| BackgroundMore evidence have shown that energy metabolism is closely related to the differentiation,function and lifespan of immune cells.When exposed to microenvironments such as tumors,microbial infections,and inflammatory,the cellular metabolic enzymes change,and immune cells reprogram energy metabolism to alter energy supply and modulate immune responses.Asthma is an inflammatory lung disease associated with innate and adaptive immune responses to abnormal allergens.Previous studies indicate that asthma manifests independent metabolic characteristics.Challenged with OVA or HDM in mouse bronchial epithelial cells and inflammatory immune cells,the important fatty acid oxidation metabolism(FAO)enzymes significantly increased.However,FAO has not been extensively studied in airway inflammation.It's suggested that inflammasomes are important components in innate immune inflammation.In the mouse model of asthma,NLRP3 inflammasome has been involved in allergic airway inflammation,however,NLRC4 inflammasome has not been studied in allergic airway inflammation.At the same time,in addition to acting as a flagellin receptor,the activation mechanism of NLRC4 inflammasome remains to be further studied.Our previous study have found that adoptive transferring different phenotypes of MDSCs derived from LPS induced by asthmatic mice could upregulate Treg cells,reduce IL-13 production,and inhibit airway inflammation in asthmatic mice.Whether MDSCs participate in the regulation of airway inflammation is related to cellular metabolism reprogramming remains unclear.Under the chronic inflammation microenvironment,whether FAO may regulate asthmatic immune response by activating NLRC4 inflammasome and regulating MDSCs may be the aim of our project.In this study,we use OVA,HDM induced asthma mouse model,and FAO inhibitor to treat asthmatic mice models and seek to analyse whether NLRC4 inflammasome and MDSCs are involved in the pathogenesis of asthma,thus study the inhibition of fatty acid oxidation in asthmatic mice NLRC4 inflammasome as well as the influence of different distributions of MDSCs,.seek to analyse the effect of FAO regulation on airway inflammation by modulating the expression of NLRC4 inflammasome and immunesuppressive MDSCs,therefore providing new ideas for the diagnosis and treatment of asthma.Objective 1)To explore Whether fatty acid oxidation inhibition can alleviate asthma airway inflammation;2)o investigate whether the NLRC4 inflammasome and downstream Caspase-1 molecules participate in the pathogenesis of asthma;the effect of fatty acid oxidation inhibition on the NLRC4 inflammasome and downstream Caspase-1 molecule in an asthma mouse model;3)To investigate whether MDSCs are involved in the pathogenesis of asthma;the effect of fatty acid oxidation inhibition on the distribution of MDSCs and their phenotypes in a mouse model of asthma.Methods 1)BALB/c mice were selected as subjects and randomly divided into control group(PBS group),OVA asthma group,HDM asthma group and FAO inhibitor intervention group.Among them,the OVA asthma group represents an ovalbumin injection to establish an asthmatic mouse model,the HDM asthma group represents the house dust mite-induced asthmatic mouse model,and the FAO inhibitor intervention group represents the addition of an FAO inhibitor to the HDM asthmatic mouse model,Rano.2)The manifestation each group during the challenging period were recorded.The airway hyperresponsiveness was reflected by the intermittent breathing value of mice(Penh value)which was dected by non-invasive lung function tester.We used semi-quantitative method to evaluate inflammatory cell infiltration in mouse lung HE stained sections,and thecondition responds to the severity of airway inflammation in mice.3)The expression of NLRC4 and Caspase-1 protein in lungs of mice was detected by immunohistochemistry.ImageJ software was used to semi-quantitatively evaluate the degree of staining of NLRC4 and CASP1 cells in immunohistochemical sections to reflect the level of protein.4)The expression of MDSCs and phenotypic PMN-MDSC and M-MDSC in the lung,spleen,bone marrow and peripheral blood of mice were measured by flow cytometry.Flowjo software was used for flow data analysis to evaluate MDSC and phenotype PMN-MDSC and M.-The proportion of MDSCs in response to different distributions of MDSCs in mice.Results1.Mice in asthma groups behaved apparent asthmatic symptoms,such as wheezing,shortness of breath,incontinence,and reduction of activity;whereas the symptoms in FAO inhibitor group were not obvious.2.Airway reactivity in mice:Mech concentrations and different modeling conditions have an effect on airway responsiveness in mice,and there is an interaction between them,that is,the use of 50mg/ml Mech concentration to induce OVA-induced asthma In the model,the airway responsiveness of mice was the highest(9.87±1.31).2).Compared with the blank control group,the Penh values of the mice in the OVA asthma group and the HDM asthma group were both increased.Among them,the P value of the OVA asthma group compared with the blank control group,P value<0.05,the difference was statistically significant;the Penh value of the mouse in the HDM asthma group compared with the blank control group,except in the concentration point of 3.125mg/ml There was no statistical difference(1.88±0.53 vs 1.09±0.24,P>0.05).There was a statistically significant difference in other Mech doses(P<0.05).At the same mech concentration,compared with the asthma group of HDM,the Penh values of the mice in the OVA asthma group were all increased,and there was a statistically significant difference between the two groups at the Mech dose of 12.5 and 25 mg/ml.After the FAO inhibitors intervened in the asthma model mice of HDM,their Penh values decreased significantly(P<0.05).Compared with the blank control group,the Penh value of the mice in the FAO inhibitor intervention group was significantly lower than the control group.There was no significant difference between the two(P>0.05).3.Airway inflammation in mice:Compared with the control group,the number of alveolar leukocytes in the OVA asthma group and the HDM asthma group increased,and the difference was statistically significant(3.14±1.08 vs 8.07±2.94,P<0.05).;3.14±1.08 vs 12.17±0.99,P<0.05).The nvumber of alveolar leukocytes in the HDM asthma group was higher than that in the OVA asthma group,with a statistically significant difference(8.07±2.94 vs 12.17±0.99,P<0.05).Compared with the asthma group of HDM,the number of alveolar leukocytes in the FAO inhibitor intervention group decreased,and the difference was statistically significant(12.17±0.99 vs 7.84±2.91,P<0.05).In mouse lung HE stained sections,the lung inflammation,central airway inflammation and peripheral airway inflammation in mice showed that the lung inflammation score was highest in the OVA asthma group,followed by the HDM asthma group.The lowest was in the control group,and there was a statistically significant difference between the three groups(P<0.05).After the intervention of FAO inhibitors in the asthmatic group of HDM,the inflammation of the whole lung,central airway inflammation and peripheral airway inflammation were significantly reduced in the three groups,and the difference was statistically significant(P<0.05).4.The expression of NLRC4 protein in mouse lung tissue:The bronchial epithelium,alveolar epithelium,and inflammatory cells(such as lung macrophages,eosinophils)of mouse lung have NLRC4 expression and are mainly expressed in cytoplasm..Among them,strong and positive expression of dark brown and yellow-brown NLRC4 proteins were observed in the OVA asthma group and the HDM asthma group,mainly in bronchial epithelium and inflammatory cells,while the FAO inhibition intervention group and the PBS group showed pale yellow and most of the sections.The cytoplasm is light blue.The H-fraction of NLRC4-positive cells in the asthma group was the highest,followed by the OVA group and the lowest in the control group.The values of the three groups were:130.94±6.34,126.18±12.37,56.37±3.85.Among them,compared with PBS group,the H scores of NLRC4 positive cells in HDM asthma group and OVA asthma group were statistically different(P<0.05);there was no significant difference between the two asthma models(P>0.05).Compared with HDM asthma group,the H score of NLRC4-positive cells in the FAO inhibition intervention group decreased,and there was a statistically significant difference(130.94±6.34 vs 76.78±4.37,P<0.05)5.Expression of Caspase-1 protein in mouse lung tissue:Caspase-1 expression was observed in bronchial epithelium,alveolar epithelium,and inflammatory cells(such as pulmonary macrophages and eosinophils)in mouse lungs.It is mainly expressed in the cytoplasm.Among them,the bronchial epithelial cells and inflammatory cells in the OVA asthmatic group and the HDM asthmatic group were all visible yellow-brown,indicating strong expression of Caspase-1 protein,while the FAO inhibition intervention group and the PBS group presented light brown.2.The H score of CASP1 positive cells in the OVA asthma group was the highest,followed by the HDM group and the lowest in the control group.The three component numbers were:94.32±7.31,84.48±3.03,56.64±1.90.Among them,compared with the PBS group,the H scores of HASP asthma group and OVA asthma group were statistically different(P<0.05);there was no significant difference between the two asthma models(P>0.05).Compared with HDM asthma group,the H score of CASP 1-positive cells in the FAO inhibition intervention group was decreased,and there was a statistically significant difference(84.48±3.03 vs 61.19±2.67,P<0.05).6.The distribution of MDSCs and their different phenotypes in mice:Compared with the control group,the proportion of MDSCs in the lungs of the OVA and HDM asthmatic mice increased,and the PMN-MDSC increased mainly,with statistical differences(P<0.05);compared with the two asthma models,the proportion of MDSCs and phenotype PMN-MDSC in the OVA group was higher(P<0.05),and the proportion of M-MDSC in the HDM asthma group was increased,and the difference was statistically significant(P<0.05).P<0.05).Compared with the asthma group of HDM,the inhibition of FAO increased the proportion of MDSCs and phenotype PMN-MDSC in lungs of mice,but the difference was not statistically significant(P>0.05).Compared with the control group,the proportion of MDSCs and phenotype PMN-MDSCs in the lung increased,and there was a statistically significant difference(P<0.05).Compared with the control group,the ratio of MDSCs in the spleen of 20VA and HDM asthma groups decreased(P<0.05),but there was no significant change in the ratio of PMN-MDSC and M-MDSC(P>0.05).There was no statistical difference between MDSCs and different phenotypes(P>0.05).Compared with the asthma group of HDM,the inhibition of FAO increased the proportion of MDSCs and phenotype PMN-MDSC in the spleen of asthmatic mice,the difference was statistically significant(P<0.05),but had no significant effect on the proportion of M-MDSC.3Compared with the control group,there was no significant change in the proportion of bone marrow and peripheral blood MDSCs and their phenotypes PMN-MDSC and M-MDSC in asthmatic mice with OVA and HDM.There was no significant difference between the two asthma models(P<0.05).Compared with the asthma group of HDM,the inhibition of FAO had no significant effect on the proportion of bone marrow,peripheral blood MDSCs and their phenotype in asthmatic mice(P>0.05).Conclusion1)OVA and HDM-induced asthma model can all induce asthma symptoms,increase airway hyperresponsiveness,and increase pulmonary inflammatory infiltration.The mouse model of asthma was successfully constructed.Among them,OVA asthma modelling method is better.2)The expression and activity of NLRC4 inflammasome in asthmatic mice increased significantly,leading to an increase in the proportion of MDSCs recruited to lung tissue and involvement in asthma.3)FAO inhibitors can significantly relieve asthma symptoms in asthmatic mouse models,reduce airway hyperresponsiveness,and reduce airway inflammation.At the same time,they can also significantly inhibit the expression and activity of NLRC4 inflammasome in asthmatic mice.The number and proportion of MDSCs have no effect.Inhibition of fatty acid oxidation may be a protective factor in the pathogenesis of asthma. |
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