The Effect Of Potassium Channel KCNJ15 On Airway Inflammation In Allergic Asthma

Objective:To explore the effect of the inward rectifying potassium channel KCNJ15 on airway inflammation in asthmatic mice.Methods:1.Potassium Channel Proteins Screening.The potassium channels with altered expression were screened by RNA sequencing in an ovalbumin（OVA）-induced allergic asthma airway inflammation model.Twelve 8-week-old female C57BL/6J mice were selected,6 mice in the control group without treatment,and the other 6 mice were sensitized and stimulated with OVA to establish an allergic asthma airway inflammation model.After challenged 2weeks,the mice were killed and the lung samples with the strongest inflammatory infiltrate were selected for RNA sequencing according to the results of Hematoxylin and eosin（H&E）staining.RNA-sequencing data was analyzed to select the potassium channel gene with the most significant changes in expression.2.Analysis of KCNJ15 expression and localization.According to the Lung GENs data library and immunohistochemical staining results,the expression and localization of KCNJ15 in the lung was determined.3.Screening of adeno-associated virus（AAV）types that efficiently infected the lungs.Four adeno-associated viruses with GFP fluorescence,including AAV2,AAV6,AAV6.2 and AAV9,were instilled in the lungs of mice by tracheal instillation,and the samples were taken 10 days later.Immunofluorescence staining of frozen sections was performed to compare the infection location and infection efficiency of the four serotypes of AAV.4.Analysis of AAV Cell Transfection.Three KCNJ15 short hairpin RNA（sh RNA）plasmids with GFP fluorescence were transfected in NIH3T3 cells using PEI transfection reagent.NIH3T3 were grouped into KCNJ15 sh RNA-1group（transfected with KCNJ15 sh RNA plasmid 1）,KCNJ15 sh RN-2 group（transfected with KCNJ15 sh RNA plasmid 2）,KCNJ15 sh RN-3 group（transfected with KCNJ15 sh RNA plasmid 3）,and sh RNA-KCNJ15 control group（transfected with KCNJ15 sh RNA plasmid NC）and blank control group（normal culture without transfection）.After 48 hours of suspension transfection of cells in each group,GFP-positive cells were screened by flow cytometry,RNA was extracted,and real-time quantitative PCR Detecting System（q PCR）technology was used to detect KCNJ15 knock down efficiency.5.Construction of the plasmid overexpressing the target gene:The KCNJ15 overexpression plasmid with GFP fluorescence was transfected in293T cells using Attractene transfection reagent.The 293T cells were divided into KCNJ15 overexpression group（transfected with KCNJ15 overexpression plasmid）and blank control group（normal culture without transfection）.After 36h of transfection,the overexpression efficiency of KCNJ15 was detected by western blotting.6.OVA-induced airway inflammation modeling and AAV instillation:488-week-old female C57BL/6J mice were randomly divided into 6 groups（8mice in each group）:NC group,OVA group,OVA+sh RNA KCNJ15 group,OVA+sh RNA KCNJ15 NC group,OVA+KCNJ15 overexpression group and OVA+KCNJ15 overexpression NC group.The NC group was sensitized and challenged with normal saline,and the other groups were sensitized and challenged with OVA to create a model of allergic asthma airway inflammation.Tracheal instillation was performed on day 11 after sensitization.The NC group did not do any drip treatment.In the OVA group,50μl of sterile normal saline was instilled.The OVA+sh RNA KCNJ15 group was instilled with 50μl of AAV6-sh RNA KCNJ15 virus at 5×10¹¹ GC.The OVA+sh RNA KCNJ15 NC group was instilled with 50μl of AAV6-sh RNA KCNJ15 NC virus at 5×10¹¹GC.The OVA+KCNJ15 overexpression group was instilled with 50μl of5×10¹¹ GC AAV6-KCNJ15 overexpression virus.The OVA+KCNJ15overexpression NC group was instilled with 50μl of 5×10¹¹ GC AAV6-KCNJ15overexpression NC virus.7.Detection of airway hyperresponsiveness.24 hours after the last OVA nebulization,a tracheal inhalation challenge test was performed to detect airway hyperresponsiveness in mice.Methacholine concentrations were 50 mg/ml,25mg/ml,12.5 mg/ml,6.25 mg/ml,3.125 mg/ml and 0 mg/ml,respectively.The spirometer was set to adapt for 1 min,nebulize for 1 min,measure for 3 min,and rest for 1 min.Export data for subsequent analysis after measurement.8.Analysis of airway inflammation in asthmatic mice.Bronchoalveolar lavage fluid was taken to detect the number of total cells and the number of eosinophils;H&E staining was used to detect the infiltration of inflammatory cells around the pulmonary airways;Detection of airway goblet cell hyperplasia by PAS staining;ELISA was used to detect white blood cells in the bronchoalveolar lavage fluid The expression of inflammatory factors such as interleukin-4（IL-4）,interleukin-5（IL-5）,interleukin-13（IL-13）;ELISA Serum OVA-specific Ig E and OVA-specific Ig G1 levels were detected.9.KCNJ15 expression detection:24 h after AHR measurement,the mice were killed and the samples were taken.The right middle lobe lung RNA was extracted from 48 mice,and q PCR was used to detect the mice in the NC group,OVA group,OVA+sh RNA KCNJ15 group,OVA+sh RNA KCNJ15 NC group,OVA+KCNJ15 overexpression group and OVA+KCNJ15 overexpression NC group m RNA expression of KCNJ15 in lungThe effect of KCNJ15 on asthmatic airway inflammation was determined by detecting the above indicators.Results:1.RNA-sequencing data showed that the expression of the inward rectifying potassium channel KCNJ15 was increased in the lungs of mice with allergic asthma airway inflammation（P<0.05）.2.Lung GENs website data and immunohistochemical techniques determined that KCNJ15 is mainly expressed in mouse airway epithelial cells.By screening four AAV serotypes AAV2,AAV6,AAV6.2 and AAV9,it was found that AAV6 infects airway epithelial cells with the highest efficiency.3.Comparing the KCNJ15 sh RNA plasmid 1,KCNJ15 sh RNA plasmid 2and KCNJ15 sh RNA plasmid 3 knockdown efficiencies of KCNJ15,the results showed that KCNJ15 sh RNA plasmid 2 had relatively the highest knockdown efficiency of KCNJ15.The constructed plasmid overexpressing KCNJ15 was verified,and the results showed that it can effectively overexpress KCNJ15 in cells.4.In the OVA model of allergic asthma airway inflammation in mice,compared with the sh RNA KCNJ15 NC group,the airway hyperresponsiveness of the KCNJ15 knockdown group was significantly enhanced（P<0.05）,and the airway hyperresponsiveness of the KCNJ15 knockdown group was significantly enhanced（P<0.05）.In contrast,the airway hyperresponsiveness of mice in the KCNJ15 overexpression group was significantly attenuated（P<0.05）.After successful knockdown or overexpression of KCNJ15 in the lungs of mice detected by q PCR,bronchoalveolar lavage fluid cell count,H&E staining,PAS staining,bronchoalveolar lavage fluid IL-4,IL-5 and IL-13 contents,serum OVA Specific Ig E and OVA-specific Ig G1 levels were used to assess airway inflammation in asthmatic mice in knockdown group.Compared with the sh RNA KCNJ15 NC group,the number of total cells and eosinophils in the bronchoalveolar lavage fluid of the mice in the sh RNA KCNJ15 knockdown group increased（P<0.05）.Compared with the KCNJ15 overexpression NC group,KCNJ15 overexpression The total number of cells and eosinophils in the bronchoalveolar lavage fluid of mice in the group decreased（P<0.05）.The results of H&E staining showed that compared with the sh RNA KCNJ15 NC group,the inflammatory cell infiltration around the airway of the sh RNA KCNJ15 knockdown group was more serious,and compared with the KCNJ15overexpression NC group,the inflammatory cells around the airway of the KCNJ15 overexpression group were Infiltration is reduced.The results of PAS staining showed that compared with the sh RNA KCNJ15 NC group,the airway goblet cell proliferation in the sh RNA KCNJ15 knockdown group was aggravated,and compared with the KCNJ15 overexpression NC group,the airway goblet cell proliferation in the KCNJ15 overexpression group was reduced.The results of ELISA showed that compared with the sh RNA KCNJ15NC group,the levels of IL-4,IL-5 and IL-13 in the bronchoalveolar lavage fluid of the mice in the sh RNA KCNJ15 knockdown group were increased（P<0.05）.Compared with the NC group,the levels of IL-4,IL-5 and IL-13 in the bronchoalveolar lavage fluid of the mice in the KCNJ15 overexpression group were decreased（P<0.05）.The results of serum ELISA showed that compared with the sh RNA KCNJ15 NC group,the serum levels of OVA-specific Ig E and OVA-specific Ig G1 in the sh RNA KCNJ15 knockdown group were increased.Compared with the KCNJ15 overexpression NC group,the KCNJ15overexpression group was less The levels of OVA-specific Ig E and OVA-specific Ig G1 in mouse serum were decreased（P<0.05）.Conclusions:KCNJ15 is specifically highly expressed in the lungs of asthmatic mice.KCNJ15 knockdown in the lungs of allergic asthma mice aggravates airway hyperresponsiveness and airway inflammation.Overexpression of KCNJ15 in the lungs of allergic asthma mice reduces airway hyperresponsiveness and airway inflammation.