摘要: Objectives A20 exerts an anti-osteoclastogenic effect through the inhibition of NF-kappa B signalling in periodontitis. It also regulates autophagy via ubiquitin modification. This study was aimed at exploring the relationship between A20 and autophagy in anti-osteoclastogenesis in human periodontal ligament cells (hPDLCs) under hypoxia. Materials and Methods Real-time PCR and Western blot were used to detect relative mRNA and protein levels. The formation of autophagosomes was measured by TEM. Osteoclastic differentiation was assessed by TRAP staining and hydroxyapatite resorption assay. The interactions between different proteins were observed by co-IP. Results Cells cultured under 2% O-2 exhibited decreased A20 expression and increased RANKL/OPG (R/O) ratio. There was a negative correlation between A20 and TRAF6, and similar results were found with autophagic flux. A20 delayed the increase in R/O ratio under hypoxia. Autophagy in hPDLCs and osteoclast differentiation and hydroxyapatite resorption areas in mouse bone marrow mononuclear cells (BMMCs) were inhibited by A20. Moreover, inhibition of autophagy using 3-MA resulted in increased expression of A20 and decreased number and function of osteoclasts. In addition, A20 inhibited polyubiquitination at K63 and enhanced that at K48 in TRAF6 to suppress autophagy under hypoxic conditions. Conclusions A20 inhibits osteoclastogenesis via inhibition of TRAF6-dependent autophagy in hPDLCs under hypoxia. These findings suggest that A20 may be a key gene target during bone loss in periodontitis via TRAF6-mediated inhibition of autophagy.

摘要: Objective Embryo implantation needs a reciprocal interaction between competent embryo and receptive endometrium. Adenosine triphosphate (ATP) produced by stressed or injured cells acts as an important signalling molecule. This study aims to investigate whether adenosine triphosphate (ATP) plays an important role in the dialogue of human blastocyst-endometrium. Materials and methods The concentration of lactate was analysed in culture medium from human embryos collected from in vitro fertilization patients. Extracellular ATP was measured by ATP Bioluminescent Assay Kit. Ishikawa cells and T-HESCs were treated with ATP, ATP receptor antagonist, ATP hydrolysis enzyme or inhibitors of ATP metabolic enzymes. The levels of gene expression were evaluated by real-time PCR and immunoassay. Results We showed that injured human endometrial epithelial cells could rapidly release ATP into the extracellular environment as an important signalling molecule. In addition, blastocyst-derived lactate induces the release of non-lytic ATP from human endometrial receptive epithelial cells via connexins. Extracellular ATP stimulates the secretion of IL8 from epithelial cells to promote the process of in vitro decidualization. Extracellular ATP could also directly promote the decidualization of human endometrial stromal cells via P2Y-purinoceptors. More importantly, the supernatants of injured epithelial cells clearly induce the decidualization of stromal cells in time-dependent manner. Conclusion Our results suggest that ATP should play an important role in human blastocyst-endometrium dialogue for the initiation of decidualization.

摘要: Objectives Hypoxia is an important risk factor for pulmonary arterial remodelling in pulmonary arterial hypertension (PAH), and the Janus kinase 2 (JAK2) is believed to be involved in this process. In the present report, we aimed to investigate the role of JAK2 in vascular smooth muscle cells during the course of PAH. Methods Smooth muscle cell (SMC)-specific Jak2 deficient mice and their littermate controls were subjected to normobaric normoxic or hypoxic (10% O-2) challenges for 28 days to monitor the development of PAH, respectively. To further elucidate the potential mechanisms whereby JAK2 influences pulmonary vascular remodelling, a selective JAK2 inhibitor was applied to pre-treat human pulmonary arterial smooth muscle cells (HPASMCs) for 1 hour followed by 24-hour hypoxic exposure. Results Mice with hypoxia-induced PAH were characterized by the altered JAK2/STAT3 activity in pulmonary artery smooth muscle cells. Therefore, induction of Jak2 deficiency in SMCs protected mice from hypoxia-induced increase of right ventricular systolic pressure (RVSP), right ventricular hypertrophy and pulmonary vascular remodelling. Particularly, loss of Jak2 significantly attenuated chronic hypoxia-induced PASMC proliferation in the lungs. Similarly, blockade of JAK2 by its inhibitor, TG-101348, suppressed hypoxia-induced human PASMC proliferation. Upon hypoxia-induced activation, JAK2 phosphorylated signal transducer and activator of transcription 3 (STAT3), which then bound to the CCNA2 promoter to transcribe cyclin A2 expression, thereby promoting PASMC proliferation. Conclusions Our studies support that JAK2 could be a culprit contributing to the pulmonary vascular remodelling, and therefore, it could be a viable target for prevention and treatment of PAH in clinical settings.

摘要: Objectives The current study aimed to investigate the mechanism by which exosomes secreted by CHB patients with PNALT and liver inflammation grade (>= A2) affected the development of liver cancer. Materials and methods Gene expression was assessed by RT-PCR, Western blotting and immunohistochemistry. CCK-8, colony formation, transwell, scratch-wound and flow cytometry assays were used to detect cell viability, proliferation, apoptosis and metastasis. The interaction of TCF21 and HHIP was assessed by co-immunoprecipitation assay. Luciferase reporter was used to detect the combination of TCF21/HHIP and miR-25-3p. Xenograft studies in nude mice manifested tumour growth ability of miR-25-3p. Bioinformatics analyses were conducted using TargetScan, EVmiRNA, TCGA, GEO, DAVID,COEXPEDIA,UALCAN, UCSC and the Human Protein Atlas databases. Results CHB-PNALT-Exo (>= A2) promoted the proliferation and metastasis of HepG2.2.15 cells. miR-25-3p was upregulated in CHB-PNALT-Exo (>= A2). miR-25-3p overexpression promoted cell proliferation and metastasis and was related to poor survival in patients with CHB-PNALT (>= A2). The cell proliferation- and metastasis-promoting functions of CHB-PNALT-Exo (>= A2) were abolished by miR-25-3p inhibitors. TCF21 directly interacted with HHIP. Inhibition of TCF21 or HHIP promoted cell proliferation and metastasis. Knockdown of TCF21 or HHIP counteracted the effects of CHB-PNALT-Exo (>= A2) containing miR-25-3p inhibitor on cell proliferation, metastasis and the expression of Ki67, E-cadherin and caspase-3/-9. Conclusions Transfer of miR-25-3p by CHB-PNALT-Exo promoted the development of liver cancer by inhibiting the co-expression of TCF21 and HHIP.

摘要: Objectives To testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion. Materials and Methods In vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek-Cre) to finally obtain the following three genotypes: YAP(f/f), Tek-Cre; YAP(f/w), Tek-Cre; and YAP(f/f). Retinal vascularization and astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC-1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes. Results In vivo, YAP(f/f);Tek-Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet-derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC-1-astrocyte coculture. The effect of YAP overexpression on LIF-LIFR axis in HMEC-1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs. Conclusions We show that EC yes-associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production.