Study Of The Lung Protective Effect Of Milk Fat Globule Epidermal Growth Factor-8 In Acute Lung Injury Model

Objective:Acute lung injury（ALI）is caused by severe infection,shock,trauma,ischemia-reperfusion,application of ventilator and other serious intra-pulmonary or extra-pulmonary pathogenic reasons.It is an acute,progressive respiratory failure,which can develop into acute respiratory distress syndrome（ARDS）when serious.The pathogenesis of ALI/ARDS is complicated which involving many pathogenic factors.Thus,ALI has very high mortality.Milk fat globular epidermal growth factor 8（MFG-E8）is a soluble glycoprotein,which is ubiquitous in different types of cells and tissues.The main function of MFG-E8 is promoting the elimination of apoptotic cells by combining phagocytes and apoptotic cells,thereby reducing inflammation.It has been proved to be an important factor in delaying the development of various inflammatory diseases.Previously,it was found by our group that MFG-E8 attenuated aberrant immune response of SLE model mice through regulating the expression of CXCR2,ultimately results in a significant blocking of neutrophils accumulation,downregulation of the type-I IFN,autoantibody（ANA,anti-ds DNA antibody,and ANCA）and reducing immune complex deposit,alleviating lupus-related tissue damage.However,there are few studies on the pro-resolution effect of MFG-E8 on ALI.Therefore,in this topic,we aim to discuss whether MFG-E8 can reduce lung injury in ALI model mice.Methods:1.Gene identification of MFG-E8^-/-mice:the gene level of MFG-E8 was detected by genomic PCR.And the protein level of MFG-E8 was detected by Western blot.2.Establishment of ALI mouse model:16 male SPF grade,8-week-old,wild-type（WT）C57BL/6J mice and 16 MFG-E8^-/-gene knockout C57BL/6J mice were randomly divided into four groups.The WT and MFG-E8^-/-mice,8 for each as control,were treated with normal saline aerosol inhalation（NS,1ml/min,20min）,named as WT+NS group and MFG-E8^-/-+NS group.Another 8 WT and 8 MFG-E8^-/-mice were used to establish ALI model by Lipopolysaccharide（LPS）aerosol inhalation（dose 200ug/ml,1ml/min,20min）.Eight hours after,the mice were anesthetized by intraperitoneal injection of pentobarbital（70mg/kg）.Then the lung tissue was dissected and fixed with formaldehyde for further pathological analysis to verify whether the model was established successfully.3.Lung histopathological examination in mice:the lung tissue was routinely treated with dehydration,paraffin embedding,slicing and staining.The thickness of the section was5μm.The staining was hematoxylin-eosin（Hematoxylinandeosin,H-E）.The pathological changes of lung tissue were observed under light microscope.4.Cell count in bronchoalveolar lavage fluid:Trachea of mice was fully exposed after intraperitoneal anesthesia.Thereafter,0.3 ml of PBS was repeatedly aspirated with a syringe 3 times and repeated lavage 3 times,and the alveolar lavage fluid was collected.Total cells were counted under a microscope.5.Wet/dry weight ratio of lung tissue（W/D）:After the mice were anesthetized with pentobarbital by intraperitoneal injection,the skin was cut from the neck to the thorax,and the right upper lobe was removed.The surface of the lung tissue was blotted with filter paper and placed in a small,dry glass beaker.After weighing,the lung was placed in oven and baked at 60℃for 48 hours until its mass no longer changed.Lung tissue dry weight was weighed and W/D was calculated to assess the degree of edema of the lung tissue.6.Determinat ion of redox state of mi tochondria in lung t issue:Eight hours after the establishment of the ALI mouse model,the mice were anesthetized by intraperitoneal injection with pentobarbital（70mg/kg）,and a small animal ventilator was connected to the trachea to maintain the normal breathing rate and extent.The lung tissues together with trachea were taken and placed in liquid nitrogen immediately.Serially sliced in the transverse direction to collect mouse mitochondrial reduced coenzyme nicotinamide adenine dinucleotide（NADH）and oxidized coenzyme yellow Flavin adenine dinucleotide（FAD）fluorescence signal.Thereafter,low-temperature fluorescence imaging（frozen imaging）technique was used to image these fluorophores.After image analysis,the mitochondrial redox status was evaluated.7.The expression of Claudin-4 protein in mouse lung tissue:The supernatant was collected after the lung tissue was homogenizated with protein lysis buffer.Then,the protein concentration was measured by BCA method,and the target protein level was detected by Western blot.8.Statistics:All data were analyzed with Graphpad Prism 6.Protein expression was calculated by gray scanning with Image J.MATLAB R2018b was applied to analyze the data and picture obtained by Cryo-micro-optical sectioning tomography.Data was presented as Means±SD.Student’s t test was used to determine the significance of difference between two groups.P<0.05 indicated a significant difference.Results:1.The knockout of MFG-E8 was verified by PCR experiment at the gene level.The results showed that the target fragments were amplified in both kinds of mice.The genome amplification product of WT mice was 474 BP,while that of MFG-E8-/-mice was 620bp fragment.Westernblot test showed that MFG-E8 protein expression was not detected in MFG-E8-/-mice.The above experimental results show that MFG-E8knockout is successful.2.The pathological results showed that the lung tissue of WT+NS mice had alveolar cavities with complete structure and vacuolar shape.The alveolar walls were thin and the alveolar micro-vessels showed no congestion or hyperemia.There was no obvious bleeding,edema,or inflammatory cell infiltration in the alveolar cavity and interstitial space.Mice with LPS inhalation showed narrowed alveolar cavity with more exudates,broken and fused alveolar wall.It could also be observed congestive,edema,or infiltration of inflammatory cells in pulmonary interstitium.Compared with WT+LPS group,MFG-E8^-/-+LPS mice had relatively more serious lung tissue damage.3.Total cell number in the bronchoalveolar lavage fluid of mice in WT+NS group was32.88±1.95×10⁴.Compared with that,cell number in both MFG-E8^-/-+NS group and WT+LPS group were significantly higher（69.38±2.96×10⁴ and 165.10±4.51×10⁴,respectively,P<0.01）.Mice in MFG-E8^-/-+LPS group had highest cell accounts(203.90±6.28×10⁴,P<0.01 vs MFG-E8^-/-+NS).4.Compared with WT+NS group,W/D value of the mice in WT+LPS group increased significantly（P<0.01）.Compared with the WT+LPS group,W/D value of lung tissue in the MFG-E8^-/-+LPS group was significantly higher（P<0.01）.5.The redox state of lung tissue was expressed by redox ratio RR.Compared with that in WT+NS group,there were less reduced coenzyme and NADH signal,more oxidized coenzyme,FAD signal and RR value in other three groups（P<0.05）.Mice in MFG-E8^-/-+LPS group had lowest RR value(P<0.05 vs MFG-E8^-/-+NS group,P<0.05 vs WT+LPS group).6.Weston blot results showed that the content of Claudin-4 protein in lung tissue of MFG-E8^-/-+LPS mice was significantly lower than that of WT+LPS mice（P<0.05）.There was no difference between WT+NS and MFG-E8^-/-+NS mice.Conclusions:1.The alveolar cavity was narrowed in mice with LPS inhalation,and alveolar atrophy,interstitial congestion and edema were also observed.MFG-E8-knockout mice had more serious lung damage with more obvious broken and fused alveolar walls and more inflammatory cells infiltrating in alveolar cavity.2.The total number of inflammatory cells in bronchoalveolar lavage fluid of MFG-E8knockout mice increased significantly.3.MFG-E8 knockout could aggravate pulmonary edema in ALI mice.4.MFG-E8 knockout aggravated oxidative status of mitochondria in ALI mice.5.MFG-E8 knockout led to the decrease of Claudin-4 protein content in lung tissue and the increase of alveolar epithelial permeability.6.MFG-E8 exerted lung protective effect on ALI mice through decreasing LPS-induced oxidative stress and reducing the hyper-permeability of alveolar epithelial.