Role And Mechanism Of DNA Methylation In Chronic Spontaneous Urticaria

Background: Chronic spontaneous urticaria(CSU)is a common skin disease,which is mainly driven by mast cells and is a hot spot of clinical attention.The classic activation of mast cells is mediated by IgE leading to the aggregation and cross-linking of FcεRI receptor,resulting in a series of cascade amplification reactions,leading to degranulation and the release of inflammatory factors.At present,the first-line drug for CSU treatment is the second-generation non sedative H1 antihistamines,but one third of patients will still have ineffective antihistamines.DNA methylation is one of the epigenetic regulation mechanisms,which affects the occurrence and development of diseases.Studies have shown that DNA methylation is one of the key mechanisms of mast cell activation.IgE receptor crosslinking decreased the overall DNA methylation level of mast cells.Recent literature suggests that DNA methylation sites are changed in CSU,but there is no in-depth study.Objective: This study aims to explore the role and mechanism of DNA methylation in CSU and antihistamine resistance.Methods:(1)The peripheral blood DNA of healthy controls and CSU patients was extracted,treated with bisulfite,and the transformed DNA was sequenced by 850 k chip.(2)Using p value and the beta value obtained by sequencing and statistical analysis,the differential methylation sites of healthy controls and CSU patients were calculated.(3)Go and KEGG analysis of differential methylation sites.(4)Pyrophosphate assay to verify differential methylation position.(5)RT-PCR was used to detect whether the annotated genes were different.(6)The level of plasma TSP-1 in CSU patients was detected by ELISA.(7)The degranulation efficiency of mast cells was detected by β-hex release test.Results:(1)There were 2142 differential methylation sites between the healthy control group and the CSU disease group.Compared with the control group,the methylation level of 1804 sites in the disease group decreased and 338 sites increased.(2)Pyrosequencing showed that the methylation level of CD47 decreased and the mRNA expression was up-regulated in the disease group.(3)The expression of TSP-1 was up-regulated in plasma and peripheral blood of CSU patients.Through biological information analysis,it was found that the expression of TSP-1 was up-regulated in CSU patients.(4)TSP-1 can promote degranulation of mast cells and up regulate cytokine mRNA levels.(5)2778 differential methylation sites were screened between the effective group and the ineffective group.Compared with the ineffective group,the methylation level of 1872 sites in the effective group decreased and 906 sites increased.(6)Pyrosequencing confirmed that the methylation levels of LCLAT1 and MICA in the effective group decreased,while the mRNA expression was up-regulated.Conclusion:(1)Compared with the control group,the methylation levels of 1804 sites decreased and 338 sites increased in the CSU disease group.Compared with the antihistamine ineffective group,the methylation level of 1872 sites decreased and 906 sites increased in the effective group.(2)Pyrosequencing verified that the methylation level of differential site cg09643587 decreased in CSU disease group,annotated to CD47,and differential sites cg17749961 and cg12182649 decreased in effective group,annotated to lclat1 and mica.(3)CD47 and TSP-1 were up-regulated in CSU patients.TSP-1 is positively correlated with UAS7 score,which can promote mast cell degranulation and cytokine mRNA expression.