Effects Of Insulin Receptor Expression On T Cell Activation And Allergic Airway Inflammation In Asthma

Objective:Helper T cells（Th cells）differentiation into Th2 is an important process of bronchial asthma.Studies have shown that insulin receptors cannot be detected on resting T lymphocytes,whereas adding insulin to T lymphocytes culturing in vitro can promote the activation and differentiation of T lymphocytes.However,the questions that whether T lymphocytes involved in asthma express insulin receptors and whether it is related to asthma inflammation have not been elucidated.The purpose of this study is to investigate the expression of insulin receptors on activated T lymphocytes in asthma and the relationship between insulin receptor expression and asthma inflammation,so as to provide a theoretical basis for further research on the mechanism of insulin receptor in the pathogenesis of asthma and the precise prevention and treatment of asthma.Methods:1.Grouping and model establishment.Twenty healthy female C57bl/6mice aged 6-8 weeks and weighing 18-20g were randomly assigned to two groups of equal numbers,including normal control group and OVA-sensitized/challenged group.On day 0,day 7,day 14 of the experiment,each mouse in the OVA-sensitized/challenged group was sensitized by intraperitoneal injection of 0.2ml OVA mixed solution which contained 50μg OVA and 20μl AL（OH）₃,and 180μl phosphate buffer（PBS buffer liquid）.On the day 21 to the day 27 of the experiment,the mice were placed in a transparent closed container and challenged with 3%OVA solution for 30 minutes each time through aerosol inhalation.The normal control group was sensitized and challenged with PBS buffer at the same time.Lung tissue,bronchoalveolar lavage fluid（BALF）and serum samples were collected within 24 hours after the last nebulized inhalation.2.Evaluation of inflammation in a mouse asthma model.（1）Determination of airway resistance in mice.A gradient concentration of acetylcholine was prepared as a bronchial stimulant,and the airway resistance value of mice was measured using a lung function instrument.（2）Pathological sections of lung tissues and hematoxylin-eosin staining（HE staining）.The left lower lobe of the mouse was collected,fixed,dehydrated,transparent,infiltrated,embedded,and pathologically sliced.Then,the HE staining method was used to observe the pathological changes of the lung tissue and evaluate the inflammation status of the lungs of the mouse.（3）Periodic acid Schiff’s staining（PAS staining）.The bronchial epithelial goblet cells in the lung tissue were stained purple with Periodic acid and Schiff reagent,so that we were able to observe the proliferation of goblet cells,and understand the severity of asthma.（4）Enzyme-linked immunosorbent assay（ELISA）was used to detect the level of Th1/Th2 cytokines in BALF and the level of immunoglobulin E（Ig E）in serum.We collected BALF and serum of mice in each group,centrifuged the supernatant,and applied ELISA kit to detect the content of Th2 type cytokine,Th1 type cytokine interferon-γ（IFN-γ）in BALF and serum Ig E of each group.3.The immunohistochemical staining and flow cytometry were used to investigate the expression of insulin receptor in the lung tissue and the expression of insulin receptors on T lymphocytes.（1）Immunohistochemical staining.Paraffin sections were repaired with antigens.Then,insulin antigen immunohistochemical antibodies and a two-step universal immunohistochemistry kit were used to bind the antigen and antibodies,and the tissues were covered with diaminobenzidine（DAB staining solution）to visualize the antigen-antibody complex.After mounting,the distribution of insulin receptors in the lung tissue were able to observe and the distribution of insulin receptors in the tissue sections of the two groups was compared.（2）Collection of single cell suspension of lung tissue specimens.Fresh lung tissue was shredded to a muddy shape.1640 medium which contained 2.5mg/ml type IV collagenase was added to the lung tissue,then placed in a 37°C constant temperature shaking box for digestion.Next,the lung tissue was gently ground on a 300 mesh nylon mesh,filtered and collected into centrifuge tubes.The red blood cells in the cell liquid were lysed with red blood cell lysate.Then the cell pellet was taken by centrifugation and resuspended in PBS buffer to count the cells.（3）Flow cytometry.The number of cells in the cell suspension were adjusted to 1×10⁶/100μl.A blank control tube,a CD4 flow-type fluorescent antibody single-stained control tube,and an IR flow-type fluorescent antibody single-stained control tube were set respectively.CD4-FITC fluorescent monoclonal antibody（FITC-conjunction CD4）was added to each flow tube and placed in a dark environment at 4°C for 20 minutes.After the cell suspension was washed three times with ice PBS,fixation/permeabilization solution was added to the cell suspension.The whole system reacted in the dark environment at 4℃for20min.Then,the permeation/washing solution was added to wash,and the antibody IR-PE fluorescent monoclonal antibody（PE-conjunction IR）were added to the cell suspension.The reaction was carried out at 4℃in the dark for 30minutes.Finally,the cell pellet was resuspended with an appropriate amount of solution containing 4%paraformaldehyde and stored in a refrigerator at 4℃waiting for detection by flow cytometry.4.Statistical methods.All data were analyzed using SPSS26.0 statistical software analysis method.The normal distribution measurement data was analyzed by two independent sample mean t test to compare the differences between groups and the statistical results were expressed as（?）±s.Linear correlation analysis was used to explore the relationship between insulin receptor expression and airway resistance,IL-4 levels,p<0.05 was considered statistically significant.Results:1.Results of determination of airway resistance in mice.After being challenged with methacholine solutions at concentrations of 6.25mg/ml,12.5mg/ml and 25mg/ml in the asthma group,the airway reactivity was significantly higher than that in the normal control group（p<0.05）.2.HE staining of lung tissue sections.Compared with mice in the normal control group,the airway epithelium in the OVA-sensitized/challenged group was significantly thickened,the lumen was narrowed,and there was a large amount of inflammatory cell infiltration around the bronchus.PAS staining results showed that the airway mucosa in the OVA-sensitized/challenged group was stained with purple-red goblet cells,and they were proliferative compared with the normal control group.3.Th1/Th2 cytokine content in the bronchial BALF of the two groups of mice.The content of Th2 cytokine IL-4 in the OVA-sensitized/challenged group was more than that in the normal control group,while the content of IFN-γwas less than that in the normal control group.The difference was statistically significant（p<0.01）.4.Serum Ig E content in two groups of mice.The asthma group had more serum Ig E content than the normal control group.The difference was statistically significant（p<0.01）.5.The expression of T lymphocyte insulin receptor in lung tissue of two groups of mice.It was observed by immunohistochemistry that the number and area of cells that positively expressed insulin receptors in the lung tissues of asthmatic mice were significantly higher than those in the normal control group.The results of flow cytometry showed that there were differences in the expression of insulin receptors of T lymphocytes in the two groups of mice.The expression of insulin receptors in the lung tissue of mice in the asthma group was significantly increased（p<0.01）.6.Correlation analysis results.The expression of insulin receptor in asthmatic mice was linearly correlated with the specific airway resistance value and IL-4level（r=0.978、0.936,p<0.01）.Conclusions:（1）T cells involved in asthma inflammation express insulin receptors,and may be one of the signs that T cells are activated;（2）When bronchial asthma develops,the level of insulin receptors expressed by T cells is positively correlated with the increase in airway resistance;（3）When bronchial asthma develops,the level of insulin receptors expressed by T cells is positively correlated with the degree of Th2 differentiation.