Inhibition Of H3K27me3 Demethylases Attenuates Asthma And The Underlying Mechanisms

Objective:By far,a shift of airway smooth muscle cells(ASMCs)phenotype between proliferation and contraction during asthma has been well documented,highlighting a crucial role of ASMCs plasticity in the pathophysiology of this disease.As a critical event involved in epigenetic post-translational modification,histone H3 lysine27(H3K27)demethylation has obtained sufficient attraction in epigenetic changes of diverse cells while little is known about its contribution to the switching of ASMCs phenotype in asthma.Methods:In brief,40 SPF female BALB/c mice were randomly divided into 5 groups Mice were intranasally instilled with droplets containing purified HDM for 5 days/week for up to 5 consecutive weeks.During the last two consecutive weeks,GSKJ4,dexamethasone or a vehicle(DMSO)was administered intraperitoneally 1 h before the HDM challenge.Lung function was assessed by direct measurement of lung resistance using the FinePointe RC system.The total number of inflammatory cells and differential cell counts in the BALF were measured,respectively.The concentration of IgE in BALF and serum and cytokines or chemokines in BALF and lung homogenates were measured using ELISA kits.We applied haematoxylin and eosin staining(H&E)to assess airway inflammation,Periodic acid-Schiff(PAS)staining to measure goblet cells hyperplasia,Masson's trichrome staining to determine fibrosis.Moreover,the levels of smooth muscle actin alpha chain(?-SMA)and proliferating cell nuclear antigen(PCNA)were assessed by immunohistochemical staining(IHC).The cytotoxicity of GSK-J4 on human primary ASMCs was determined by cell counting kit-8(CCK-8)assay.The impact of GSK-J4 on PDGF induced ASMCs proliferation was measured by CCK-8 assay,EdU assay and flow cytometry,respectively.The impact of GSK-J4 on PDGF-elicited ASMCs synthesis and migration was measured utilizing ELISA assay,transwell assay and wound healing assay.Immunofluorescence and western blotting were used to determine the expression of ?-SMA and MLCK.The phosphorylation of JNK,ERK,p38,Akt and Smad3 was detected by western blotting.Results: Using the lungs harvested from the mice exposed to house dust mite(HDM),we found a significant demethylation in trimethylated H3K27(H3k27me3)sites.Administration with a selective inhibitor of H3K27 demethylase(GSK-J4)could notably ameliorate the classical hallmarks in asthmatic mice,such as airway hyperresponsiveness(AHR),airway inflammation and remodeling.Furthermore,we established a proliferative as well as a contractive model in human ASMCs to explore the impacts of an inhibition of H3K27 demethylase activity on ASMCs phenotype.The results indicated that GSK-J4 markedly alleviated ASMCs proliferation and migration elicited by platelet-derived growth factor(PDGF),probably via Akt/JNK signaling.GSK-J4 also prevented ASMCs from the upregulation of contractile proteins induced by transforming growth factor-?(TGF-?)with an inhibition of Smad3 pathway.Conclusion: Our findings demonstrated for the first time that H3K27me3 demethylation is pivotal for ASMCs phenotype variation.Inhibition of H3K27me3 demethylation suppressed proliferation,migration and contraction of ASMCs probabaly through modulating Akt/JNK and Smad3 signaling pathway,respectively.