Correlation Between LncRNA MALAT1 And Th17/Treg Cell Balance In Bronchial Asthma Mice

Background:Bronchial asthma,a complex and heterogeneous disease,which characterized by chronic airway inflammation in which multiple cells(eosinophils,mast cells,neutrophils,etc.)and cytokines(IFN-γ,IL-4,and IL-2)play important roles.The pathogenesis of bronchial asthma has attracted much attention,but there is still a lack of complete explanation.It is now widely accepted that the pathogenesis of bronchial asthma is that in the process of bronchial asthma,T lymphocytes will differentiate to Th2,release Th2 type cytokines,and reduce the differentiation to Th1 cells,the secretion of Th1 type cytokines is reduced,and Th1/Th2 cell dysfunction is unbalanced.However,studies have found that the bronchoalveolar lavage fluid in patients with moderate and severe bronchial asthma has increased neutrophilia,Th17-related cytokines(IL-17 A,IL-17F)and IL-22.Recently,it has been found that Th17 cell/Treg cell imbalance plays a very significant role in the progression of bronchial asthma,but its specific mechanism has not been clarified.The function of non-coding Rnas(lncrnas)has become a focus of research.Metastasis associated with human lung denocarcinoma transcript 1(MALAT1),as a member of lnc RNA,has close relationship with the occurrence and development of many allergic diseases.Its regulatory effect has received extensive attention in the study of various allergic diseases.Objective:This study aims to establish a bronchial asthma model,detect Th17/Treg cell level,expression level of related cytokines and relative expression level of lnc RNA MALAT1 in spleen cells of bronchial asthma mice,and study the influence of lnc RNA MALAT1 on Th17/Treg balance.By clarifying the relationship between the two,To explore the potential mechanism and relationship between lnc RNA MALAT1 and Th17 and Treg cell balance as well as the expression level of related cytokines.Methods:1.Establishment of bronchial asthma mouse model.The BALB/c mice were divided into two groups.At day 0,7 and 14,mice in the control group were intraperitoneally injected with0.2m L phosphate buffer(PBS)solution,and mice in the asthma group were intraperitoneally injected with 0.2m L Ovalbumin(OVA)mixture.The asthma group was given 2.5%OVA solution for asthma stimulation from the 21 st day,once a day,40 min each time,lasting until the27 th day for 7 days.All mice were killed the day after the final atomization.The changes of lung inflammation were observed by HE staining and PAS staining under microscope.2.Under microscope,HE and PAS staining were used to observe the Pulmonary inflammatory changes in mice.3.The expressions of IL-17 and Foxp3 in mouse airway epithelial cells were detected by immunohistochemistry.4.Mouse spleen single cell suspension was prepared.After staining with CD4,CD25,CD45,Forkhead/winged helix transcription factor 3(Foxp3),Retinoic acid receptor related orphan C(Rorc)and IL-17 antibody,the proportions of Th17 and Treg cells were determined by flow cytometry.5.Serum ovalbumin specific immunoglobulin E(Ig E)was detected by ELISA.6.Total RNA was extracted from mouse spleen tissue,and reverse transcription RNA is c DNA to detect the expression of lnc RNA MALAT1,IL-17,IL-4,Rorc and Foxp3,and the expression of MALAT1,Rorc/Foxp3 was then standardized with U6.Results1.HE staining results showed,the airway inflammation changes were significant in OVAinduced bronchial asthma mice.Inflammatory cells infiltration could be found near the bronchus and blood vessels,the wall of the bronchial wall was narrowed and thickened,and the airway epithelial cells were arranged irregularly.PAS staining showed that the bronchial goblet cells and mucus secretion increased in asthma group.Meanwhile,the proportion of type 2 natural lymphocytes(ILC2)in spleen cells of asthmatic mice increased(P < 0.0001)and the content of obumin specific Ig E increased(P < 0.01).2.Compared with the control group,the number of Th17 cells and Treg cells in spleen single-cell suspension in asthma group was significantly increased(P < 0.0001),and the proportion of Th17/Treg was significantly increased(P < 0.01).Correspondingly,the expression of Rorc in Th17 cells was up-regulated(P<0.01),while the expression of Foxp3 in Treg cells was down-regulated(P<0.05).3.In the asthma group,the average IL-17 positive area of airway epithelial cells was significantly greater than in the control group(P<0.01),while the Foxp3 positive area of airway epithelial cells was lower in the asthma group(P<0.01).4.Compare with control group,The relative expression level of IL-17 m RNA(P<0.0001)and IL-4 m RNA(P<0.0001)in lung tissue of asthmatic group was significantly increased.5.Compare with control group,The relative expression level of lnc RNA MALAT1 in spleen tissue of asthma group was significantly increased(P<0.0001).6.With the increase of lnc RNA MALAT1 expression and Th17 cell expression in spleen tissue of mice,there was a positive correlation(r = 0.64,P<0.05),and the decrease of Treg cell expression was a negative correlation(r =-0.73,P<0.01),and the proportion of Th17/Treg increased.There was a positive correlation(r = 0.85,P<0.001).7.With the increase of lnc RNA MALAT1 expression,the expression of key transcription factor Rorc in Th17 cells was increased,which was positively correlated(r = 0.65,P<0.05).Treg cells exhibited a decrease in the expression of Foxp3,a critical transcription factor,with a negative correlation(r =-0.60,P<0.05).8.With the increase of lnc RNA MALAT1 expression in spleen tissue of mice,the expression of IL-4,a key cellular factor in Th2 cells,was increased,with positive correlation(r =0.84,P<0.01).The expression of IL-17,a key cytokine in Th17 cells,was positively correlated with that in Th17 cells(r = 0.89,P<0.0001).Conclusion:1.Dysregulation of Th17/Treg cells was found in bronchial asthma mice,and lnc RNA MALAT1 was highly expressed in spleen tissue of bronchial asthma mice.2.High expression of lnc RNA MALAT1 is closely related to Th17/Treg cell balance,Rorc/Foxp3,IL-4 and IL-17 in bronchial asthma mice.lnc RNA MALAT1 was positively correlated with Th17 cells,related transcription factor Rorc,its related cytokine IL-17 and Th17/Treg ratio,and negatively correlated with Treg cells and related transcription factor Foxp3.