The Role Of AKT Inhibitor MK2206 On Airway Inflammation And Airway Remodeling And Its Mechanism In TDI-induced Asthmatic Mouse Model

Background and Objective:Toluene diisocyanate(TDI)is a chemical intermediate used to make a variety of synthetic materials.TDI is also an important toxic chemicals that induces asthma in daily life.Previous studies have shown that AKT signaling pathway is of great importance in the pathogenesis of TDI-induced asthma,but its role in airway inflammation and remodeling of TDI-induced asthma is still unclear.MK2206 is a novel type of inhibitor of AKT activation.Whether it can relieve airway inflammation and airway remodeling in asthma and its related mechanism are still confused.Therefore,this study was to evaluate potential effects of MK2206 on airway inflammation and airway remodeling and related molecular mechanism in a TDI-induced asthmatic mouse model.Methods:1.In vivo experiments1.1 Male BALB/c mice were sensitized and challenged with TDI to establish a chemical-induced asthmatic model.Mice were distributed into 4 groups:(1)AOO group:mice were sensitized and challenged with acetone and olive oil;(2)TDI group;(3)TDI+MK2206 group:mice were sensitized and challenged with TDI and AKT inhibitor MK2206 were administered by gavage before each challenge;(4)TDI+DEX group:mice were sensitized and challenged with TDI,and before each challenge,mice were injected by dexamethasone intraperitoneally.1.2(1)Airway hyperresponsiveness of mice in each group was detected after stimulation with different concentrations of acetylmethyl choline;(2)Total serum IgE and inflammatory cytokine in BALF were detected by Elisa;(3)BALF was collected for total cells counting and assessment of differential cell percentages;(4)Stained with H&E staining,lung sections were planned to evaluate the infiltration of inflammatory cells;(5)Western blotting(WB)was used for detecting expression of HMGB1 in lung tissue.1.3(1)The proportion of goblet cells and the thickness of airway epithelial reticular basement membrane in lung tissue sections of mice in each group were detected by Periodic acid-Schiff stain(PAS);(2)Immunohistochemistry was used to detect not only the expression of α-SMA,but also expression of phosphorylated AKT(p-AKT)in lung tissue of mice;(3)WB was used to measure expression of Collagen I and smooth muscle actin(α-SMA),which were airway remodeling marker.2.In vitro experiment2.1 Stimulate by TDI-HSA complex,human airway epithelial cell line 16HBEs were used to detect expression of inflammatory molecule HMGB1 and airway remodeling molecule α-SMA.by WB..2.2 After pretreatment with AKT inhibitor MK2206 and DEX,16HBEs was stimulated with TDI-HSA,and expression of HMGB1 and α-SMA in cells was detected by western blot.The expression of HMGB1 and α-SMA in 16HBEs were assessed by immunofluorescence(IF)staining.3.Statistical methodsStatistical analysis of continuous or ordered data was performed using SPSS 20.0 or GraphPad Prism 8,respectively.The experimental data were expressed as mean±SEM.The ranking data is represented by quarterback spacing.The differences of continuous data between multiple groups was compared,and the Bonferroni post hoc test was performed after one-way ANOVA.Kruskal-wallis test and Dunn’s multiple comparison test were used for ordered data.P<0.05 was considered statistically significant.Results:1.In vivo experiments:L1 TDI-induced asthma mouse model was successfully constructed:Compared with the control group,airway reactivity in TDI-induced asthma mice was significantly increased,and inflammatory factors(IL-4,IL-5,IL-6,and IL-13)and serum total IgE levels in bronchalveolar lavage fluid were significantly increased.Lung inflammatory cell infiltration increased significantly.Lung tissue sections stained with HE staining showed that around airway and blood vessels,there were plenty of inflammatory cells in TDI group,and scores of peribronchial inflammation and perivascular inflammation were higher than those in control group.Meanwhile,HMGB1 in lung tissue of TDI group was up-regulated.After pretreatment with MK2206 or DEX,airway hyperresponsiveness of TDI asthmatic mice decreased,and inflammatory factors(IL-4,IL-5,IL-6,and IL-13)in BALF and serum total IgE levels decreased.And IL-6 in DEX group decreased more significantly than that in MK2206 group.MK2206 can reduce inflammatory cells in BALF and effectively relieve infiltration of inflammatory cells in lung tissue.The peri-vascular inflammation score of the MK2206 group are lower than those of the TDI group.It was shown that MK2206 or DEX markedly reduced HMGB1 in lung homogenates of TDI asthmatic mice,and PHMGB1 in MK2206 group was decreased evidently than that in DEX group.L2 Compared with mice in control group,PAS staining showed that aggravated goblet cell metaplasia and airway epithelial reticular basement membrane thickness were increased in TDI-induced asthmatic mice.Immunohistochemistry showed expression of α-SMA under the airway epithelium was obviously increased in TDI group,and phosphorylation of AKT in airway epithelium of also increased.WB fund that collagen I and α-SMA in lung of TDI group was up-regulated,which was consistent with the increasing phosphorylation of AKT.After pretreatment with MK2206 or DEX,airway epithelial goblet cell metaplasia and thickness of airway epithelial reticular basement membrane in TDI asthmatic mice were decreased.The decrease of thickness of airway epithelial reticular basement membrane was less in TDI+MK2206 group than TDI+DEX group.Immunohistochemistry showed that MK2206 or DEX treatment reduced expression of α-SMA and phosphorylation of AKT in the airway epithelium of TDI-induced asthma,and efficacy of the two was equivalent.WB showed that in inhibiting expression of α-SMA in lung tissue of TDI-induced asthmatic mice,MK2206 and DEX showed the similar efficacy.MK2206 was more effective than DEX in inhibiting HMGB1,collagen I and AKT phosphorylation.2.In vitro experiments:2.1 HMGB1 and α-SMA in TDI-HSA group had higher expressions than those in control group,so does the phosphorylation of AKT.MK2206 or DEX pretreatment can reduce the up-regulation of HMGB1 induced by TDI-HSA in 16HBEs,but MK2206 is less effective than DEX in vitro.2.2 The intracellular HMGB1 and α-SMA in 16HBEs induced by TDI-HSA were more than those of control group.MK2206 or DEX pretreatment weakened the fluorescence signal intensity in the cytoplasm of 16HBEs,and diminished the expression of HMGB1 and α-SMA.Conclusion:MK2206 relieves airway inflammation and airway remodeling in TDI-induced asthmatic mice.The mechanism may be related to restraining AKT signaling pathway,suppressing the amount of HMGB1 and limiting epithelial-mesenchymal transition(EMT).