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Objective The aetiology of Behçet's disease (BD), known as a systemic vasculitis, is not completely understood. Increasing evidence suggests that aberrant DNA methylation may contribute to the pathogenesis of BD. The aim of this epigenome-wide association study was to identify BD-associated methylation loci in Han Chinese. Methods Genome-wide DNA methylation profiles were compared between 60 BD patients and 60 healthy controls using the Infinium Human Methylation 450 K Beadchip. BD-associated methylation loci were validated in 100 BD patients and 100 healthy controls by pyrosequencing. Gene expression and cytokine production was quantified by real-time PCR and ELISA. Results A total of 4332 differentially methylated CpG sites were associated with BD. Five differentially methylated CpG sites (cg03546163, cg25114611, cg20228731, cg23261343 and cg14290576) revealed a significant hypomethylation status across four different genes (FKBP5 , FLJ43663 , RUNX2 and NFIL3) and were validated by pyrosequencing. Validation results showed that the most significant locus was located in the 5'UTR of FKBP5 (cg03546163, P = 3.81E-13). Four CpG sites with an aberrant methylation status, including cg03546163, cg25114611, cg23261343 and cg14290576, may serve as a diagnostic marker for BD (area under the receiver operating curve curve = 83.95%, 95% CI 78.20, 89.70%). A significantly inverse correlation was found between the degree of methylation at cg03546163 as well as cg25114611 and FKBP5 mRNA expression. Treatment with a demethylation agent, 5-Aza-2'-deoxycytidine resulted in an increase of FKBP5 mRNA expression and a stimulated IL-1β production. Conclusion Our findings suggest that aberrant DNA methylation, independently of previously known genetic variants, plays a vital role in the pathogenesis of BD. Trial registration Chinese Clinical Trial Registry, chictr.org.cn, ChiCTR-CCC-12002184. [ABSTRACT FROM AUTHOR]

As a traditional Chinese medicine, Artemisia argyi has been used medicinally and eaten for more than 2000 years in China. It is widely reported in treating inflammatory diseases such as eczema, dermatitis, arthritis, allergic asthma and colitis. Although several studies claim that its volatile oil and organic reagent extracts have certain anti-inflammatory effects, the water-soluble fractions and molecular mechanisms have not been studied. To evaluate the therapeutic effect of A. argyi water extract (AAWE) on lipopolysaccharide (LPS)-induced inflammatory responses and to identify the most effective water-soluble subfractions. Moreover, the relevant pharmacological and molecular mechanisms by which the active subfraction mitigates inflammation were further investigated. Firstly, RAW 264.7 cells stimulated with LPS were treated with AAWE (50, 100, and 200 μg/mL) or the water-soluble subfractions separated by D101 macroporous resin (AAWE1-AAWE4, 100 μg/mL), and NO production and mRNA levels of inflammatory genes were evaluated to determine the most effective water-soluble subfractions. Secondly, the chemical components of the active subfraction (AAWE4) were analyzed by UPLC-QTOF-MS. Thirdly, transcriptome and network pharmacology analysis, RT-qPCR and Western blotting assays were conducted to explore the underlying anti-inflammatory mechanism and active compounds of AAWE4. Subsequently, the binding ability of the potential active components in AAWE4 to the core targets was further determined by molecular docking. Eventually, the in vivo anti-inflammatory activity of AAWE4 (1.17, 2.34 and 4.68 g/kg, administered per day for 7 d) was evaluated in mice with LPS-induced systemic inflammation. In this study, AAWE showed excellent anti-inflammatory effects, and its water-soluble subfraction AAWE4 exhibited the strongest inhibitory effect on NO concentration and inflammatory gene mRNA expression after LPS stimulation, indicating that it was the most effective subfraction. Thereafter, four main compounds in AAWE4 were confirmed or tentatively identified by UPLC-QTOF-MS, including three flavonoid glycosides and one phenolic acid. Furthermore, the transcriptome and network pharmacology analysis showed that AAWE4 inhibited inflammation via multiple pathways and multiple targets. Based on the RT-qPCR and Western blotting results, AAWE4 downregulated not only the p38, PI3K, CCL5, MMP9, AP-1, and BCL3 mRNA expression levels activated by LPS but also their upstream and downstream protein expression levels and protein phosphorylation (p-AKT/AKT, p-p38/p38, p-ERK/ERK, p-JNK/JNK). Moreover, four identified compounds (isochlorogenic acid A, vicenin-2, schaftoside and isoschaftoside) could significantly inhibit NO content and the overexpression of inflammatory factors TNF-α, IL-1β, iNOS and COX-2 mRNA induced by LPS, and the molecular docking confirmed the high binding activity of four active compounds with selected core targets (p38, AKT1, MMP9, and CCL5). In addition, the mRNA expression and immunohistochemical analysis showed that AAWE44 could inhibit lung inflammation via multiple pathways and multiple targets in vivo. The findings of this study suggest that the water-soluble subfraction AAWE4 from A. argyi ameliorated the inflammation caused by LPS through multiple pathways and multiple targets in vitro and in vivo , providing scientific support for the medicinal use of A. argyi. Importantly, it shows that the A. argyi subfraction AAWE4 can be developed as an anti-inflammatory drug. [Display omitted] • A. argyi water extract (AAWE) alleviated LPS-induced inflammatory responses. • AAWE4, a water-soluble subfraction, showed the most effective anti-inflammatory effect. • AAWE4 ameliorated inflammation via regulating the MAPK/PI3K-AKT signaling pathways. • The effects of AAWE4 were attributed to flavonoid glycosides and phenolic acids. • AAWE4 inhibited lung inflammation via the MAPK/PI3K-AKT signaling pathways in vivo. [ABSTRACT FROM AUTHOR]

The key transcription factors of T helper cell subpopulations, including T-bet, GATA3, RORγt, and Foxp3 are involved in various autoimmune diseases. Whether methylation of these master transcription factors is associated with the development of experimental autoimmune uveitis (EAU) and the possible epigenetic regulatory mechanisms involved has however not yet been addressed. In our study, significant methylation changes in both Tbx21 and Rorc were observed in one CpG site in the retinas of EAU mice. Two CpG sites of Tbx21 and one CpG site of Rorc showed significant dynamic methylation changes in the RPE-choroid complex during EAU. The mRNA expressions of Tbx21 and Rorc in both the retinas and RPE-choroid complexes correlated with the methylation changes at the various time points during EAU development. The methylation changes were associated with the production of the Th1/Th17 cells’ signature cytokines, IFN-γ and IL-17. Dynamic changes in mRNA expression of DNA methyltransferases (DNMT1) were also noted, which may be related to the observed methylation changes of these genes. The present study provides evidence that DNA methylation of Tbx21 and Rorc may be associated with the development of EAU. DNMT1 activation may have an important effect on regulating DNA methylation dynamics. [ABSTRACT FROM AUTHOR]

Hepatitis B virus X protein (HBx) has been previously demonstrated to be associated with the regulation of cell proliferation; however, the exact mechanisms underlying this effect remain unclear. The present study aimed to investigate the regulatory mechanism of HBx on the cycle progression of primary renal tubular epithelial cells. Primary renal tubular epithelial cells of Sprague Dawley (SD) rats were separated and cultured. The morphology of cultured cells was characterized by immunohistochemical analysis and the results demonstrated that primary renal tubular epithelial cells with the expected morphology and distribution were successfully separated and cultured from SD rats. HBx gene pcDNA3.1/myc vector and empty vector were constructed and transfected into cells as HBx and empty groups, respectively. Following transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were determined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The results demonstrated that following HBx gene transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were significantly upregulated, compared with the empty control group (P<0.05). Furthermore, cell apoptosis and the cell cycle were evaluated by Annexin V‑fluorescein isothiocyanate/propidium iodide staining and flow cytometry. HBx gene transfection significantly inhibited the cell apoptosis (P<0.05), promoted cell cycle progression from the G1 to S phase and arrested the cell cycle in the S phase. Therefore, the results of the present study indicated that HBx gene transfection may regulate the apoptosis and cell cycle of primary renal tubular epithelial cells by affecting the expression of cyclins. The results of the present study may improve the understanding of pathogenesis associated with HBV‑associated glomerulonephritis, and may also provide insight and theoretical support for the future design and development of drugs for the treatment of hepatitis B virus. [ABSTRACT FROM AUTHOR]