content,myeloperoxidase activity in lung tissue,etc.Lung injury was evaluated after treatment with different doses of recombinant FGF21 protein（r FGF21）in a mechanical ventilation mouse model.Total antioxidant capacity,ATP content,mitochondrial DNA expression,immunofluorescence and m RNA expression of the endothelial marker VE-cadherin,as well as the mesenchymal markerα-SMA and Vimentin in lung tissue were assessed.Primary lung microvascular endothelial cells were cultured and a cyclic mechanical stretch model was established.The cell viability,lactate dehydrogenase release,superoxide dismutase activity,reactive oxygen species,ATP content,mitochondrial DNA level,mitochondrial membrane potential and apoptosis were assessed after treatment with different concentrations of r FGF21.Finally,caspase-1 activity in lung tissues and cells was assessed,and m RNA and protein expression levels of key molecules in NLRP3 pathway were assessed by real-time quantitative PCR and Western Blot.Results:We first analyzed serum samples from 69 patients undertaken mechanical ventilation during general anesthesia,and found that the average serum FGF21 levels increased after mechanical ventilation.Correlation analysis suggested that the elevated levels of FGF21 was positively proportional to the duration of ventilation.Increased levels of FGF21 and serum inflammatory cytokines IL-18 and IL-1βwere also observed in mechanical ventilation mouse models.We used Fgf21knockout mice for VILI modeling,and found that lung injury and intrapulmonary inflammation in Fgf21^-/-mice were more severe than those in wild-type mice,suggesting that FGF21 is likely to play a protective role in VILI.In a wild-type mouse model of mechanical ventilation,different doses of r FGF21 were administered,and it was found that r FGF21 could reduce lung lesion,pulmonary edema,intrapulmonary inflammation,mitochondrial dysfunction and endothelial-mesenchymal transition.In the mechanical stretch model of cells,r FGF21 could reduce cell damage,oxidative stress and mitochondrial dysfunction.Finally,r FGF21 was found to reduce caspase-1activity in the cell stretch model and mouse model.Real-time PCR and Western blot analysis of mouse lung tissue showed that the m RNA levels and protein expression levels of key members of NLRP3 inflammasome pathway were down-regulated after r FGF21 treatment.Conclusion:The lung injuries got worse in mice subjected to mechanical ventilation after the Fgf21 gene was knocked out.Treatment with r FGF21 was found to play a protective role on both mechanical ventilation model in mice and mechanical stretch model in lung microvascular endothelial cells,which may be related to the inhibiting of the NLRP3 inflammatory pathway.