Effect Of Influenza Virus Hemagglutinin On Tissue Factor Expression In Endothelial Cells

Objective:To detect the effect of influenza virus(H1N1)hemagglutinin on the expression of tissue factor(TF)of endothelial cells,and to explore the effect of influenza virus hemagglutinin on the levels of tissue factor genes and proteins of vascular endothelial cells to further explore the effect of influenza virus hemagglutinin on mice pathological changes in the lungs.Methods:1 in vitro experiment:1.1 The Bend3 cell line was purchased and cultured.Three groups were randomly divided and starved for 12 hours.The first group: saline(0?g/ml)stimulated Bend3 cells as a control group;the second and third groups: Bend3 cells stimulated by influenza virus hemagglutinin at different concentrations(2.5?g/ml and 5?g/ml)were used as experimental groups.After 12 hours of action,the total protein of the three groups of cells were collected,and the changes in TF protein levels after 12 hours were verified by Western Blot experiments.The gray values of the results obtained by Western blot were measured for statistical analysis,with P <0.05 as the difference was statistically significant.1.2 Continue to culture Bend3 cells,and then randomly divide three groups,starve the cells for 12 hours.The first group: normal saline(0?g/ml)stimulated Bend3 cells as a control group;the second and third groups:different concentrations(2.5?g/ml and 5?g/ml)influenza virus hemagglutinin stimulated Bend3 cells as an experimental group.After 12 hours of action,the total RNA of the three groups of cells were collected separately,and the changes of TF at the gene level after 12 hours were verified by Real-time PCR experiments.P <0.05 was considered statistically significant.2 In vivo experiments:Eighteen SPF-grade BABL / c mice(male and female,9-week-old male,8-week-old female,and consistent body weight)were randomly divided into three groups,six in each group.The first group: normal saline(0?g/ml)control group,the second group and the third group: different concentrations(2.5?g/ml and 5?g/ml)influenza virus hemagglutinin group.According to the volume of 2ml of mice,physiological saline and different concentrations(2.5?g /ml and 5?g/ml)of influenza virus hemagglutinin were injected into the mice along the tail vein.Feed and feed freely in the cage.During the 12-hour observation period,the behavioral changes of the mice and the post-mortem examination of the mouse lung tissue were observed and the mouse lung tissue was retained to observe the pathological changes of the mouse lung tissue after HE staining by an electron microscope.Results:1 in vitro experiment:1.1 Western blot resultsThe experimental results showed that TF was expressed in each group of Bend3 cells.The protein levels of TF in Bend3 cells were up-regulated under the stimulation of influenza virus hemagglutinin at different concentrations(2.5?g /ml and 5?g/ml),which were significantly higher than those of normal saline(0?g/ml)control group,in which the ratio of TF expression in the influenza virus hemagglutinin(2.5?g/ml)experimental group to the normal saline(0?g/ml)control group was P = 0.009(P <0.05),and the influenza virus hemagglutinin(5?g/ml)ml)The expression of TF in the experimental group was compared with that of the normal saline(0?g/ml)control group at P =0.000(P <0.05).1.2 Real-time PCR resultsThe experimental results showed that: TF was expressed in Bend3 cells of each group,and the ratio of TF expression in the experimental group of influenza virus hemagglutinin(2.5?g/ml)to that of the normal saline(0?g/ml)control group was P = 0.586(P> 0.05),the difference was significant.Therewas no statistical significance;the expression of TF in the influenza virus hemagglutinin(5?g/ml)experimental group was compared with the normal saline(0?g/ml)control group at P = 0.019(P <0.05).Under the stimulation of influenza virus hemagglutinin,TF of Bend3 cells was up-regulated at the gene level,which was significantly higher than that of the normal saline(0?g/ml)control group.2 In vivo experiments:Animal experiment results: We performed behavioral observations during the 12 hours after the influenza virus hemagglutinin was injected into the mice.Immediately after 12 hours,the three groups of mice were sacrificed and anesthetized and sacrificed.The lung tissue was examined by autopsy and the lung tissue was retained by HE staining Electron microscope observation was then performed.2.1 Behavioral observations:During the 12-hour injection of three groups of mice with different concentrations of viral hemagglutinin(0?g/ml,2.5?g/ml,and 5?g / ml),the status of the mice was observed once every 10 minutes.Observed significant behavioral changes;2.2 Post-mortem observation:Lung tissue of mice in the normal saline(0?g / ml)control group showed a pale pink color,soft texture,and smooth and moisturized lung leaves;lungs of mice with different influenza virus hemagglutinin(2.5?g / ml and 5?g / ml)in the experimental group All showed dark red,the surface of lung lobes appeared rough,and some parts showed slight consolidation,with spot-like hyperemia and exudation.2.3 Microscope observation:Assessment of pathological damage to lung tissue of mice by HE staining.Alveolar epithelial cells in normal saline(0?g/ml)control group had basically normal morphology,no area of consolidation,and no alveolar atrophy or expansion,no hyperemia,Exudation and inflammatory cell infiltration;mice with different influenza virus hemagglutinin(2.5?g/ml and 5?g/ml)experimental group showed obvious pathological changes in lung tissue,including destruction of alveolar structure,widening of alveolar space and lung Diffuse tissue damage,congestion,coagulation,and edema accompanied by pathological changes such as infiltration of inflammatory factors.Conclusion:1.Influenza virus hemagglutinin can upregulate tissue factor genes and proteins of endothelial cells.2.In vivo animal experiments have further confirmed that influenza virus hemagglutinin causes changes in the lung coagulation system and tissue inflammatory pathological damage in mice.