RAGE Mediates TDI-induced Airway Inflammation By Regulating Circadian Transcription Factor BMAL1 In Airway Epithelial

Background and purposeToluene diisocyanate(TDI)is one of the vital factors inducing asthma.In our previous research,we have reported that HMGB1/RAGE axis has abnormal regulation in airway epithelium in the development of TDI-induced asthma,but its mechanism has not yet been elucidated.The clinical manifestations of asthmatic patients have obvious circadian rhythm fluctuations.In recent years,more and more attention has been paid to the role of circadian clock in asthma,but exact mechanism of how circadian protein participates in airway inflammation induced by TDI has not been reported.This study hypothesizes that RAGE participates in the regulation of airway inflammation by mediating circadian clock function that is closely related to circadian rhythm of asthma.Methods1.In vivo experiments1.1 Male BALB/c mice were sensitized and challenged with TDI to establish a chemical-induced asthmatic model,and their lung tissue were used to detect differential gene expression by transcriptome profiling analyses,including Quality Control Testing,Hierarchical Clustering and so on.Gene set enrichment analysis,Gene co-expression analysis,Ingenuity Pathway Analysis were used to explore functions and pathways of differential gene expression.We selected the target genes and verified the differentially expressed target genes in control group and TDI group by real-time quantitative PCR.1.2 Male BALB/c mice were sensitized and challenged with TDI to establish a chemical-induced asthmatic model,and were divided into three groups:①AOO group,②TDI group,③TDI+FPS-ZM1 group.RAGE inhibitor(FPS-ZM1)were given intraperitoneally 2 hours before each challenge in group ③ while mice in group①and②were given 2%DMSO before each challenge.1.3(1)Blood samples were collected to measure total serum IgE and HMGB1 by ELISA;(2)Mice’s lymphocytes were taken off for culture,and the supernatants of cultured lymphocytes were harvested for detection of IL-4,IL-5 and IL-13 by ELISA;(3)Bronchoalveolar lavage fluid(BALF)was collected for total cells counting,and cytospin samples were made and stained with haematoxylin and eosin(H&E)for assessment of differential cell percentages in BALF;(4)Left lung sections were stained with H&E and periodic acid-Schiff(PAS)for histopathological assessment and were detected for BMAL1 by immunohistochemistry;(5)Right lung tissues were used to detect expression of RAGE,BMAL1 by western blot.2.In vitro experiments2.1 Wild-type human bronchial epithelial cells(16HBEs)and a RAGE knock-out 16HBEs were cultured and stimulated with TDI-HSA and rhHMGB1 respectively.Western blotting was for the detection of BMAL1 in 16HBEs.3.Data and statistical analysisStatistical analysis was performed using SPSS 22.0.Data are expressed as mean±Standard Deviation(SD)and were normally distributed.Comparisons among groups were analysed by Student’s t test or one-way ANOVA,followed by Bonferonni(equal variances assumed)or Dunnett’s T3(equal variances not assumed)post hoc test for multiple comparisons.P<0.05 was considered statistically significant.Key Results1.In vivo experiments1.1 Fold Change>1.5 and P<0.05 were used as the screening criteria for differentially expressed genes.In TDI-exposed mice,589 genes were up-regulated and 398 genes were down-regulated compared with control group.Pathways annotation and enrichment analysis based on KEGG&BioCarta revealed that these differentially expressed genes are mainly involved in mammal circadian rhythm,cytokine receptor interaction,intestinal immune network for IgA production and so on.The circadian rhythm pathway in mice was most affected among the pathways.1.2 The circadian clock genes were screened out according to Pathways enrichment analysis.Cluster analysis showed that expression of circadian genes in lung was obviously different from control group in TDI-induced mice,expression of BMAL1 and NPAS2 were significantly reduced while PERI,PER2,PER3 and NR1D1 were increased.Quantitative PCR showed that BMAL1 was down-regulated in lung of TDI-induced mice,and PER1 was abnormally increased.1.3 In TDI-exposed mice,Th2 cytokines from supernatants of cultured lymphocytes,serum IgE and HMGB1 increased significantly,and airway inflammation and goblet cell metaplasia were pronounced compared with AOO group,all of above can be blunted by FPS-ZM1.1.4 TDI exposure decreased expression of BMAL1 in airway epithelium,which was inhibited by FPS-ZM1 in immunohistochemistry and western blot analysis.2.In vitro experiments2.1 Compared with wild-type human bronchial epithelial cells,decrease of BMAL1 was attenuated in RAGE knockdown 16HBEs,which was caused by rhHMGB1.ConclusionsAbnormal expression of circadian clock can be found in TDI-induced asthmatic mice.HMGB1/RAGE axis may regulate airway inflammation by undermining expression of BMAL1.